AU2021283199B2 - Methods of treating aging-related disorders - Google Patents

Abstract

Provided herein are methods of killing or reducing the number of naturally-occurring and/or treatment-induced senescent cells and diseased cells in a subject in need thereof, decreasing the accumulation of naturally-occurring and/or treatment-induced senescent cells and diseased cells in a subject in need thereof, that include administering to the subject a therapeutically effective amount of one or more common gamma-chain family cytokine receptor activating agent(s) and/or one or more agent(s) that result(s) in a decrease in the activation of a TGF-β receptor.

Description

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METHODS OF TREATING AGING-RELATED DISORDERS CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application Serial No.

63/032,933, filed on June 1, 2020, International Patent Application No.

PCT/US2020/035598, filed on June 1, 2020, and U.S. Provisional Patent Application

Serial No. 63/118,536, filed on November 25, 2020, which is incorporated herein by

reference in its entirety.

TECHNICAL FIELD The present disclosure relates to the field of immunology and cell biology.

BACKGROUND Senescence is a form of irreversible growth arrest accompanied by phenotypic

changes, resistance to apoptosis, and activation of damage-sensing signaling pathways.

Cellular senescence was first described in cultured human fibroblast cells that lost their

ability to proliferate, reaching permanent arrest after about 50 population doublings

(referred to as the Hayflick limit). Senescence is considered a stress response that can be

induced by a wide range of intrinsic and extrinsic insults, including oxidative and

genotoxic stress, DNA damage, telomere attrition, oncogenic activation, mitochondrial

dysfunction, or chemotherapeutic agents.

Senescent cells remain metabolically active and can influence tissue hemostasis,

disease, and aging through their secretory phenotype. Senescence is considered as a

physiologic process and is important in promoting wound healing, tissue homeostasis,

regeneration, and regulation of fibrosis. For instance, transient induction of senescent

cells is observed during would healing and contributes to wound resolution. Senescence

also plays a role in tumor suppression. The accumulation of senescent cells also drives

aging and aging-related diseases and conditions. The senescent phenotype also can

trigger chronic inflammatory responses and consequently augment chronic inflammatory

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conditions to promote tumor growth. The connection between senescence and aging was

initially based on the observation that senescent cells accumulate in aged tissue. The use

of transgenic models has enabled the detection of senescent cells systematically in many

aging-related disorders. Strategies to selectively eliminate senescent cells have

demonstrated that senescent cells play a causal role in aging-related disorders.

Cellular senescence is a series of progressive and phenotypically diverse cellular

states that are acquired after initial growth arrest (van Deursen, Nature 509(7501):439-

446, 2014) Thus, senescent cells are heterogeneous populations of cells with few shared

core properties (Dou et al., Nature 550(7676):402-406 2017). Identifying common

senolytic drug targets, therefore, is difficult. This further precludes the achievement of a

goal of developing senolytics that selectively, safety, and effectively eliminate senescent

cells upon systemic administration. As described above, immune cells are the effector

cells to remove senescent cells naturally after the fulfillment of senescent-cell

physiological roles. (Brighton et al., Elife 6, 2017) The weakening of the immune system

during the aging process allows the accumulation of senescent cells. (Karin et al., Nat.

Comm. 10(1):5495, 2019; Chambers et al., J. Allergy Clin. Immunol. 145(5):1323-1331,

2020).

SUMMARY SUMMARY The present invention is based on the discovery that subcutaneous administration

of an agent that results in a decrease in the activation of a TGF-B receptor or a common

gamma-chain family cytokine receptor activating agent (e.g., complexes of gamma-chain

cytokines and their cognate receptors) to a mammal promotes and activates immune cells

to regain their capabilities of reducing senescent cells in vivo effectively, selectively, and

safely. In view of this discovery, provided herein are methods of killing or reducing the

number of naturally-occurring and/or treatment-induced senescent cells in a subject that

include administering to the subject a therapeutically effective amount of one or more

agent(s) that result(s) in a decrease in the activation of a TGF-B receptor. Also provided

herein are methods of decreasing the accumulation of naturally-occurring and/or

treatment-induced senescent cells in a subject that include administering to the subject a

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therapeutically effective amount of one or more agent(s) that result(s) in a decrease in the

activation of a TGF-B receptor. Also provided herein are methods of decreasing a level

of a marker of naturally-occurring and/or treatment-induced senescent cells in a subject

that include administering to the subject a therapeutically effective amount of one or

more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor. Also

provided herein are methods of reducing the activity of naturally-occurring and/or

treatment-induced senescent cells in a subject that include administering to the subject a

therapeutically effective amount of one or more agent(s) that result(s) in a decrease in the

activation of a TGF-B receptor. Also provided herein are methods of decreasing levels

and/or activity of one or more senescence associated secretory phenotype(*SASP")

factor(s) derived from naturally-occurring and/or treatment-induced senescent cells in a

subject that include administering to the subject a therapeutically effective amount of one

or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor.

Also provided herein are methods of killing and reducing the number of naturally-

occurring and/or treatment-induced senescent cells (and methods of decreasing the

accumulation or reducing markers of senescent cells) in a subject that include

administering to the subject a therapeutically effective amount of one or more common

gamma-chain family cytokine receptor activating agent(s) (e.g., complexes of gamma-

chain cytokines and their cognate receptors). Also provided herein are methods of

reducing the activity of naturally-occurring and/or treatment-induced senescent cells in a

subject that include administering to the subject a therapeutically effective amount of one

or more common gamma-chain family cytokine receptor activating agent(s). Also

provided herein are methods of decreasing levels and/or activity of one or more SASP

factor(s) derived from naturally-occurring and/or treatment-induced senescent cells in a

subject that include administering to the subject a therapeutically effective amount of one

or more common gamma-chain family cytokine receptor activating agent(s).

The present invention is also based on the discovery that administration of NK

cell activating agents to a mammal having a cancer resulted in a tumor inhibition and

administration of NK cell activating agents to a diabetic animal model demonstrated

improved skin and hair appearance and texture, and decreased blood glucose levels. In

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view of this discovery provided herein are methods of treating an aging-related disease or

condition in a subject in need thereof that include administering to a subject identified as

having an aging-related disease or condition a therapeutically effective amount of one or

more natural killer (NK) cell activating agent (s) and/or a therapeutically effective

number of activated NK cells. Also provided herein are methods of killing or reducing

the number of senescent cells in a subject in need thereof that include administering to

the subject a therapeutically effective amount of one or more NK cell activating agent(s)

and/or or a therapeutically effective number of activated NK cells. Also provided herein

are methods of improving the texture and/or appearance of skin and/or hair in a subject in

need thereof over a period of time that include administering to the subject a

therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s) and/or a therapeutically effective number of activated NK cells. Also provided

herein are methods of assisting in the treatment of obesity in a subject in need thereof

over a period of time that include administering to the subject a therapeutically effective

amount of one or more natural killer (NK) cell activating agent(s) and/or a therapeutically

effective number of activated NK cells.

Provided herein are methods of killing or reducing the number of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing the accumulation of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing a level of a marker of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of reducing the activity of naturally-occurring

and/or treatment-induced senescent cells in a subject that include administering to the

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subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing levels and/or activity of one or

more SASP factor(s) derived from naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more agent(s) that result(s) in a decrease in the activation of a

TGF-B receptor.

In some embodiments of any of the methods described herein, the subject has

been previously diagnosed or identified as having an aging-related disease or an

inflammatory disease. In some embodiments of any of the methods described herein, the

aging-related disease is inflamm-aging related. In some embodiments of any of the

methods described herein, the aging-related disease is selected from the group of:

Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,

hypertension, inflammatory bowel disease, intervertebral disc degeneration,

osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis,

cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis,

loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia,

cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-associated loss of

lung tissue elasticity, age-related macular degeneration, cachexia, glomerulosclerosis,

liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's

disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer,

dementia, vascular disease, infection susceptibility, chronic inflammation, and renal

dysfunction. In some embodiments of any of the methods described herein, the aging-

related disease is a cancer selected from the group consisting of: solid tumor,

hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma,

B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic

lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia

(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),

cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung

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cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate

cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma,

squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver

cancer, and hepatocellular carcinoma. In some embodiments of any of the methods

described herein, the inflammatory disease is selected from the group of: rheumatoid

arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic

nephropathy, CNS injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral

sclerosis, Crohn's disease, multiple sclerosis, Guillain-Barre syndrome, psoriasis,

Grave's disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders and

cancer treatment-related cognitive impairment.

In some embodiments of any of the methods described herein, the treatment-

induced senescent cells are chemotherapy-induced senescent cells. In some embodiments

of any of the methods described herein, the administration of the one or more agent(s)

that result(s) in a decrease in the activation of a TGF-B receptor results in a decrease in

the number or activity of naturally-occurring senescent cells and/or treatment-induced

senescent cells in a target tissue in the subject. In some embodiments of any of the

methods described herein, the target tissue is selected from the group of: adipose tissue,

pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone

tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and

secondary lympho-organ tissue.

In some embodiments of any of the methods described herein, the TGFß receptor

is a TGF-B receptor II (TGFBRII). In some embodiments of any of the methods

described herein, the TGFB receptor is a TGFBRIII.

In some embodiments of any of the methods described herein, at least one of the

one or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor is a

soluble TGF-B receptor, an extracellular domain of TGF-B receptor, an antibody that

binds specifically to TGF-B, an antagonistic antibody that binds to a TGF-B receptor, an

agent that binds to a latency-associated peptide ("LAP"), or an agent that binds to a TGF-

B/LAP complex. In some embodiments of any of the methods described herein, the one

or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor

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decrease(s) the activation of a TGF-B receptor through binding to a LAP, or to a TGF-

B/LAP complex.

In some embodiments of any of the methods described herein, at least one of the

one or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor is a

multi-chain chimeric polypeptide including: (a) a first chimeric polypeptide comprising:

(i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first

domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a

second domain of a pair of affinity domains; and (ii) a second target-binding domain,

where one or both of the first target-binding domain and the second target-binding

domain binds specifically to a ligand of a TGF-B receptor; or one or both of the first

target-binding domain and the second target-binding domain is an antagonistic antigen-

binding domain that binds specifically to a TGF-B receptor. In some embodiments of any

of the methods described herein, the TGF-B receptor is TGFBRII. In some embodiments

of any of the methods described herein, the TGF-B receptor is TGFBRIII.

In some embodiments of any of the methods described herein, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some embodiments of any of the methods described herein, the

first chimeric polypeptide further includes a linker sequence between the first target-

binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain and the first domain of the pair of affinity domains directly abut each other

in the first chimeric polypeptide. In some embodiments of any of the methods described

herein, the first chimeric polypeptide further includes a linker sequence between the

soluble tissue factor domain and the first domain of the pair of affinity domains in the

first chimeric polypeptide.

In some embodiments of any of the methods described herein, the second domain

of the pair of affinity domains and the second target-binding domain directly abut each

other in the second chimeric polypeptide. In some embodiments of any of the methods

described herein, the second chimeric polypeptide further includes a linker sequence

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between the second domain of the pair of affinity domains and the second target-binding

domain in the second chimeric polypeptide.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

antigen. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to different

antigens. In some embodiments of any of the methods described herein, the first

chimeric polypeptide further comprises one or more additional target-binding domain(s).

In some embodiments of any of the methods described herein, the second chimeric

polypeptide further comprises one or more additional target-binding domain(s).

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is a soluble human tissue factor domain. In some embodiments of any of

the methods described herein, the soluble human tissue factor domain comprises a

sequence that is at least 80% identical to SEQ ID NO: 93.

In some embodiments of any of the methods described herein, the pair of affinity

domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a

soluble IL-15. In some embodiments of any of the methods described herein, the soluble

IL-15 has a D8N or D8A amino acid substitution. In some embodiments of any of the

methods described herein, the soluble IL-15 comprises a mutation to reduce or eliminate

IL-15 activity.

In some embodiments of any of the methods described herein, the pair of affinity

domains is selected from the group of: barnase and barnstar, a PKA and an AKAP,

adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules

based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and

SNAP25. In some embodiments of any of the methods described herein, the first domain

or the second domain of a pair of affinity domains is a soluble common gamma-chain

family cytokine or an antigen-binding domain that binds specifically to a common

gamma-chain family cytokine receptor.

In some embodiments of any of the methods described herein, the first target-

binding domain and/or the second target-binding domain include a soluble TGF-B

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receptor. In some embodiments of any of the methods described herein, the soluble TGF-

receptor is a soluble TGFBRII. In some embodiments of any of the methods described

herein, the soluble TGFßRII includes a first sequence that is at least 80% identical to

SEQ ID NO: 183, and a second sequence that is at least 80% identical to SEQ ID NO:

183, wherein the first and second sequence are separated by a linker. In some

embodiments of any of the methods described herein, the soluble TGFßRII comprises a

first sequence that is at least 90% identical to SEQ ID NO: 183, and a second sequence

that is at least 90% identical to SEQ ID NO: 183. In some embodiments of any of the

methods described herein, the soluble TGFßRII includes a first sequence of SEQ ID NO:

183, and a second sequence of SEQ ID NO: 183. In some embodiments of any of the

methods described herein, the linker includes a sequence of SEQ ID NO: 102. In some

embodiments of any of the methods described herein, the soluble TGFßRII includes a

sequence that is at least 80% identical to SEQ ID NO: 188. In some embodiments of any

of the methods described herein, the soluble TGFBRII includes a sequence that is at least

90% identical to SEQ ID NO: 188. In some embodiments of any of the methods

described herein, the soluble TGF-BRII includes a sequence of SEQ ID NO: 188.

In some embodiments of any of the methods described herein, the first chimeric

polypeptide includes a sequence that is at least 80% identical to SEQ ID NO: 236. In

some embodiments of any of the methods described herein, the first chimeric polypeptide

includes a sequence that is at least 90% identical to SEQ ID NO: 236. In some

embodiments of any of the methods described herein, the first chimeric polypeptide

includes a sequence of SEQ ID NO: 236. In some embodiments of any of the methods

described herein, the second chimeric polypeptide includes a sequence that is at least

80% identical to SEQ ID NO: 193. In some embodiments of any of the methods

described herein, the first chimeric polypeptide includes a sequence that is at least 80%

identical to SEQ ID NO: 236. In some embodiments of any of the methods described

herein, the second chimeric polypeptide includes a sequence that is at least 90% identical

to SEQ ID NO: 193. In some embodiments of any of the methods described herein, the

second chimeric polypeptide includes a sequence of SEQ ID NO: 193. In some

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embodiments of any of the methods described herein, the first chimeric polypeptide

comprises a sequence of SEQ ID NO: 236.

In some embodiments of any of the methods described herein, at least one of the

one or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor is a

single-chain chimeric polypeptide including: (i) a first target-binding domain; (ii) a

soluble tissue factor domain; and (iii) a second target-binding domain, wherein one or

both of the first target-binding domain and the second target-binding domain binds

specifically to a ligand of a TGF-B receptor; or one or both of the first target-binding

domain and the second target-binding domain is an antagonistic antigen-binding domain

that binds specifically to a TGF-B receptor. In some embodiments of any of the methods

described herein, the TGF-B receptor is TGF-BRII. In some embodiments of any of the

methods described herein, the TGF-B receptor is TGF3RIII.

In some embodiments of any of the methods described herein, the first target-

binding domain and the soluble tissue factor domain directly abut each other. In some

embodiments of any of the methods described herein, the single-chain chimeric

polypeptide further includes a linker sequence between the first target-binding domain

and the soluble tissue factor domain. In some embodiments of any of the methods

described herein, the soluble tissue factor domain and the second target-binding domain

directly abut each other. In some embodiments of any of the methods described herein,

the single-chain chimeric polypeptide further includes a linker sequence between the

soluble tissue factor domain and the second target-binding domain.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

antigen. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to different

antigens.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is a soluble human tissue factor domain. In some embodiments of any of

the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 80% identical to SEQ ID NO: 93.

In some embodiments of any of the methods described herein, the single-chain

chimeric polypeptide further includes one or more additional target-binding domains at

its N- and/or C-terminus. In some embodiments of any of the methods described herein,

the first target-binding domain and/or the second target-binding domain comprise a

soluble TGF-B receptor. In some embodiments of any of the methods described herein,

the soluble TGF-B receptor is a soluble TGF-BRII.

In some embodiments of any of the methods described herein, the soluble TGF-

BRII includes a first sequence that is at least 80% identical to SEQ ID NO: 183, and a

second sequence that is at least 80% identical to SEQ ID NO: 183, wherein the first and

second sequence are separated by a linker. In some embodiments of any of the methods

described herein, the soluble TGFßRII includes a first sequence that is at least 90%

identical to SEQ ID NO: 183, and a second sequence that is at least 90% identical to SEQ

ID NO: 183. In some embodiments of any of the methods described herein, the soluble

TGFßRII includes a first sequence of SEQ ID NO: 183, and a second sequence of SEQ

ID NO: 183. In some embodiments of any of the methods described herein, the linker

includes a sequence of SEQ ID NO: 102. In some embodiments of any of the methods

described herein, the soluble TGFßRII includes a sequence that is at least 80% identical

to SEQ ID NO: 188. In some embodiments of any of the methods described herein, the

soluble TGFßRII includes a sequence that is at least 90% identical to SEQ ID NO: 188.

In some embodiments of any of the methods described herein, the soluble TGFßRII

includes a sequence of SEQ ID NO: 188.

In some embodiments of any of the methods described herein, the method

includes administering two or more doses of the one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor to the subject. In some embodiments of

any of the methods described herein, any two consecutive doses of the two or more doses

are administered about 1 week to about one year apart. In some embodiments of any of

the methods described herein, any two consecutive doses of the two or more doses are

administered about 1 week to about 6 months apart. In some embodiments of any of the

methods described herein, any two consecutive doses of the two or more doses are

administered about 1 week to about 2 months apart. In some embodiments of any of the

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methods described herein, any two consecutive doses of the two or more doses are

administered about 1 week to about 1 month apart.

In some embodiments of any of the methods described herein, the two or more

doses are administered by subcutaneous administration. In some embodiments of any of

the methods described herein, the two or more doses are administered by intramuscular

administration.

In some embodiments of any of the methods described herein, the two or more

doses are administered over a period of time of about 1 year to about 60 years. In some

embodiments of any of the methods described herein, the two or more doses are

administered over a period of time of about 1 year to about 50 years. In some

embodiments of any of the methods described herein, the two or more doses are

administered over a period of time of about 1 year to about 40 years. In some

embodiments of any of the methods described herein, the two or more doses are

administered over a period of time of about 1 year to about 30 years. In some

embodiments of any of the methods described herein, the two or more doses are

administered over a period of time of about 1 year to about 20 years. In some

embodiments of any of the methods described herein, the two or more doses are

administered over a period of time of about 1 year to about 10 years.

In some embodiments of any of the methods described herein, a first dose of the

one or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor

begins when the subject reaches an age of at least 30 years. In some embodiments of any

of the methods described herein, a first dose of the one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor begins when the subject reaches an age of

at least 40 years. In some embodiments of any of the methods described herein, a first

dose of the one or more agent(s) that result(s) in a decrease in the activation of a TGF-B

receptor begins when the subject reaches an age of at least 50 years. In some

embodiments of any of the methods described herein, a first dose of the one or more

agent(s) that result(s) in a decrease in the activation of a TGF-B receptor begins when the

subject reaches an age of at least 60 years.

In some embodiments of any of the methods described herein, each of the two or

more doses are administered at a dosage of about 0.01 mg of each agent that results in a

decrease in the activation of a TGF-B receptor/kg to about 10 mg of each agent that

results in a decrease in the activation of a TGF-B receptor/kg. In some embodiments of

any of the methods described herein, each of the two or more doses are administered at a

dosage of about 0.02 mg of each agent that results in a decrease in the activation of a

TGF-B receptor/kg to about 5 mg of each agent that results in a decrease in the activation

of a TGF-B receptor/kg.

In some embodiments of any of the methods described herein, the subject is not

diagnosed or identified as having an aging-related disease or an inflammatory disease. In

some embodiments of any of the methods described herein, the subject has not been

previously treated with a chemotherapeutic agent. In some embodiments of any of the

methods described herein, the subject has not been previously treated with a therapeutic

agent that induces cellular senescence.

Provided herein are methods of treating an aging-related disease or condition in a

subject in need thereof that include administering to a subject identified as having an

aging-related disease or condition a therapeutically effective amount of one or more

natural killer (NK) cell activating agent(s).

Also provided herein are methods of killing or reducing the number of senescent

cells in a subject in need thereof that include administering to the subject a

therapeutically effective amount of one or more NK cell activating agent(s). In some

embodiments of any of the methods described herein, the senescent cells are senescent

cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes,

senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent dermal

fibroblasts, senescent keratinocytes, or other differentiated tissue-specific dividing

functional cells. In some embodiments of any of the methods described herein, the

senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced

senescent cells. In some embodiments of any of the methods described herein, the

subject has been identified or diagnosed as having an aging-related disease or condition.

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In some embodiments of any of the methods described herein, the aging-related

disease or condition is selected from the group of: a cancer, an autoimmune disease, a

metabolic disease, a neurodegenerative disease, a cardiovascular disease, a skin disease, a

progeria disease, and a fragility disease. In some embodiments of any of the methods

described herein, the cancer is selected from the group of: solid tumor, hematological

tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma,

B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic

lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia

(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),

cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung

cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate

cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma,

squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver

cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the autoimmune

disease is type-1 diabetes.

In some embodiments of any of the methods described herein, the metabolic

disease is selected from the group of: obesity, a lipodystrophy, and type-2 diabetes

mellitus.

In some embodiments of any of the methods described herein, the

neurodegenerative disease is selected from the group of: Alzheimer's disease,

Parkinson's disease, and dementia.

In some embodiments of any of the methods described herein, the cardiovascular

disease is selected from the group of: coronary artery disease, atherosclerosis, and

pulmonary arterial hypertension.

In some embodiments of any of the methods described herein, the skin disease is

selected from the group of: wound healing, alopecia, wrinkles, senile lentigo, skin

thinning, xeroderma pigmentosum, and dyskeratosis congenita.

In some embodiments of any of the methods described herein, the progeria

disease is selected from the group of: progeria and Hutchinson-Gilford Progeria

Syndrome.

In some embodiments of any of the methods described herein, the fragility disease

is selected from the group of: frailty, responsiveness to vaccination, osteoporosis, and

sarcopenia.

In some embodiments of any of the methods described herein, the aging-related

disease or condition is selected from the group of: osteoarthritis, adipose atrophy,

idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,

sarcopenia, age-associated loss of lung tissue elasticity, osteoporosis, age-associated renal

dysfunction, and chemical-induced renal dysfunction.

In some embodiments of any of the methods described herein, the aging-related

disease or condition is type-2 diabetes or atherosclerosis.

In some embodiments of any of the methods described herein, the administering

results in a decrease in the number of senescent cells in a target tissue in the subject. In

some embodiments of any of the methods described herein, the target tissue is selected

from the group of: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature,

bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue,

and secondary lympho-organ tissue.

In some embodiments of any of the methods described herein, the administering

results in an increase in the expression levels of CD25, CD69, mTORC1, SREBP1, IFN-

Y, and granzyme B in activated NK cells.

Also provided herein are methods of treating an aging-related disease or condition

in a subject in need thereof that include administering to a subject identified as having an

aging-related disease or condition a therapeutically effective number of activated NK

cells.

Also provided herein are methods of killing or reducing the number of senescent

cells in a subject in need thereof that include administering to the subject a

therapeutically effective number of activated NK cells. In some embodiments of any of

the methods described herein, the senescent cells are senescent cancer cells, senescent

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monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent

neurons, senescent tissue fibroblasts, senescent dermal fibroblasts, senescent

keratinocytes, or other differentiated tissue-specific dividing functional cells. In some

embodiments of any of the methods described herein, the senescent cancer cells are

chemotherapy-induced senescent cells or radiation-induced senescent cells. In some

embodiments of any of the methods described herein, the subject has been identified or

diagnosed as having an aging-related disease or condition.

In some embodiments of any of the methods described herein, the aging-related

disease or condition is selected from the group of: a cancer, an autoimmune disease, a

metabolic disease, a neurodegenerative disease, a cardiovascular disease, a skin disease, a

progeria disease, and a fragility disease. In some embodiments of any of the methods

described herein, the cancer is selected from the group of: solid tumor, hematological

tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,

rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma,

B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic

lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia

(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),

cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung

cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate

cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma,

squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver

cancer, and hepatocellular carcinoma.

In some embodiments of any of the methods described herein, the autoimmune

disease is type-1 diabetes.

In some embodiments of any of the methods described herein, the metabolic

disease is selected from the group of: obesity, a lipodystrophy, and type-2 diabetes

mellitus.

In some embodiments of any of the methods described herein, the

neurodegenerative disease is selected from the group of: Alzheimer's disease,

Parkinson's disease, and dementia.

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In some embodiments of any of the methods described herein, the cardiovascular

disease is selected from the group of: coronary artery disease, atherosclerosis, and

pulmonary arterial hypertension.

In some embodiments of any of the methods described herein, the skin disease is

selected from the group of: wound healing, alopecia, wrinkles, senile lentigo, skin

thinning, xeroderma pigmentosum, and dyskeratosis congenita.

In some embodiments of any of the methods described herein, the progeria

disease is selected from the group of: progeria and Hutchinson-Gilford Progeria

Syndrome.

In some embodiments of any of the methods described herein, the fragility disease

is selected from the group of: frailty, responsiveness to vaccination, osteoporosis, and

sarcopenia.

In some embodiments of any of the methods described herein, the aging-related

disease or condition is selected from the group of: age-related macular degeneration,

osteoarthritis, adipose atrophy, idiopathic pulmonary fibrosis, kidney transplant failure,

liver fibrosis, loss of bone mass, sarcopenia, age-associated loss of lung tissue elasticity,

osteoporosis, age-associated renal dysfunction, and chemical-induced renal dysfunction.

Some embodiments of any of the methods described herein further include:

obtaining a resting NK cell; and contacting the resting NK cell in vitro in a liquid culture

medium including one or more NK cell activating agent(s), where the contacting results

in the generation of the activated NK cells that are subsequently administered to the

subject. In some embodiments of any of the methods described herein, the resting NK

cell is an autologous NK cell obtained from the subject. In some embodiments of any of

the methods described herein, the resting NK cell is an allogeneic resting NK cell. In

some embodiments of any of the methods described herein, the resting NK cell is an

artificial NK cell. In some embodiments of any of the methods described herein, the

resting NK cell is a haploidentical resting NK cell. In some embodiments of any of the

methods described herein, the resting NK cell is a genetically-engineered NK cell

carrying a chimeric antigen receptor or recombinant T cell receptor. Some embodiments

of any of the methods described herein further include isolating the activated NK cells

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before the activated NK cells are administered to the subject. Some embodiments of any

of the methods described herein further include introducing a nucleic acid that encodes a

chimeric antigen receptor or a recombinant T cell receptor into the resting NK cell or the

activated NK cell prior to administration to the subject.

Also provided herein are methods of improving the texture and/or appearance of

skin and/or hair in a subject in need thereof over a period of time that include

administering to the subject a therapeutically effective amount of one or more natural

killer (NK) cell activating agent(s).

Also provided herein are methods of improving the texture and/or appearance of

skin and/or hair in a subject in need thereof over a period of time that include

administering to the subject a therapeutically effective number of activated NK cells.

Some embodiments of any of the methods described herein further include: obtaining a

resting NK cell; and contacting the resting NK cell in vitro in a liquid culture medium

including one or more NK cell activating agent(s), where the contacting results in the

generation of the activated NK cells that are subsequently administered to the subject. In

some embodiments of any of the methods described herein, the resting NK cell is an

autologous NK cell obtained from the subject. In some embodiments of any of the

methods described herein, the resting NK cell is an allogeneic resting NK cell. In some

embodiments of any of the methods described herein, the resting NK cell is an artificial

NK cell. In some embodiments of any of the methods described herein, the resting NK

cell is a haploidentical resting NK cell. In some embodiments of any of the methods

described herein, the resting NK cell is a genetically-engineered NK cell carrying a

chimeric antigen receptor or recombinant T cell receptor. Some embodiments of any of

the methods described herein further include isolating the activated NK cells before the

activated NK cells are administered to the subject.

In some embodiments of any of the methods described herein, the method

provides for an improvement in the texture and/or appearance of skin of the subject over

the period of time. In some embodiments of any of the methods described herein, the

method results in a decrease in the rate of formation of wrinkles in the skin of the subject

over the period of time. In some embodiments of any of the methods described herein,

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the method results in an improvement in the coloration of skin of the subject over the

period of time. In some embodiments of any of the methods described herein, the

method results in a reduction of age spots on skin of the subject over the period of time.

In some embodiments of any of the methods described herein, the method results in an

improvement in the texture of skin of the subject over the period of time. In some

embodiments of any of the methods described herein, the method provides for an

improvement in the texture and/or appearance of hair of the subject over the period of

time. In some embodiments of any of the methods described herein, the method results

in a decrease in the rate of formation of gray hair in the subject over the period of time.

In some embodiments of any of the methods described herein, the method results in a

decrease in the number of gray hairs of the subject over the period of time. In some

embodiments of any of the methods described herein, the method results in a decrease in

the rate of hair loss in the subject over time. In some embodiments of any of the methods

described herein, the method results in an improvement in the texture of hair of the

subject over the period of time.

In some embodiments of any of the methods described herein, the period of time

is between about one month and about 10 years. In some embodiments of any of the

methods described herein, the method results in a decrease in the number of senescent

dermal fibroblasts in the skin of the subject over the period of time.

Also provided herein are methods of assisting in the treatment of obesity in a

subject in need thereof over a period of time that include administering to the subject a

therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s).

Also provided herein are methods of assisting in the treatment of obesity in a

subject in need thereof over a period of time that include administering to the subject a

therapeutically effective number of activated NK cells. Some embodiments of any of the

methods described herein further include: obtaining a resting NK cell; and contacting the

resting NK cell in vitro in a liquid culture medium including one or more NK cell

activating agent(s), where the contacting results in the generation of the activated NK

cells that are subsequently administered to the subject. In some embodiments of any of

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the methods described herein, the resting NK cell is an autologous NK cell obtained from

the subject. In some embodiments of any of the methods described herein, the resting

NK cell is an allogeneic resting NK cell. In some embodiments of any of the methods

described herein, the resting NK cell is an artificial NK cell. In some embodiments of

any of the methods described herein, the resting NK cell is a haploidentical resting NK

cell. In some embodiments of any of the methods described herein, the resting NK cell is

a genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor. Some embodiments of any of the methods described herein further include

isolating the activated NK cells before the activated NK cells are administered to the

subject.

In some embodiments of any of the methods described herein, the method results

in a decrease in the mass of the subject over the period of time. In some embodiments of

any of the methods described herein, the method results in a decrease in the body mass

index (BMI) of the subject over the period of time. In some embodiments of any of the

methods described herein, the method results in a decrease in the rate of progression from

pre-diabetes to type-2 diabetes in the subject. In some embodiments of any of the

methods described herein, the method results in a decrease in fasting serum glucose level

in the subject. In some embodiments of any of the methods described herein, the method

results in an increase in insulin sensitivity in the subject. In some embodiments of any of

the methods described herein, the method results in a decrease in the severity of

atherosclerosis in the subject. In some embodiments of any of the methods described

herein, the period of time is between about two weeks and about 10 years.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) results in activation of one or more of: a receptor

for IL-2, a receptor for IL-7, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

18, a receptor for IL-21, a receptor for IL-33, CD16, CD69, CD25, CD59, CD352,

NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1.

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In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-2

is a soluble IL-2 or an agonistic antibody that binds specifically to an IL-2 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-7

is a soluble IL-7 or an agonistic antibody that binds specifically to an IL-7 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

12 is a soluble IL-12 or an agonistic antibody that binds specifically to an IL-12 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

15 is a soluble IL-15 or an agonistic antibody that binds specifically to an IL-15 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

21 is a soluble IL-21 or an agonistic antibody that binds specifically to an IL-21 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

33 is a soluble IL-33 or an agonistic antibody that binds specifically to an IL-33 receptor.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD16 is an agonistic antibody that binds specifically to a CD16.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD69 is an agonistic antibody that binds specifically to a CD69.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD25 or CD59 is an agonistic antibody that binds specifically to CD25 or CD59.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD352 is an agonistic antibody that binds specifically to a CD352.

PCT/US2021/035285

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp80 is an agonistic antibody that binds specifically to an NKp80.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

DNAM-1 is an agonistic antibody that binds specifically to a DNAM-1.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for 2B4

is an agonistic antibody that binds specifically to a 2B4.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp30 is an agonistic antibody that binds specifically to an NKp30.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp44 is an agonistic antibody that binds specifically to an NKp44.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp46 is an agonistic antibody that binds specifically to an NKp46.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKG2D is an agonistic antibody that binds specifically to an NKG2D.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS1 is an agonistic antibody that binds specifically to a KIR2DS1.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS2/3 is an agonistic antibody that binds specifically to a KIR2DS2/3.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DL4 is an agonistic antibody that binds specifically to a KIR2DL4.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS4 is an agonistic antibody that binds specifically to a KIR2DS4.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS5 is an agonistic antibody that binds specifically to a KIR2DS5.

In some embodiments of any of the methods described herein, the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR3DS1 is an agonistic antibody that binds specifically to a KIR3DS1.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) results in a decrease in the activation of one or

more of: PD-1, a TGF-B receptor, TIGIT, CD1, TIM-3, Siglec-7, IRP60, Tactile, IL 1R8,

NKG2A/KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2. In some embodiments of any of the methods described herein, the at least

one of the one or more NK cell activating agent(s) that results in a decrease in the

activation of PD-1 is an antagonistic antibody that binds specifically to PD-1, a soluble

PD-1, a soluble PD-L1, or an antibody that binds specifically to PD-L1. In some

embodiments of any of the methods described herein, at least one of the one or more NK

cell activating agent(s) that results in a decrease in the activation of a TGF-B receptor is a

soluble TGF-B receptor, an antibody that binds specifically to TGF-B, or an antagonistic

antibody that binds specifically to a TGF-B receptor.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

TIGIT is an antagonistic antibody that binds specifically to TIGIT, a soluble TIGIT, or an

antibody that binds specifically to a ligand of TIGIT.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of CD1

is an antagonistic antibody that binds specifically to CD1, a soluble CD1, or an antibody

that binds specifically to a ligand of CD1.

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In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

TIM-3 is an antagonistic antibody that binds specifically to TIM-3, a soluble TIM-3, or

an antibody that binds specifically to a ligand of TIM-3.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

Siglec-7 is an antagonistic antibody that binds specifically to Siglec-7 or an antibody that

binds specifically to a ligand of Siglec-7.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

IRP60 is an antagonistic antibody that binds specifically to IRP60 or an antibody that

binds specifically to a ligand of IRP60.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

Tactile is an antagonistic antibody that binds specifically to Tactile or an antibody that

binds specifically to a ligand of Tactile.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

IL1R8 is an antagonistic antibody that binds specifically to IL1R8 or an antibody that

binds specifically to a ligand of IL 1R8.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

NKG2A/KLRD1 is an antagonistic antibody that binds specifically to NKG2A/KLRD1

or an antibody that binds specifically to a ligand of NKG2A/KLRD1.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR2DL1 is an antagonistic antibody that binds specifically to KIR2DL1 or an antibody

that binds specifically to a ligand of KIR2DL1.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

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KIR2DL2/3 is an antagonistic antibody that binds specifically to KIR2DL2/3 or an

antibody that binds specifically to a ligand of KIR2DL2/3.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR2DL5 is an antagonistic antibody that binds specifically to KIR2DL5 or an antibody

that binds specifically to a ligand of KIR2DL5.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR3DL1 is an antagonistic antibody that binds specifically to KIR3DL1 or an antibody

that binds specifically to a ligand of KIR3DL1.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR3DL2 is an antagonistic antibody that binds specifically to KIR3DL2 or an antibody

that binds specifically to a ligand of KIR3DL2.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

ILT2/LIR-1 is an antagonistic antibody that binds specifically to ILT2/LIR-1 or an

antibody that binds specifically to a ligand of ILT2/LIR-1.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

LAG-2is an antagonistic antibody that binds specifically to LAG-2 or an antibody that

binds specifically to a ligand of LAG-2.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) is a single-chain chimeric polypeptide that

includes: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a

second target-binding domain. In some embodiments of any of the methods described

herein, the first target-binding domain and the soluble tissue factor domain directly abut

each other. In some embodiments of any of the methods described herein, the single-

chain chimeric polypeptide further includes a linker sequence between the first target-

binding domain and the soluble tissue factor domain. In some embodiments of any of the

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methods described herein, the soluble tissue factor domain and the second target-binding

domain directly abut each other. In some embodiments of any of the methods described

herein, the single-chain chimeric polypeptide further includes a linker sequence between

the soluble tissue factor domain and the second target-binding domain. In some

embodiments of any of the methods described herein, the first target-binding domain and

the second target-binding domain directly abut each other. In some embodiments of any

of the methods described herein, the single-chain chimeric polypeptide further includes a

linker sequence between the first target-binding domain and the second target-binding

domain. In some embodiments of any of the methods described herein, the second target-

binding domain and the soluble tissue factor domain directly abut each other. In some

embodiments of any of the methods described herein, the single-chain chimeric

polypeptide further includes a linker sequence between the second target-binding domain

and the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

antigen. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

epitope. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain include the same amino acid

sequence.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to different

antigens.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is an antigen-binding

domain. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain are each an antigen-binding

domain. In some embodiments of any of the methods described herein, the antigen-

binding domain includes a scFv or a single domain antibody.

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In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain bind to a target selected

from the group of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1,

TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200,

IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-

DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B

receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAMI, a ligand of NKp46,

a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a

ligand for a scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a receptor for stem

cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a

receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a

receptor for CD155, and a receptor for CD122.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

or cytokine protein. In some embodiments of any of the methods described herein, the

soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-

7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

or cytokine receptor. In some embodiments of any of the methods described herein, the

soluble interleukin or cytokine receptor is a soluble TGF-B receptor II (TGF-BRII) a

soluble TGF-BRIII, a soluble receptor for TNFa, a soluble receptor for IL-4, or a soluble

receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is a soluble human tissue factor domain. In some embodiments of any of

the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 80% identical to SEQ ID NO: 93. In some embodiments of any

of the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 90% identical to SEQ ID NO: 93. In some embodiments of any

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of the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 95% identical to SEQ ID NO: 93. In some embodiments of any

of the methods described herein, the soluble human tissue factor domain does not include

one or more of: a lysine at an amino acid position that corresponds to amino acid position

20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid

position that corresponds to amino acid position 22 of mature wildtype human tissue

factor protein; a tryptophan at an amino acid position that corresponds to amino acid

position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino

acid position that corresponds to amino acid position 58 of mature wildtype human tissue

factor protein; a tyrosine at an amino acid position that corresponds to amino acid

position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid

position that corresponds to amino acid position 135 of mature wildtype human tissue

factor protein; and a phenylalanine at an amino acid position that corresponds to amino

acid position 140 of mature wildtype human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble human

tissue factor domain does not include any of: a lysine at an amino acid position that

corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an

isoleucine at an amino acid position that corresponds to amino acid position 22 of mature

wildtype human tissue factor protein; a tryptophan at an amino acid position that

corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an

aspartic acid at an amino acid position that corresponds to amino acid position 58 of

mature wildtype human tissue factor protein; a tyrosine at an amino acid position that

corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an

arginine at an amino acid position that corresponds to amino acid position 135 of mature

wildtype human tissue factor protein; and a phenylalanine at an amino acid position that

corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is not capable of binding Factor VIIa. In some embodiments of any of the

methods described herein, the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide does not blood stimulate coagulation in a mammal.

In some embodiments of any of the methods described herein, the single-chain chimeric

polypeptide further includes one or more additional target-binding domains at its N-

and/or C-terminus.

In some embodiments of any of the methods described herein, the single-chain

chimeric polypeptide includes one or more additional target-binding domains at its N-

terminus. In some embodiments of any of the methods described herein, one or more

additional target-binding domains directly abuts the first target-binding domain, the

second target-binding domain, or the soluble tissue factor domain. In some embodiments

of any of the methods described herein, the single-chain chimeric polypeptide further

includes a linker sequence between one of the at least one additional target-binding

domains and the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain.

In some embodiments of any of the methods described herein, the single-chain

chimeric polypeptide includes one or more additional target-binding domains at its C-

terminus. In some embodiments of any of the methods described herein, one of the one

or more additional target-binding domains directly abuts the first target-binding domain,

the second target-binding domain, or the soluble tissue factor domain. In some

embodiments of any of the methods described herein, the single-chain chimeric

polypeptide further includes a linker sequence between one of the at least one additional

target-binding domains and the first target-binding domain, the second target-binding

domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the single-chain

chimeric polypeptide includes one or more additional target binding domains at its N-

terminus and the C-terminus. In some embodiments of any of the methods described

herein, one of the one or more additional antigen binding domains at the N-terminus

directly abuts the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain. In some embodiments of any of the methods described

herein, the single-chain chimeric polypeptide further includes a linker sequence between

one of the one or more additional antigen-binding domains at the N-terminus and the first

WO wo 2021/247604 PCT/US2021/035285

target-binding domain, the second target-binding domain, or the soluble tissue factor

domain. In some embodiments of any of the methods described herein, one of the one or

more additional antigen binding domains at the C-terminus directly abuts the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the single-chain chimeric

polypeptide further includes a linker sequence between one of the one or more additional

antigen-binding domains at the C-terminus and the first target-binding domain, the

second target-binding domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, two or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same antigen. In some

embodiments of any of the methods described herein, two or more of the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains bind specifically to the same epitope. In some embodiments of any of

the methods described herein, two or more of the first target-binding domain, the second

target-binding domain, and the one or more additional target-binding domains include the

same amino acid sequence. In some embodiments of any of the methods described

herein, the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same antigen. In

some embodiments of any of the methods described herein, the first target-binding

domain, the second target-binding domain, and the one or more additional target-binding

domains each bind specifically to the same epitope. In some embodiments of any of the

methods described herein, the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains each include the same

amino acid sequence.

In some embodiments of any of the methods described herein, the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains bind specifically to different antigens.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

WO wo 2021/247604 PCT/US2021/035285

target-binding domains is an antigen-binding domain. In some embodiments of any of

the methods described herein, the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains are each an antigen-

binding domain. In some embodiments of any of the methods described herein, the

antigen-binding domain includes a scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

target-binding domains bind specifically to a target selected from the group of: CD16a,

CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA,

MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC,

MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3,

AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a

ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a

ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a

ligand for a scTCR, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a

receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a

receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, and a

receptor for CD122.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains is a soluble interleukin or cytokine protein. In some

embodiments of any of the methods described herein, the soluble interleukin or cytokine

protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,

IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains is a soluble interleukin or cytokine receptor. In some

embodiments of any of the methods described herein, the soluble receptor is a soluble

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TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor for TNFa, a

soluble receptor for IL-4, or a soluble receptor for IL-10.

In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) is a multi-chain chimeric polypeptide that

includes: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a

soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; and (b)

a second chimeric polypeptide including: (i) a second domain of a pair of affinity

domains; and (ii) a second target-binding domain, where the first chimeric polypeptide

and the second chimeric polypeptide associate through the binding of the first domain

and the second domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some embodiments of any of the methods described herein, the

first chimeric polypeptide further includes a linker sequence between the first target-

binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In

some embodiments of any of the methods described herein, the soluble tissue factor

domain and the first domain of the pair of affinity domains directly abut each other in the

first chimeric polypeptide. In some embodiments of any of the methods described herein,

the first chimeric polypeptide further includes a linker sequence between the soluble

tissue factor domain and the first domain of the pair of affinity domains in the first

chimeric polypeptide. In some embodiments of any of the methods described herein, the

second domain of the pair of affinity domains and the second target-binding domain

directly abut each other in the second chimeric polypeptide. In some embodiments of

any of the methods described herein, the second chimeric polypeptide further includes a

linker sequence between the second domain of the pair of affinity domains and the

second target-binding domain in the second chimeric polypeptide.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

antigen. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

WO wo 2021/247604 PCT/US2021/035285

epitope. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain include the same amino acid

sequence.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to different

antigens.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is an antigen-binding

domain. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain are each antigen-binding domains.

In some embodiments of any of the methods described herein, the antigen-binding

domain includes a scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain bind specifically to a

target selected from the group of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a

receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand

(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding

protein, a receptor for CD155, and a receptor for CD122.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

or cytokine protein. In some embodiments of any of the methods described herein, the

soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-

7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

or cytokine receptor. In some embodiments of any of the methods described herein, the

soluble receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a

soluble receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the first chimeric

polypeptide further includes one or more additional target-binding domain(s), where at

least one of the one or more additional antigen-binding domain(s) is positioned between

the soluble tissue factor domain and the first domain of the pair of affinity domains. In

some embodiments of any of the methods described herein, the first chimeric polypeptide

further includes a linker sequence between the soluble tissue factor domain and the at

least one of the one or more additional antigen-binding domain(s), and/or a linker

sequence between the at least one of the one or more additional antigen-binding

domain(s) and the first domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the first chimeric

polypeptide further includes one or more additional target-binding domains at the N-

terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments

of any of the methods described herein, at least one of the one or more additional target-

binding domains directly abuts the first domain of the pair of affinity domains in the first

chimeric polypeptide. In some embodiments of any of the methods described herein, the

first chimeric polypeptide further includes a linker sequence between the at least one of

the one or more additional target-binding domains and the first domain of the pair of

affinity domains. In some embodiments of any of the methods described herein, the at

least one of the one or more additional target-binding domains directly abuts the first

target-binding domain in the first chimeric polypeptide. In some embodiments of any of

the methods described herein, the first chimeric polypeptide further includes a linker

sequence between the at least one of the one or more additional target-binding domains

and the first target-binding domain.

In some embodiments of any of the methods described herein, at least one of the

one or more additional target-binding domains is disposed at the N- and/or C-terminus of

WO wo 2021/247604 PCT/US2021/035285

the first chimeric polypeptide, and at least one of the one or more additional target-

binding domains is positioned between the soluble tissue factor domain and the first

domain of the pair of affinity domains in the first chimeric polypeptide. In some

embodiments of any of the methods described herein, the at least one additional target-

binding domain of the one or more additional target-binding domains disposed at the N-

terminus directly abuts the first target-binding domain or the first domain of the pair of

affinity domains in the first chimeric polypeptide. In some embodiments of any of the

methods described herein, the first chimeric polypeptide further includes a linker

sequence disposed between the at least one additional target-binding domain and the first

target-binding domain or the first domain of the pair of affinity domains in the first

chimeric polypeptide. In some embodiments of any of the methods described herein, the

at least one additional target-binding domain of the one or more additional target-binding

domains disposed at the C-terminus directly abuts the first target-binding domain or the

first domain of the pair of affinity domains in the first chimeric polypeptide. In some

embodiments of any of the methods described herein, the first chimeric polypeptide

further includes a linker sequence disposed between the at least one additional target-

binding domain and the first target-binding domain or the first domain of the pair of

affinity domains in the first chimeric polypeptide. In some embodiments of any of the

methods described herein, the at least one of the one or more additional target-binding

domains positioned between the soluble tissue factor domain and the first domain of the

pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first

domain of the pair of affinity domains. In some embodiments of any of the methods

described herein, the first chimeric polypeptide further includes a linker sequence

disposed (i) between the soluble tissue factor domain and the at least one of the one or

more additional target-binding domains positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains, and/or (ii) between the first

domain of the pair of affinity domains and the at least one of the one or more additional

target-binding domains positioned between the soluble tissue factor domain and the first

domain of the pair of affinity domains.

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In some embodiments of any of the methods described herein, the second

chimeric polypeptide further includes one or more additional target-binding domains at

the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some

embodiments of any of the methods described herein, at least one of the one or more

additional target-binding domains directly abuts the second domain of the pair of affinity

domains in the second chimeric polypeptide. In some embodiments of any of the

methods described herein, the second chimeric polypeptide further includes a linker

sequence between at least one of the one or more additional target-binding domains and

the second domain of the pair of affinity domains in the second chimeric polypeptide. In

some embodiments of any of the methods described herein, at least one of the one or

more additional target-binding domains directly abuts the second target-binding domain

in the second chimeric polypeptide. In some embodiments of any of the methods

described herein, the second chimeric polypeptide further includes a linker sequence

between at least one of the one or more additional target-binding domains and the second

target-binding domain in the second chimeric polypeptide.

In some embodiments of any of the methods described herein, two or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same antigen. In some

embodiments of any of the methods described herein, two or more of the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains bind specifically to the same epitope. In some embodiments of any of

the methods described herein, two or more of the first target-binding domain, the second

target-binding domain, and the one or more additional target-binding domains include the

same amino acid sequence. In some embodiments of any of the methods described

herein, the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same antigen. In

some embodiments of any of the methods described herein, the first target-binding

domain, the second target-binding domain, and the one or more additional target-binding

domains each bind specifically to the same epitope. In some embodiments of any of the

methods described herein, the first target-binding domain, the second target-binding

PCT/US2021/035285

domain, and the one or more additional target-binding domains each include the same

amino acid sequence.

In some embodiments of any of the methods described herein, the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains bind specifically to different antigens. In some embodiments of any of

the methods described herein, one or more of the first target-binding domain, the second

target-binding domain, and the one or more target-binding domains is an antigen-binding

domain. In some embodiments of any of the methods described herein, the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains are each an antigen-binding domain. In some embodiments of any of

the methods described herein, the antigen-binding domain includes a scFv.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

target-binding domains bind specifically to a target selected from the group of: CD16a,

CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA,

MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC,

MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3,

AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a

ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a

ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a

ligand for a scTCR, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a

receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a

receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, and a

receptor for CD122.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains is a soluble interleukin or cytokine protein. In some

embodiments of any of the methods described herein, the soluble interleukin or cytokine

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protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,

IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the methods described herein, one or more of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains is a soluble interleukin or cytokine receptor. In some

embodiments of any of the methods described herein, the soluble receptor a soluble TGF-

receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor for TNFa, a soluble

receptor for IL-4, or a soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is a soluble human tissue factor domain. In some embodiments of any of

the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 80% identical to SEQ ID NO: 93. In some embodiments of any

of the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 90% identical to SEQ ID NO: 93. In some embodiments of any

of the methods described herein, the soluble human tissue factor domain includes a

sequence that is at least 95% identical to SEQ ID NO: 93.

In some embodiments of any of the methods described herein, the soluble human

tissue factor domain does not include one or more of: a lysine at an amino acid position

that corresponds to amino acid position 20 of mature wildtype human tissue factor

protein; an isoleucine at an amino acid position that corresponds to amino acid position

22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position

that corresponds to amino acid position 45 of mature wildtype human tissue factor

protein; an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position

that corresponds to amino acid position 94 of mature wildtype human tissue factor

protein; an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid

position that corresponds to amino acid position 140 of mature wildtype human tissue

factor protein.

In some embodiments of any of the methods described herein, the soluble human

tissue factor domain does not include any of: a lysine at an amino acid position that

corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an

isoleucine at an amino acid position that corresponds to amino acid position 22 of mature

wildtype human tissue factor protein; a tryptophan at an amino acid position that

corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an

aspartic acid at an amino acid position that corresponds to amino acid position 58 of

mature wildtype human tissue factor protein; a tyrosine at an amino acid position that

corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an

arginine at an amino acid position that corresponds to amino acid position 135 of mature

wildtype human tissue factor protein; and a phenylalanine at an amino acid position that

corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is not capable of binding to Factor VIIa. In some embodiments of any of

the methods described herein, the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa. In some embodiments of any of the methods described herein,

the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some embodiments of any of the methods described herein, the pair of affinity

domains is a sushi domain from an alpha chain of human IL-15 receptor (IL-15Ra) and a

soluble IL-15 In some embodiments of any of the methods described herein, the soluble

IL-15 has a D8N or D8A amino acid substitution. In some embodiments of any of the

methods described herein, the human IL-15Ra is a mature full-length IL-15Ra.

In some embodiments of any of the methods described herein, the pair of affinity

domains is selected from the group of: barnase and barnstar, a PKA and an AKAP,

adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules

based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and

SNAP25. In some embodiments of any of the methods described herein, at least one of the

one or more NK cell activating agent(s) is a multi-chain chimeric polypeptide that

includes: (a) a first and second chimeric polypeptides, where each includes: (i) a first

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target-binding domain; (ii) a Fc domain; and (iii) a first domain of a pair of affinity

domains; and (b) a third and fourth chimeric polypeptide, where each includes: (i) a

second domain of a pair of affinity domains; and (ii) a second target-binding domain,

where the first and second chimeric polypeptides and the third and fourth chimeric

polypeptides associate through the binding of the first domain and the second domain of

the pair of affinity domains, and the first and second chimeric polypeptides associate

through their Fc domains.

In some embodiments of any of the methods described herein, the first target-

binding domain and the Fc domain directly abut each other in the first and second

chimeric polypeptides. In some embodiments of any of the methods described herein, the

first and second chimeric polypeptides further include a linker sequence between the first

target-binding domain and the Fc domain in the first and second chimeric polypeptides.

In some embodiments of any of the methods described herein, the Fc domain and the first

domain of the pair of affinity domains directly abut each other in the first and second

chimeric polypeptides. In some embodiments of any of the methods described herein, the

first chimeric polypeptide further includes a linker sequence between the Fc domain and

the first domain of the pair of affinity domains in the first and second chimeric

polypeptides.

In some embodiments of any of the methods described herein, the second domain

of the pair of affinity domains and the second target-binding domain directly abut each

other in the third and fourth chimeric polypeptides. In some embodiments of any of the

methods described herein, the third and fourth chimeric polypeptides further include a

linker sequence between the second domain of the pair of affinity domains and the

second target-binding domain in the third and fourth chimeric polypeptides.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

antigen. In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to the same

epitope. In some embodiments of any of the methods described herein, the first target-

WO wo 2021/247604 PCT/US2021/035285

binding domain and the second target-binding domain include the same amino acid

sequence.

In some embodiments of any of the methods described herein, the first target-

binding domain and the second target-binding domain bind specifically to different

antigens. In some embodiments of any of the methods described herein, one or both of

the first target-binding domain and the second target-binding domain is an antigen-

binding domain. In some embodiments of any of the methods described herein, the first

target-binding domain and the second target-binding domain are each antigen-binding

domains. In some embodiments of any of the methods described herein, the antigen-

binding domain includes a scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain bind specifically to a

target selected from the group of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a

receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand

(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding

protein, a receptor for CD155, and a receptor for CD122.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

or cytokine protein. In some embodiments of any of the methods described herein, the

soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-

7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the methods described herein, one or both of the

first target-binding domain and the second target-binding domain is a soluble interleukin

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or cytokine receptor. In some embodiments of any of the methods described herein, the

soluble receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a

soluble receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue

factor domain is a soluble human tissue factor domain that does not stimulate blood

coagulation. In some embodiments of any of the methods described herein, the soluble

tissue factor domain comprises or consists of a sequence from a wildtype soluble human

tissue factor.

Provided herein are methods of killing or reducing the number of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more common gamma-chain

family cytokine receptor activating agent(s).

Also provided herein are methods of decreasing the accumulation of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more common gamma-chain

family cytokine receptor activating agent(s).

Also provided herein are methods of decreasing a level of a marker of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more common gamma-chain

family cytokine receptor activating agent(s).

Also provided herein are methods of reducing the activity of naturally-occurring

and/or treatment-induced senescent cells in a subject that include administering to the

subject a therapeutically effective amount of one or more common gamma-chain family

cytokine receptor activating agent(s).

Also provided herein are methods of decreasing levels and/or activity of one or

more SASP factor(s) derived from naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more common gamma-chain family cytokine receptor

activating agent(s).

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In some embodiments, the subject has been previously diagnosed or identified as

having an aging-related disease or an inflammatory disease. In some embodiments, the

aging-related disease is inflamm-aging related.

In some embodiments, the aging-related disease is selected from the group of:

Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,

hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral

disc degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy,

atherosclerosis, cataracts, COPD, kidney transplant failure, liver fibrosis, loss of bone

mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte

hypertrophy, osteoarthritis, Parkinson's disease, age-associated loss of lung tissue

elasticity, age-related macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis,

NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease,

spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia,

vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

In some embodiments, the aging-related disease is a cancer selected from the

group of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,

neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell

neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,

Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia

(AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL),

myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma,

stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and

esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer,

ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma,

endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments, the inflammatory disease is selected from the group of:

rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis,

diabetic nephropathy, CNS injury, Alzheimer's disease, Parkinson's disease, amyotrophic

lateral sclerosis, Crohn's disease, multiple sclerosis, Guillain-Barre syndrome, psoriasis,

Grave's disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders and

cancer treatment-related cognitive impairment.

In some embodiments, the treatment-induced senescent cells are chemotherapy-

induced senescent cells. In some embodiments, the administration of the one or more

common gamma-chain family cytokine receptor activating agent(s) results in a decrease

in the number of naturally-occurring senescent cells and/or treatment-induced senescent

cells in a target tissue in the subject. In some embodiments, the target tissue is selected

from the group of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue,

vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue,

muscle tissue, and secondary lympho-organ tissue.

In some embodiments, at least one of the one or more common gamma-chain

family cytokine receptor activating agent(s) is a complex of a common gamma-chain

family cytokine or a functional fragment thereof and an antibody or antibody fragment

that binds specifically to the common gamma-chain family cytokine or the functional

fragment thereof.

In some embodiments, at least one of the one or more common gamma-chain

family cytokine receptor activating agent(s) is a single-chain chimeric polypeptide

comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii)

a second target-binding domain, wherein one or both of the first target-binding domain

and the second target-binding domain is a soluble common gamma-chain family

cytokine, an agonistic antigen-binding domain that binds specifically to a common

gamma-chain family cytokine receptor, a soluble common gamma-chain family cytokine

receptor, or an antigen-binding domain that binds specifically to a common gamma-chain

family cytokine.

In some embodiments, one or both of the first target-binding domain and the

second target-binding domain comprises a soluble common gamma-chain family

cytokine. In some embodiments, the soluble common gamma-chain family cytokine is

selected from the group consisting of: soluble IL-2, soluble IL-4, soluble IL-7, soluble

IL-9, soluble IL-15, and soluble IL-21. In some embodiments, one or both of the first

target-binding domain and the second target-binding domain comprises an agonistic

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antigen-binding domain that binds specifically to a common gamma-chain family

cytokine receptor. In some embodiments, the common gamma-chain family cytokine

receptor is a receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some

embodiments, the agonistic antigen-binding domain is an scFv, a VHH, or a VNAR.

In some embodiments, the first target-binding domain and the soluble tissue factor

domain directly abut each other. In some embodiments, the single-chain chimeric

polypeptide further comprises a linker sequence between the first target-binding domain

and the soluble tissue factor domain. In some embodiments, the soluble tissue factor

domain and the second target-binding domain directly abut each other. In some

embodiments, the single-chain chimeric polypeptide further comprises a linker sequence

between the soluble tissue factor domain and the second target-binding domain. In some

embodiments, the first target-binding domain and the second target-binding domain bind

specifically to the same antigen. In some embodiments, the first target-binding domain

and the second target-binding domain bind specifically to the same epitope. In some

embodiments, the first target-binding domain and the second target-binding domain

comprise the same amino acid sequence. In some embodiments, the first target-binding

domain and the second target-binding domain bind specifically to different antigens.

In some embodiments, the soluble tissue factor domain is a soluble human tissue

factor domain. In some embodiments, the soluble human tissue factor domain comprises

a sequence that is at least 80% identical to SEQ ID NO: 93. In some embodiments, the

single-chain chimeric polypeptide further comprises one or more additional target-

binding domains at its N- and/or C-terminus.

In some embodiments, at least one of the one or more common gamma-chain

family cytokine receptor activating agent(s) is a multi-chain chimeric polypeptide

comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity

domains; and (ii) a second target-binding domain, wherein one or both of the first target-

binding domain and the second target-binding domain is a soluble common gamma-chain

family cytokine, an agonistic antigen-binding domain that binds specifically to a common

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gamma-chain family cytokine receptor, a soluble common gamma-chain family cytokine

receptor, or an antigen-binding domain that binds specifically to a common gamma-chain

family cytokine.

In some embodiments, one or both of the first target-binding domain and the

second target-binding domain comprises a soluble common gamma-chain family

cytokine. In some embodiments, the soluble common gamma-chain family cytokine is

selected from the group of: soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble

IL-15, and soluble IL-21. In some embodiments, one or both of the first target-binding

domain and the second target-binding domain comprises an agonistic antigen-binding

domain that binds specifically to a common gamma-chain family cytokine receptor. In

some embodiments, the common gamma-chain family cytokine receptor is a receptor for

one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, the

agonistic antigen-binding domain is an scFv, a VHH, or a VNAR.

In some embodiments, the first target-binding domain and the soluble tissue factor

domain directly abut each other in the first chimeric polypeptide. In some embodiments,

the first chimeric polypeptide further comprises a linker sequence between the first

target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide. In some embodiments, the soluble tissue factor domain and the first domain

of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

In some embodiments, the first chimeric polypeptide further comprises a linker sequence

between the soluble tissue factor domain and the first domain of the pair of affinity

domains in the first chimeric polypeptide. In some embodiments, the second domain of

the pair of affinity domains and the second target-binding domain directly abut each other

in the second chimeric polypeptide. In some embodiments, the second chimeric

polypeptide further comprises a linker sequence between the second domain of the pair of

affinity domains and the second target-binding domain in the second chimeric

polypeptide.

In some embodiments, the first target-binding domain and the second target-

binding domain bind specifically to the same antigen. In some embodiments, the first

target-binding domain and the second target-binding domain bind specifically to different

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antigens. In some embodiments, the first chimeric polypeptide further comprises one or

more additional target-binding domain(s). In some embodiments, the second chimeric

polypeptide further comprises one or more additional target-binding domains.

In some embodiments, the soluble tissue factor domain is a soluble human tissue

factor domain. In some embodiments, the soluble human tissue factor domain comprises

a sequence that is at least 80% identical to SEQ ID NO: 93. In some embodiments, the

pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor

(IL15Ra) and a soluble IL-15. In some embodiments, the pair of affinity domains is

selected from the group of: barnase and barnstar, a PKA and an AKAP, adapter/docking

tag modules based on mutated RNase I fragments, and SNARE modules based on

interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

In some embodiments, the first domain or the second domain of a pair of affinity

domains is a soluble common gamma-chain family cytokine or an antigen-binding

domain that binds specifically to a common gamma-chain family cytokine receptor. In

some embodiments, at least one of the one or more common gamma-chain family

cytokine receptor activating agent(s) is soluble IL-15 or an IL-15 agonist. In some

embodiments, the soluble IL-15 is at least 90% identical to SEQ ID NO: 82. In some

embodiments, the IL-15 agonist comprises a complex of IL-15 and all or a portion of a

soluble IL-15 receptor (IL-15R). In some embodiments, the portion of the soluble IL-

15R is a portion of IL-15Ra. In some embodiments, the portion of the soluble IL-15Ra is

a sushi domain of IL-15Ra. In some embodiments, the IL-15 agonist further comprises

an Fc domain. In some embodiments, the IL-15 agonist comprises a fusion protein

comprising IL-15 and a sushi domain from an IL-15Ra. In some embodiments, one of

the one or more common gamma-chain family cytokine receptor activating agent(s) is a

soluble IL-2 or an IL-2 agonist. In some embodiments, one of the one or more common

gamma-chain family cytokine receptor activating agent(s) is an antibody or an antigen-

binding antibody fragment that binds specifically to a common gamma-chain family

cytokine.

In some embodiments, the method comprises administering one, two or more

doses of the one or more common gamma-chain family cytokine receptor activating

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agent(s) to the subject. In some embodiments, any two consecutive doses of the two or

more doses are administered about 1 week to about one year apart. In some

embodiments, any two consecutive doses of the two or more doses are administered

about 1 week to about 6 months apart. In some embodiments, any two consecutive doses

of the two or more doses are administered about 1 week to about 2 months apart. In

some embodiments, any two consecutive doses of the two or more doses are administered

about 1 week to about 1 month apart.

In some embodiments, the one, two or more doses are administered by

subcutaneous administration. In some embodiments, the two or more doses are

administered by intramuscular administration. In some embodiments, the two or more

doses are administered over a period of time of about 1 year to about 60 years. In some

embodiments, the two or more doses are administered over a period of time of about 1

year to about 50 years. In some embodiments, the two or more doses are administered

over a period of time of about 1 year to about 40 years. In some embodiments, the two or

more doses are administered over a period of time of about 1 year to about 30 years. In

some embodiments, the two or more doses are administered over a period of time of

about 1 year to about 20 years. In some embodiments, the two or more doses are

administered over a period of time of about 1 year to about 10 years.

In some embodiments, each of the two or more doses are administered at a dosage

of about 0.01 mg of each common gamma-chain family cytokine receptor activating

agent/kg to about 10 mg of each common gamma-chain family cytokine receptor

activating agent/kg. In some embodiments, each of the two or more doses are

administered at a dosage of about 0.02 mg of each common gamma-chain family

cytokine receptor activating agent/kg to about 5 mg of each common gamma-chain

family cytokine receptor activating agent/kg.

In some embodiments, a first dose of the one or more common gamma-chain

family cytokine receptor activating agent(s) begins when the subject reaches an age of at

least 30 years. In some embodiments, a first dose of the one or more common gamma-

chain family cytokine receptor activating agent(s) begins when the subject reaches an age

of at least 40 years. In some embodiments, a first dose of the one or more common

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gamma-chain family cytokine receptor activating agent(s) begins when the subject

reaches an age of at least 50 years. In some embodiments, a first dose of the one or more

common gamma-chain family cytokine receptor activating agent(s) begins when the

subject reaches an age of at least 60 years.

In some embodiments, the subject is not diagnosed or identified as having an

aging-related disease or an inflammatory disease. In some embodiments, the subject has

not been previously treated with a chemotherapeutic agent. In some embodiments, the

subject has not been previously treated with a therapeutic agent that induces cellular

senescence. In some embodiments, the method further comprises administering to the

subject at least one or more agent(s) that results in a decrease in the activation of a TGF-B

receptor. In some embodiments, the agent that results in a decrease in the activation of a

TGF-B receptor is a soluble TGF-B receptor, an extracellular domain of TGF-B receptor,

an antibody that binds specifically to TGF-B, an antagonistic antibody that binds to a

TGF-B receptor, an agent that binds to a LAP, or an agent that binds to a TGF-B/LAP

complex. In some embodiments, the one or more agent(s) that result(s) in a decrease in

the activation of a TGF-B decrease(s) the activation of a TGF-B receptor through binding

to a LAP, or to a TGF-B/LAP complex.

In some embodiments, the soluble human tissue factor domain does not initiate

blood coagulation. In some embodiments, the method further comprises administering an

additional therapeutic agent selected from the group of: combinations of agents, such as

checkpoint inhibitors, chemotherapy drugs, and therapeutic antibodies.

In some embodiments of any of the methods described herein, the single-chain

chimeric polypeptide is stable in human serum for at least 10 days at 37 °C. In some

embodiments of any of the methods described herein, the multi-chain chimeric

polypeptide is stable in human serum for at least 10 days at 37 °C. In some embodiments

of any of the methods described herein, the single-chain chimeric polypeptide does not

have significant clotting activity. In some embodiments of any of the methods described

herein, the multi-chain chimeric polypeptide does not have significant clotting activity.

In some embodiments of any of the methods described herein, the method results

in rejuvenation of aged immune cells in the subject. In some embodiments of any of the methods described herein, the rejuvenation of the aged immune cells results in a reduction of number of diseased cells or infectious agents in the subject. In some embodiments of any of the methods described herein, the aged immune cells include one or more of aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, aged Kupffer cells, and aged microgial cells. In some embodiments of any of the methods described herein, the diseased cells include cancer cells, virally-infected cells, and intracellularly- bacterially-infected cells. In some embodiments of any of the methods described herein, the infectious agents include virus, bacterium, fungus, and parasite.

As used herein, the term "chimeric" refers to a polypeptide that includes amino

acid sequences (e.g., domains) originally derived from two different sources (e.g., two

different naturally-occurring proteins, e.g., from the same or different species). For

example, a chimeric polypeptide can include domains from at least two different

naturally occurring human proteins. In some examples, a chimeric polypeptide can

include a domain that is a synthetic sequence (e.g., a scFv) and a domain that is derived

from a naturally-occurring protein (e.g., a naturally-occurring human protein). In some

embodiments, a chimeric polypeptide can include at least two different domains that are

synthetic sequences (e.g., two different scFvs).

An "activated NK cell" is a NK cell demonstrating increased expression levels of

two or more (e.g., three, four, five, or six) of CD25, CD69, MTOR-C1, SREBP, IFN-y,

and a granzyme (e.g., granzyme B), e.g., as compared to a resting NK cell. Exemplary

methods for identifying the expression levels of CD25, CD69, MTOR-C1, SREBP, IFN-

Y, and a granzyme (e.g., granzyme B) are described herein.

A "resting NK cell" is a NK cell that has a reduced expression of two or more

(e.g., three, four, five, or six) of CD25, CD69, MTOR-C1, SREBP, IFN-y, and a granzyme (e.g., granzyme B), e.g., as compared to an activated NK cell.

An "NK cell activating agent" is an agent that induces or promotes (alone or in

combination with additional NK cell activating agents) a resting NK cell to develop into

an activated NK cell. Non-limiting examples and aspects of NK cell activating agents are

described herein.

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An "antigen-binding domain" is one or more protein domain(s) (e.g., formed from

amino acids from a single polypeptide or formed from amino acids from two or more

polypeptides (e.g., the same or different polypeptides) that is capable of specifically

binding to one or more different antigen(s). In some examples, an antigen-binding

domain can bind to an antigen or epitope with specificity and affinity similar to that of

naturally-occurring antibodies. In some embodiments, the antigen-binding domain can

be an antibody or a fragment thereof. In some embodiments, an antigen-binding domain

can include an alternative scaffold. Non-limiting examples of antigen-binding domains

are described herein. Additional examples of antigen-binding domains are known in the

art.

A "soluble tissue factor domain" refers to a polypeptide having at least 70%

identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least

90% identity, at least 95% identity, at least 99% identity, or 100% identical) to a segment

of a wildtype mammalian tissue factor protein (e.g., a wildtype human tissue factor

protein) that lacks the transmembrane domain and the intracellular domain. Non-limiting

examples of soluble tissue factor domains are described herein.

The term "soluble interleukin protein" is used herein to refer to a mature and

secreted interleukin protein or a biologically active fragment thereof. In some examples,

a soluble interleukin protein can include a sequence that is at least 70% identical, at least

75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at

least 95% identical, at least 99% identical, or 100% identical to a wildtype mature and

secreted mammalian interleukin protein (e.g., a wildtype human interleukin protein) and

retains its biological activity. Non-limiting examples of soluble interleukin proteins are

described herein.

The term "soluble cytokine protein" is used herein to refer to a mature and

secreted cytokine protein or a biologically active fragment thereof. In some examples, a

soluble cytokine protein can include a sequence that is at least 70% identical, at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical to a wildtype mature and secreted

mammalian interleukin protein (e.g., a wildtype human interleukin protein) and retains its

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biological activity. Non-limiting examples of soluble cytokine proteins are described

herein.

The term "soluble interleukin receptor" is used herein in the broadest sense to

refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular

domain) that is capable of binding one or more of its natural ligands (e.g., under

physiological conditions, e.g., in phosphate buffered saline at room temperature). For

example, a soluble interleukin receptor can include a sequence that is at least 70%

identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at

least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to an

extracellular domain of wildtype interleukin receptor and retains its ability to specifically

bind to one or more of its natural ligands, but lacks its transmembrane domain (and

optionally, further lacks its intracellular domain). Non-limiting examples of soluble

interleukin receptors are described herein.

The term "soluble cytokine receptor" is used herein in the broadest sense to refer

to a polypeptide that lacks a transmembrane domain (and optionally an intracellular

domain) that is capable of binding one or more of its natural ligands (e.g., under

physiological conditions, e.g., in phosphate buffered saline at room temperature). For

example, a soluble cytokine receptor can include a sequence that is at least 70% identical

(e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90%

identical, at least 95% identical, at least 99% identical, or 100% identical) to an

extracellular domain of wildtype cytokine receptor and retains its ability to specifically

bind to one or more of its natural ligands, but lacks its transmembrane domain (and

optionally, further lacks its intracellular domain). Non-limiting examples of soluble

cytokine receptors are described herein.

The term "antibody" is used herein in its broadest sense and includes certain types

of immunoglobulin molecules that include one or more antigen-binding domains that

specifically bind to an antigen or epitope. An antibody specifically includes, e.g., intact

antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific

antibodies. One example of an antigen-binding domain is an antigen-binding domain

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formed by a VH -VL dimer. Additional examples of an antibody are described herein.

Additional examples of an antibody are known in the art.

"Affinity" refers to the strength of the sum total of non-covalent interactions

between an antigen-binding site and its binding partner (e.g., an antigen or epitope).

Unless indicated otherwise, as used herein, "affinity" refers to intrinsic binding affinity,

which reflects a 1:1 interaction between members of an antigen-binding domain and an

antigen or epitope. The affinity of a molecule X for its partner Y can be represented by

the dissociation equilibrium constant (KD). The kinetic components that contribute to the

dissociation equilibrium constant are described in more detail below. Affinity can be

measured by common methods known in the art, including those described herein.

Affinity can be determined, for example, using surface plasmon resonance (SPR)

technology (e.g., BIACORE or biolayer interferometry (e.g., FORTEBIOR).

Additional methods for determining the affinity for an antigen-binding domain and its

corresponding antigen or epitope are known in the art.

A "single-chain polypeptide" as used herein to refers to a single protein chain.

A "multi-chain polypeptide" as used herein to refers to a polypeptide comprising

two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at

least a first chimeric polypeptide and a second polypeptide), where the two or more

proteins chains associate through non-covalent bonds to form a quaternary structure.

The term "pair of affinity domains" is two different protein domain(s) that bind

specifically to each other with a KD of less than of less than 1 X 10-7 M (e.g., less than 1 X

10-8 M, less than 1 X 10-9 M, less than 1 X 10-10 M, or less than 1 X 10-11 M). In some

examples, a pair of affinity domains can be a pair of naturally-occurring proteins. In

some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Non-

limiting examples of pairs of affinity domains are described herein.

The term "epitope" means a portion of an antigen that specifically binds to an

antigen-binding domain. Epitopes can, e.g., consist of surface-accessible amino acid

residues and/or sugar side chains and may have specific three-dimensional structural

characteristics, as well as specific charge characteristics. Conformational and non-

conformational epitopes are distinguished in that the binding to the former but not the

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latter may be lost in the presence of denaturing solvents. An epitope may comprise

amino acid residues that are directly involved in the binding, and other amino acid

residues, which are not directly involved in the binding. Methods for identifying an

epitope to which an antigen-binding domain binds are known in the art.

The term "treatment" means to ameliorate at least one symptom of a disorder. In

some examples, the disorder being treated is cancer and to ameliorate at least one

symptom of cancer includes reducing aberrant proliferation, gene expression, signaling,

translation, and/or secretion of factors. Generally, the methods of treatment include

administering a therapeutically effective amount of a composition that reduces at least

one symptom of a disorder to a subject who is in need of, or who has been determined to

be in need of such treatment.

Unless otherwise defined, all technical and scientific terms used herein have the

same meaning as commonly understood by one of ordinary skill in the art to which this

invention belongs. Methods and materials are described herein for use in the present

invention; other, suitable methods and materials known in the art can also be used. The

materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patent applications, patents, sequences, database entries, and other

references mentioned herein are incorporated by reference in their entirety. In case of

conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the

following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS Figures 1A-1B show the results of immunostimulation of an exemplary multi-

chain polypeptide in C57BL/6 mice. Figure 1A shows the spleen weight of mice treated

with increasing dosage of the exemplary multi-chain polypeptide as compared to mice

treated with the control solution. Figure 1B shows the percentages of immune cell types

present in the spleen of mice treated with increasing dosage of the exemplary multi-chain

polypeptide as compared to mice treated with the control solution.

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Figures 2A-2B show the duration of immunostimulation of an exemplary multi-

chain polypeptide in C57BL/6 mice. Figure 2A shows the spleen weight over a period of

92 hours in mice treated with 3 mg/kg of the exemplary multi-chain polypeptide. Figure

2B shows the percentages of immune cell types present in the spleen over a period of 92

hours in mice treated with 3 mg/kg of the exemplary multi-chain polypeptide.

Figures 3A-3B show the expression of Ki67 and Granzyme B in immune cells

induced by the exemplary multi-chain polypeptide. Figure 3A shows the expression of

Ki67 in CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and CD19+ B cells at

various time points post-treatment with the multi-chain polypeptide. Figure 3B shows the

expression of Granzyme B in CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and

CD19+ B cells at various time points post-treatment with the multi-chain polypeptide.

Figure 4 shows the effect of tumor inhibition by splenocytes prepared from mice

treated with an exemplary multi-chain polypeptide at various time points after treatment.

Figures 5A-5B show the percentages and the proliferation rate of CD4+ T cells,

CD8+ T cells, Natural Killer (NK) cells, and CD19+B cells in the blood of B6.129P2-

ApoE tmlUnc/J mice (purchased from The Jackson Laboratory) fed a control diet, a high fat

diet and untreated, and mice fed a high fat diet and treated with TGFRt15-TGFRs, 2t2, or

21t15-TGFRs. Figure 5A shows the percentages of the different cell types in each control

and experimental group. Figure 5B shows the proliferation rate of the of the different cell

types in each control and experimental group.

Figures 6A-6E show exemplary physical appearance of mice fed either a control

or high fat diet and were either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-

TGFRs. Figure 7 shows the fasting body weight of mice fed either a control or a high fat

diet and were either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs.

Figure 8 shows the fasting blood glucose levels of mice fed either a control or a

high fat diet and were either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-

TGFRs. Figures 9A-9F show chemotherapy-induced senescent B16F10 cells and

expression of senescent genes. Figure 9A shows chemotherapy induction of senescent

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B16F10 cells visualized using SA B-gal staining. Figures 9B-9F show expression of

p21CIP1, IL6, DPP4, RATE1E, and ULBP1 over time in the chemotherapy-induced

senescent B16F10 cells.

Figures 10A-10F show colony formation and expression of stem cell markers by

chemotherapy-induced senescent B16F10 cells. Figure 10A shows colony formation by

chemotherapy-induced senescent B16F10 cells. Figures 10B and 10C show expression of

Oct4 mRNA and Notch4 mRNA by chemotherapy-induced senescent B16F10 cells as

compared to control B16F10 cells. Figures 10D-10F show percentage of chemotherapy-

induced senescent B16F10 cells double-positive for two out of the three stem cell

markers including CD44, CD24, and CD133.

Figures 11A-11C show migratory and invasive properties of chemotherapy-

induced senescent B16F10 cells. Figure 11A shows the results of a migration assay

comparing chemotherapy-induced senescent cells with stem cell properties (B16F10-

SNC-CSC) with control B16F10 cells. Figures 11B and 11C show the results of an

invasion assay comparing chemotherapy-induced senescent cells with stem cell

properties (B16F10-SNC-CSC) with control B16F10 cells.

Figures 12A and 12B show in vitro expanded NK cells and their cytotoxicity

against chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-

CSC) or control B16F10 cells. Figure 12A shows an exemplary schematic of a process of

obtaining in vitro expanded NK cells. Figure 12 B shows cytotoxicity of the expanded

NK cells against chemotherapy-induced senescent cells with stem cell properties

(B16F10-SNC-CSC) or control B16F10 cells.

Figures 13A-13C show results of combination treatment using a mouse melanoma

model. Figure 13A shows an exemplary schematic for treating melanoma in a mouse

model. Figures 13B and 13C show the change in tumor volume over time with

combination treatments including TGFRt15-TGFRs as compared to chemotherapy or

TA99 treatment alone.

Figure 14 shows induction of senescence in the human pancreatic tumor cell line

SW1990 and expression of CD44 and CD24 in senescent SW 1990 cells as compared to

control SW1990 cells.

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Figure 15 shows expression of senescent markers by chemotherapy-induced

senescent SW1990 cells

Figure 16 shows the cytotoxicity of in vitro activated human NK cells against

chemotherapy-induced senescent SW1990 cells or control SW1990 cells.

Figure 17 shows a schematic diagram of an exemplary IL-12/IL-15RaSu DNA

construct.

Figure 18 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNA

construct.

Figure 19 shows a schematic diagram of the interaction between the exemplary

IL-12/IL-15RaSu and IL-18/TF/IL-15 DNA constructs.

Figure 20 shows a schematic diagram of the interaction between the exemplary

IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins resulting in IL-18/TF/IL-15:IL-

12/IL-15RaSu complex (18t15-12s).

Figure 21 shows a chromatograph of 18t15-12s purification elution from an anti-

TF antibody affinity column.

Figure 22 shows an exemplary chromatographic profile of anti-TF Ab /SEC-

purified 18t15-12s protein following elution on an analytical size exclusion column,

demonstrating separation of monomeric multiprotein 18t15-12s complexes from protein

aggregates.

Figure 23 shows an example of a 4-12% SDS-PAGE of the 18t15-12s complex

following disulfide bond reduction. Lane 1: SeeBlue Plus2 marker; Lane 2: anti-TF Ab-

purified 18t15-12s (0.5 ug); Lane 3: anti-TF Ab-purified 18t15-12s (1 ug).

Figure 24 shows SDS PAGE analysis of deglycosylated and non-deglycosylated

18t15-12s. Lane 1: anti-TF Ab-purified 18t15-12s (0.5 ug), non-deglycosylated; Lane 2:

anti-TF Ab -purified 18t15-12s (1 ug), non-deglycosylated; Lane 3: 18t15-12s (1 ug),

deglycosylated, Lane 4: Mark12 unstained maker.

Figure 25 shows a sandwich ELISA for the 18t15-12s complex, comprising an

anti-human tissue factor antibody capture and a biotinylated anti-human IL-12 detection

antibody (BAF 219).

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Figure 26 shows a sandwich ELISA for the 18t15-12s complex, comprising an

anti-human tissue factor antibody capture and a biotinylated anti-human IL-15 detection

antibody (BAM 247).

Figure 27 shows a sandwich ELISA for the 18t15-12s complex, comprising an

anti-human tissue factor antibody capture and a biotinylated anti-human IL-18 detection

antibody (D045-6).

Figure 28 shows a sandwich ELISA for the 18t15-12s complex, comprising an

anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection

antibody.

Figure 29 shows proliferation of IL-15-dependent 32DB cells mediated by the

18t15-12s complex (open squares) and recombinant IL-15 (black squares).

Figure 30 shows biological activity of IL-18 within the 18t15-12s complex (open

squares), where recombinant IL-18 (black squares) and recombinant IL-12 (black circles)

serve as positive and negative controls, respectively.

Figure 31 shows biological activity of IL-12 within the 18t15-12s complex (open

squares), where recombinant IL-12 (black circles) and recombinant IL-18 (open squares)

serve as positive and negative controls, respectively.

Figures 32A and 32B show cell-surface expression of CD25 on NK cells induced

by the 18t15-12s complex and cell-surface CD69 expression of NK cells induced by the

18t15-12s complex.

Figure 33 shows a flow cytometry graph of intracellular IFN-y expression of NK

cells induced by the 18t15-12s complex.

Figure 34 shows cytotoxicity of 18t15-12s induced human NK cells against K562

cells.

Figure 35 shows a schematic diagram of an exemplary IL-12/IL-15RaSu/aCD16

DNA construct. DNA construct

Figure 36 shows a schematic diagram of an exemplary IL-18/TF/IL-15DNA

construct.

Figure 37 shows a schematic diagram of the interaction between the exemplary

IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 DNA constructs.

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Figure 38 shows a schematic diagram of an exemplary 18t15-12s/aCD16 protein

complex.

Figure 39 shows a sandwich ELISA for the 18t15-12s16 complex, comprising an

anti-human tissue factor antibody capture antibody and a biotinylated anti-human IL-12

(BAF 219) (dark line) or an anti-human tissue factor detection antibody (light line).

Figure 40 shows a schematic diagram of an exemplary TGF3RII/IL-15RoSu

DNA construct.

Figure 41 shows a schematic diagram of an exemplary IL-21/TF/IL-15 construct.

Figure 42 shows a schematic diagram of the interaction between the exemplary

IL- IL-21/TF/IL-15 and TGF3RII/IL-15RaSu constructs.

Figure 43 shows a schematic diagram of the interaction between the exemplary

TGFBRII/IL-15RaSu and IL-21/TF/IL-15 fusion proteins, resulting in an IL-21/TF/IL-

15/TGFBRII/IL-15RaSu complex (21t15-TGFRs).

Figure 44 shows a chromatograph of 21t15-TGFRs purification elution from an

anti-TF antibody affinity column.

Figure 45 shows an exemplary 21t15-TGFRs size exclusion chromatograph

showing a main protein peak and a high molecular weight peak

Figure 46 shows an example of a 4-12% SDS-PAGE of the 21t15-TGFRs

complex following disulfide bond reduction. Lane 1: Mark12 unstained marker (numbers

on the left side indicate molecular weights in kDa); Lane 2: 21t15-TGFRs (0.5 ug); Lane

3: 21t15-TGFRs (1 ug); Lane 4: 21t15-TGFRs, deglycosylated (1 ug), wherein the MW

was the expected size of 53kDa and 39.08 kDa.

Figure 47 shows a sandwich ELISA for the 21t15-TGFRs complex, comprising an

anti-human tissue factor capture and a biotinylated anti-human IL-21 detection antibody

(13-7218-81, BioLegend).

Figure 48 shows a sandwich ELISA for the 21t15-TGFRs complex, comprising an

anti-human tissue factor antibody capture and a biotinylated anti-human IL-15 detection

antibody (BAM 247, R&D Systems).

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Figure 49 shows a sandwich ELISA for the 21t15-TGFRs complex, comprising an

anti-human tissue factor antibody capture and a biotinylated anti-human TGFßRII

detection antibody (BAF241, R&D Systems).

Figure 50 shows a sandwich ELISA for the 21t15-TGFRs complex, comprising an

anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection

antibody.

Figure 51 shows IL-15-dependent proliferation of 32D cells mediated by the

21t15-TGFRs complex (open squares) compared to IL-15 (black squares).

Figure 52 shows biological activity of the TGFßRII domain within the 21t15-

TGFRs complex (open squares). TGF3RII/Fc (black squares) served as a positive

control.

Figure 53 shows a flow cytometry graph of cell-surface CD25 expression of NK

cells induced by the 21t15-TGFRs complex.

Figure 54 shows a flow cytometry graph of cell-surface CD69 expression of NK

cells induced by the 21t15-TGFRs complex.

Figure 55 shows a flow cytometry graph of intracellular IFN-y expression of NK

cells induced by the 21t15-TGFRs complex.

Figure 56 shows cytotoxicity of 21t15-TGFRs-induced human NK cells against

K562 cells.

Figure 57 are schematic diagrams of an exemplary aCD3scFv/TF/aCD28scFv

single-chain chimeric polypeptide.

Figure 58 is a chromatograph showing the elution of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide from an anti-tissue factor

affinity column.

Figure 59 is a chromatograph showing the elution of a Superdex 200 Increase

10/300 GL gel filtration column loaded with an exemplary aCD3scFv/TF/aCD28scFv

single-chain chimeric polypeptide.

Figure 60 is a sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-

Tris gel) of an exemplary aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide

purified using an anti-tissue factor affinity column.

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Figure 61 is a graph showing the ELISA quantitation of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide performed using the

methods described in Example 1. Purified tissue factor was used as the control.

Figure 62 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD25

expression in CD4+ T-cells isolated from blood from two donors. The experiments were

performed as described in Example 2.

Figure 63 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD25

expression in CD8+ T-cells isolated from blood from two donors. The experiments were

performed as described in Example 2.

Figure 64 is a graph showing the ability of an exemplary

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide to stimulate CD69

expression in CD4+ T-cells isolated from blood from two donors. The experiments were

performed as described in Example 2.

Figure 65 shows a schematic diagram of an exemplary IL-7/IL-15RaSu DNA

construct.

Figure 66 shows a schematic diagram of an exemplary IL-21/TF/IL-151 DNA

construct.

Figure 67 shows a schematic diagram of the interaction between the exemplary

IL-7/IL-15RaSu and IL-21/TF/IL-15 DNA constructs.

Figure 68 shows a schematic diagram of the interaction between the exemplary

IL-7/IL-15RoSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-21/TF/IL-15:IL-

7/IL-15Ra.Su complex (21t15-7s).

Figure 69 shows a schematic diagram of an exemplary IL-21/IL-15RaSu DNA

construct.

Figure 70 shows a schematic diagram of an exemplary IL-7/TF/IL-15 DNA

construct.

Figure 71 shows a schematic diagram of the interaction between the exemplary

IL-21/IL-15RaSu and IL-7/TF/IL-15 DNA constructs.

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Figure 72 shows a schematic diagram of the interaction between the exemplary

IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins resulting in an IL-7/TF/IL-15:IL-

21/IL-15RaSU complex (7t15-21s).

Figure 73 shows the oxygen consumption rate (OCR) in pmoles/min for human

NK cells isolated from blood (2 X 106 cells/mL) of two different donors.

Figure 74 shows the extracellular acidification rate (ECAR)

in mpH/minute for human NK cells isolated from blood (2 X 106 cells/mL) of two

different donors.

Figure 75 shows a schematic of the 7t15-16s21 construct.

Figure 76 shows an additional schematic of the 7t15-16s21 construct.

Figures 77A and 77B show binding of 7t15-16s21 to CHO cells expressing

human CD16b as compared to a control protein.

Figures 78A-78C are results from ELISA experiments using antibodies against

IL-15, IL-21, and IL-7 in detecting 7t15-16s21.

Figure 79 shows results of the 32DB cell proliferation assay with 7t15-16s21 or

recombinant IL-15.

Figure 80 shows the chromatographic profile of 7t15-16s21 protein containing

cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 81 shows the analytical SEC Profile of 7t15-16s21.

Figure 82 shows a schematic of the TGFRt15-16s21 construct.

Figure 83 shows an additional schematic of the TGFRt15-16s21 construct.

Figures 84A and 84B show binding affinity of TGFRT15-16S21 and 7t15-21s

with CHO cells expressing human CD16b. Figure 84A shows binding affinity of

TGFRT15-16S21 with CHO cells expressing human CD16b. Figure 84B shows binding

affinity of 7t15-21s with CHO cells expressing human CD16b.

Figure 85 shows results of TGFß1 inhibition by TGFRt15-16s21 and TGFR-Fc.

Figure 86 shows results of 32DB cell proliferation assay with TGFRt15-16s21 or

recombinant IL-15.

Figures 87A-87C show results of detecting IL-15, IL-21, and TGFßRII in

TGFRt15-16s21 with corresponding antibodies using ELISA.

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Figure 88 shows the chromatographic profile of TGFRt15-16s21 protein

containing cell culture supernatant following binding and elution on anti-TF antibody

resin.

Figure 89 shows results of a reduced SDS-PAGE analysis of TGFRt15-16s21.

Figure 90 shows a schematic of the 7t15-7s construct.

Figure 91 shows an additional schematic of the 7t15-7s construct.

Figure 92 shows the chromatographic profile of 7t15-7s protein containing cell

culture supernatant following binding and elution on anti-TF antibody resin.

Figure 93 shows detection of TF, IL-15 and IL-7 in 7t15-7s using ELISA.

Figures 94A and 94B show spleen weight and the percentages of immune cell

types in 7t15-7s -treated and control-treated mice. Figure 94A shows spleen weight in

mice treated with 7t15-7s as compared to PBS control. Figure 94B shows the percentage

of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with 7t15-7s as compared to

PBS control.

Figure 95 shows a schematic of the TGFRt15-TGFRs construct.

Figure 96 shows an additional schematic of the TGFRt15-TGFRs construct.

Figure 97 shows results of TGFß1 inhibition by TGFRt15-TGFRs and TGFR-Fc.

Figure 98 shows results of 32D cell proliferation assay with TGFRt15-TGFRs or

recombinant IL-15

Figures 99A and 99B show results of detecting IL-15 and TGFßRII in TGFRt15-

TGFRs with corresponding antibodies using ELISA.

Figure 100 is a line graph showing the chromatographic profile of TGFRt15-

TGFRs protein containing cell culture supernatant following binding and elution on anti-

TF antibody resin.

Figure 101 shows the analytical SEC profile of TGFRt15-TGFRs.

Figure 102 shows TGFR115-TGFRs before and after deglycosylation as analyzed

by reduced SDS-PAGE.

Figures 103A and 103B show spleen weight and the percentages of immune cell

types in TGFRt15-TGFRs-treated and control-treated mice. Figure 103A shows spleen

weight in mice treated with TGFRt15-TGFRs as compared to PBS control. Figure 103B

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shows the percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with

TGFRt15-TGFRs as compared to PBS control.

Figure 104A and 104B show the spleen weight and immunostimulation over 92

hours in mice treated with TGFRt15-TGFRs. Figure 104A shows spleen weight of mice

treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure

104B shows the percentages of immune cells in mice treated with TGFRt15-TGFRs at

16, 24, 48, 72, and 92 hours after treatment.

Figure 105A and 105B show Ki67 and Granzyme B expression in mice treated

with TGFRt15-TGFRs over time.

Figure 106 shows enhancement of cytotoxicity of splenocytes by TGFRt15-

TGFRs in C57BL/6 Mice.

Figure 107 shows changes in tumor size in response to PBS treatment,

chemotherapy alone, TGFRt15-TGFRs alone, or chemotherapy and TGFRt15-TGFRs

combination, in a pancreatic cancer mouse model.

Figure 108 shows the cytotoxicity of NK cells isolated from mice treated with

TGFRt15-TGFRs. Figure 109 shows a schematic of the 7t15-21s137L (long version) construct.

Figure 110 shows an additional schematic of the 7t15-21s137L (long version)

construct.

Figure 111 is a line graph showing the chromatographic profile of 7t15-21s137L

(long version) protein containing cell culture supernatant following binding and elution

on anti-TF antibody resin.

Figure 112 shows the analytical SEC profile of 7t15-21s137L (long version).

Figure 113 shows binding of 7t15-21s137L (short version) to CD137L (4.1BBL)

Figures 114A-114C show detection of IL-15, IL21, and IL7 in 7t15-21s137L

(short version) with ELISA. Figure 114A shows detection of IL-15 in 7t15-21s137L

(short version) with ELISA. Figure 114B shows detection of IL21 in 7t15-21s137L

(short version) with ELISA. Figure 114C shows detection of IL7 in 7t15-21s137L (short

version) with ELISA.

Figure 115 shows results from a CTLL-2 cell proliferation assay.

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Figure 116 shows the activity of 7t15-1s137L (short version) in promoting IL21R

containing B9 cell proliferation.

Figure 117 shows a schematic of the 7t15-TGFRs construct.

Figure 118 shows an additional schematic of the 7t15-TGFRs construct.

Figure 119 shows results of TGFB1 inhibition by 7t15-TGFRs and TGFR-Fc.

Figures 120A-120C show detection of IL-15, TGFßRII, and IL-7 in 7t15-TGFRs

with ELISA.

Figure 121 shows results of a 32D cell proliferation assay with 7t15-TGFRs or

recombinant IL-15.

Figure 122 is a line graph showing the chromatographic profile of 7t15-TGFRs

protein containing cell culture supernatant following binding and elution on anti-TF

antibody resin.

Figure 123 shows 7t15-TGFRs before and after deglycosylation as analyzed using

reduced SDS-PAGE.

Figure 124 shows ELISA detection of IL-7, IL-15 and TGFßRII in the 7t15-

TGFRs protein.

Figures 125A and 125B show spleen weight and the percentages of immune cell

types in 7t15-TGFRs-treated and control-treated mice. Figure 125A shows spleen weight

in mice treated with 7t15-TGFRs at various dosages, as compared to PBS control. Figure

125B shows the percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated

with 7t15-TGFRs at various dosages, as compared to PBS control.

Figures 126A and 126B show upregulation of CD44 expression of CD4+ and

CD8+ T cells by 7t15-TGFRs in C57BL/6 mice.

Figures 127A and 127B show upregulation of Ki67 expression and Granzyme B

expression of CD8+ T cells and NK Cells by 7t15-TGFRs in C57BL/6 mice.

Figure 128 shows enhancement of cytotoxicity of splenocytes by 7t15-TGFRs in

C57BL/6 mice.

Figure 129 shows a schematic of the TGFRt15-21s137L construct.

Figure 130 shows an additional schematic of the TGFRt15-21s137L construct.

PCT/US2021/035285

Figure 131 is a line graph showing the chromatographic profile of

TGFRt15-21s137L protein containing cell culture supernatant following binding

and elution on anti-TF antibody resin.

Figure 132 shows a schematic of the TGFRt15-TGFRs21 construct.

Figure 133 shows an additional schematic of the TGFRt15-TGFRs21

construct.

Figure 134 is a line graph showing the chromatographic profile of TGFRt15-

TGFRs21 protein containing cell culture supernatant following binding and elution on

anti-TF antibody resin.

Figure 135 shows TGFRt15-TGFRs21 before and after deglycosylation as

analyzed by reduced SDS-PAGE.

Figures 136A and 136B show detection of components of TGFRt15-TGFRs21

using ELISA.

Figures 137A and 137B show the percentages and proliferation of CD4+ T cells,

CD8+ T cells, and natural killer (NK) cells present in the spleen of control-treated and

TGFRt15-TGFRs21-treated mice.

Figure 138 shows upregulation of Granzyme B expression of splenocytes in mice

treated with TGFRt15-TGFRs21.

Figure 139 shows enhancement of cytotoxicity of splenocytes by TGFRt15-TGFRs21

in C57BL/6 Mice.

Figure 140 shows a schematic of the TGFRt15-TGFRs16 construct.

Figure 141 shows an additional schematic of the TGFRt15-TGFRs16 construct.

Figure 142 shows a schematic of the TGFRt15-TGFRs137L construct.

Figure 143 shows an additional schematic of the TGFRt15-TGFRs137L construct.

Figure 144 are schematic diagrams of an exemplary 2t2 single-chain chimeric

polypeptide.

Figure 145 shows IL-2 activity in 2t2 as compared to recombinant IL-2 using a

32D cell proliferation assay.

Figure 146 shows IL-2 activity in 2t2 as compared to recombinant IL-2 using a

CTLL-2 cell proliferation assay.

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Figure 147 shows the fasting blood glucose levels in ApoE- mice fed with

standard chow or a high fat diet and treated with a PBS control (untreated) or with 2t2.

Figure 148 shows the ratio of CD4*CD25*FoxP3* T regulatory cells in blood

lymphocytes from ApoE- mice fed with standard chow or a high fat diet and treated with

a PBS control (untreated) or with 2t2.

Figure 149 is a line graph showing the chromatographic profile of 2t2 protein

containing cell culture supernatant following binding and elution on anti-TF antibody

resin.

Figure 150 shows an analytical SEC profile of 2t2.

Figures 151A and 151B show reduced SDS-PAGE analysis of 2t2 before and

after deglycosylation. Figure 151A shows reduced SDS-PAGE analysis of 2t2 before

deglycosylation. Figure 151B shows reduced SDS-PAGE analysis of 2t2 after

deglycosylation.

Figures 152A and 152B show results of immunostimulation in C57BL/6 mice

using 2t2. Figure 152A shows spleen weight following treatment with 2t2. Figure 152B

shows the percentages of immune cell types following 2t2 treatment.

Figure 153 shows upregulation of CD25 expression of CD4+ T cells in mice

treated with 2t2.

Figure 154 shows the pharmacokinetics of 2t2 in C57BL/6 mice.

Figures 155A and 155B show effects of 2t2 in attenuating the formation of high

fat-induced atherosclerotic plaques in ApoE- mice. Figure 155A shows a representative

view of atherosclerotic plaques from ApoE- mice fed with standard chow or a high fat

diet and treated with either PBS control or 2t2. Figure 155B shows the results of

quantitative analysis of atherosclerotic plaques of each group.

Figure 156 shows fasting glucose levels in 2t2 treated-mice as compared to

control-treated mice.

Figure 157 shows the percentage of CD4+CD25*FoxP3+ Tregs in blood

lymphocytes from mice treated with 2t2 and control-treated mice.

Figure 158 are schematic diagrams of an exemplary 15t15 single-chain chimeric

polypeptide.

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Figure 159 shows the IL-15 activity of 15t15 as compared to recombinant IL-15

in a 32DB cell proliferation assay.

Figure 160 is a line graph showing the chromatographic profile of 15t15 protein

containing cell culture supernatant following binding and elution on anti-TF antibody

resin.

Figures 161A and 161B show reduced SDS-PAGE analysis of 15t15 before and

after deglycosylation. Figure 161A shows reduced SDS-PAGE analysis of 15t15 before

deglycosylation. Figure 161B shows reduced SDS-PAGE analysis of 15t15 after

deglycosylation.

Figures 162A and 162B is a set of histograms (Figure 162A) and a set of graphs

(Figure 162B) showing the change in the surface phenotype of NK cells after stimulation

with 18t15-12s, 18t15-12s16, and 7t15-21s + anti-TF antibody.

Figure 163 is a set of graphs showing changes in the surface phenotype of

lymphocyte populations after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s.

Figure 164 is a set of graphs showing an increase in glycolysis in NK cells

following treatment with 18t15-12s.

Figure 165 is a set of graphs showing an increase in phospho-STAT4 and

phospho-STAT5 levels in NK cells after stimulation with 18t15-12s.

Figure 166 is a set of graphs showing that overnight stimulation of NK cells with

18t15-12s enhances cell metabolism.

Figure 167A-C is a set of graphs showing immunostimulation in C57BL/6 mice

following treatment with 2t2.

Figure 168A-B is a set of graphs showing immunostimulation in C57BL/6 mice

following treatment with TGFRt15-TGFRs.

Figure 169A-C is a set of graphs showing in vivo stimulation of Tregs, NK cells,

and CD8+ T cells in ApoE- mice fed with a Western diet and treated with TGFRt15-

TGFRs or 2t2.

Figure 170A-B is a set of graphs showing induction of splenocyte proliferation by

2t2 in C57BL/6 mice.

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Figure 171A-C is a set of graphs showing immunostimulation in C57BL/6 mice

following treatment with TGFRt15-TGFRs.

Figure 172A-B is a set of graphs showing in vivo induction of proliferation of NK

cells and CD8+ T cells in ApoE- mice fed with a Western diet and treated with

TGFRt15-TGFRs or 2t2.

Figure 173 is a schematic and a set of graphs showing the persistence of 7t15-21s

and anti-TF antibody-expanded NK cells in NSG mice following treatment with 7t15-21,

TGFRt15-TGFRs or 2t2.

Figure 174A-B is a set of graphs showing enhancement of cytotoxicity of NK

cells following treatment of NK cells with TGFRt15-TGFRs.

Figure 175A-B is a set of graphs showing enhancement of ADCC activity of NK

cells following treatment of NK cells with TGFRt15-TGFRs.

Figure 176 is a graph of in vitro killing of senescent B16F10 melanoma cells by

TGFRt15-TGFRs/2t2-activated mouse NK cells.

Figure 177A-H is a set of graphs showing antitumor activity of TGFRt15-TGFRs

plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma

mouse model.

Figure 178A-C is a set of graphs showing amelioration of the Western diet-

induced hyperglycemia in ApoE- mice by 2t2.

Figure 179 is a set of graphs showing cell surface staining summarizing the

differentiation of NK cells into cytokine-induced memory like NK cells (CIML-NK

Cells) after stimulation with 18t15-12s and cultured in rhIL-15.

Figure 180 shows upregulation of CD44 memory T cells. The upper panel shows

upregulation of CD44 memory T cells upon treatment with TGFRt15-TGFRs The lower

panel shows upregulation of CD44 memory T cells upon treatment with 2t2.

Figures 181A and 181B show improvement in hair regrowth following depilation

in mice treated with 2t2 or IL-2. Figure 181A shows skin pigmentation 10 days after

depilation in PBS-, 2t2-, or IL-2-treated mice. Figure 181B shows percent pigmentation

in PBS-, 2t2-, or IL-2-treated mice as analyzed using the ImageJ software.

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Figure 182 shows skin pigmentation 14 days after depilation in PBS-, 2t2-, or IL-

2-treated mice.

Figure 183 shows a graph of Factor X (FX) activation following treatment with

single-chain or multi-chain chimeric polypeptides.

Figure 184 shows clotting time for a buffer with varying concentrations of

Innovin in a prothrombin time (PT) test.

Figure 185 shows clotting time for multi-chain chimeric polypeptides in a PT

Assay.

Figure 186 shows clotting time of the multi-chain chimeric polypeptides in a PT

assay when mixed with 32DB cells.

Figure 187 shows clotting time of multi-chain chimeric polypeptides in a PT

assay when mixed with human PBMC.

Figure 188 shows binding of 7t15-21s137L (long version) and 7t15-21s137L

(short version) to CD137 (4.1BB).

Figure 189A-189D show detection of IL7, IL21, IL15, and 4.1BBL in 7t15-

21s137L (long version) by the respective antibodies using ELISA.

Figure 190 shows IL-15 activity of 7t15-21s137L (long version) and 7t15-

21s137L (short version) as evaluated by an IL2Ro.By-containing CTLL2 cell proliferation

assay.

Figures 191A-191C show human blood lymphocyte pStat5a responses in

CD4*CD25"Treg cells, CD4*CD25*Tcon cells, or in CD8+ Tcon cells in response to 2t2 or

IL2 treatment. Figure 191A shows pSTAT5 responses in CD4*CD25"brT cells. Figure

C191B shows pSTAT5 responses in CD4*CD25*Tcon cells. Figure 191C shows pSTAT5

responses in CD8+ Tcon cells.

Figures 192A-192E is a set of imaging showing that treatment with an IL-2 based

molecule (2t2) can induce formation of hair follicles following depilation in mouse

model. Figure 192A is an image from a control mouse - only depilation done after hair

was shaved, Figure 192B is an image from a mouse where depilation was followed by

low dose IL-2 (1 mg/kg) administration, and Figures 192C-192E show images from mice

where depilation was followed by 2t2 at 0.3 mg/kg, (Figure 192C), 1 mg/kg (Figure

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192D), and (Figure 192E) 3 mg/kg. Black arrows indicate anagen-phase hair follicles

that will later extend into dermis and facilitate hair growth.

Figure 193 shows the total number of anagen phase hair follicles counted per 10

fields for each treatment group.

Figure 194 is a graph showing the percentage different in DNA demethylation in

NK cells (relative to unexposed NK cells) from two different donors following expansion

with 7t15-21s+ anti-tissue factor (TF)-antibody (IgG1) (50 n MM.

Figure 195 is a set of graphs showing the immune-phenotype from peripheral

blood analysis after 4 days post single dose treatment with TGFRt15-TGFRs.

Figure 196 is a set of graphs showing the immune-phenotype from peripheral

blood analysis after 4 days post single dose treatment with TGFRt15-TGFRs.

Figure 197 is a graph showing B-Gal staining analysis by FACS at seven days

after the second administration with TGFRt15-TGFRs.

Figure 198 is a set of graphs showing the levels of senescence markers in liver

tissue determined using qPCR at 7 days after the second administration with TGFRt15-

TGFRs. Figure 199 is a set of graphs showing the levels of senescence markers in kidney

tissue determined using qPCR at 7 days after the second administration with TGFRt15-

TGFRs.

Figure 200 is a set of graphs showing the levels of senescence markers in skin

tissue determined using qPCR at 7 days after the second administration with TGFRt15-

TGFRs. Figure 201 is a set of graphs showing the levels of senescence markers in lung

tissue determined using qPCR at 7 days after the second administration with TGFRt15-

TGFRs. Figure 202 is a set of histological images showing B-Gal staining on kidney tissue

at 7 days post second treatment with TGFRt15-TGFRs.

Figures 203A-203C show chemotherapy induces p21clP1p21 senescence-

associated gene expression in C57BL/6 mice. Figure 203A is an exemplary schematic showing the experimental treatment regimen. Figures 203B and 203C are graphs showing expression of p21clPlp21 in lung (B) and liver (C) tissues respectively.

Figure 204 is a set of graphs showing immune-phenotype and cell proliferation

following treatment with IL-15-based agents at day 3 post treatment.

Figures 205A-205C are graphs showing TGFRt15-TGFRs treatment reduces

senescence-associated gene expression in C57BL/6 mice. The graphs show expression of

p21clPlp21 and CD26 in lung (A and B) and p21clPlp21 in liver (C) tissues respectively.

Figure 206 is a set of graphs showing CD4+, CD8+, and Treg cell percentages and

proliferation.

Figure 207 is a set of graphs showing NK, CD19+ and monocyte cell percentages

and proliferation.

Figures 208A-208C are graphs showing evaluation of senescence markers

p21clPlp21 and CD26 in lung and liver tissues. Figures 208A and 208B show lung

p21clP1p21 (A) and lung CD26 (B) senescence markers. Figure 208C shows liver

p21clP1p21 senescence marker.

Figure 209 shows a schematic diagram of the interaction between the exemplary

TGFBRII/IL-15RaSu and TGFBRII/TF/IL-15Mut proteins resulting in TGFRt15*-TGFRs

complex.

Figure 210 shows a schematic diagram of the interaction between the exemplary

TGF3RII/IL-15RaSu and TGFBRII/TF/IL-15Mut proteins.

Figures 211A is a graph showing the binding activity of TGFRt15-TGFRs to

TGF-B1 and LAP.

Figure 211B is a graph showing the binding activity of TGFRII/Fc to TGF-31 and

LAP.

Figure 211C is a graph showing the binding activity of TGFRt15-TGFRs to TGF-

B1 and LAP.

Figure 211D is a graph showing the binding activity of TGFRt15*-TGFRs to

TGF-B1 and LAP.

Figure 211E is a graph showing the binding activity of TGFRt15-TGFRs,

TGFRt15*-TGFRs, and 7t15-21s to CTLL-2 cells.

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Figure 212A is a graph of TGF- B 1 blocking activity of TGFRt15-TGFRs and

TGFRt15*-TGFRs. Figure 212B is a graph of the IL-15 biological activity of TGFRt15-TGFRs and

TGFRt15*-TGFRs. Figure 212C is a graph showing that TGF-B1, TGF-B2, and TGF-B3 each

similarly inhibit IL-4-induced CTLL-2 growth in the absence of TGFRt15*-TGFRs.

Figure 212D is a graph showing that TGFRt15*-TGFRs significantly reversed the

inhibition of TGF-B1 and TGF-B3 of IL-4-induced CTLL-2 cell growth.

Figure 213A shows that there is no significant damage to the IL-15 domain of

TGFRt15-TGFRs following 10-day incubation 4°C, 25 °C, or 37 °C.

Figure 213B shows that there is no significant damage to the TGFB-RII domain of

TGFRt15-TGFRs following 10-day incubation 4°C, 25 °C, or 37 °C.

Figure 213C is a graph showing TGF-B1 neutralizing activity of TGFRt15-

TGFRs following incubation in human serum for 10 days at 4°C, 25 °C, or 37 °C.

Figure 213D is a graph showing IL-15 activity of TGFRt15-TGFRs following

incubation in human serum for 10 days at 4 °C, 25 °C, or 37°C.

Figure 214A is a graph showing cell-mediated cell cytotoxicity in an assay using

NK cells and the constructs shown.

Figure 214B is a graph showing cell-mediated cell cytotoxicity in an assay using

PMBCs and the constructs shown.

Figure 214C is a graph showing intracellular granzyme B production in an assay

using NK cells and the constructs shown.

Figure 214D is a graph showing intracellular granzyme B production in an assay

using PBMCs and the constructs shown.

Figure 214E is a graph showing interferon-gamma production in an assay using

NK cells and the constructs shown.

Figure 214F is a graph showing interferon-gamma production in an assay using

PMBCs and the constructs shown.

Figure 215 is a graph showing the pharmacokinetics (half-life, t1/2) of TGFRt15-

TGFRs evaluated in female C57BL/6 mice.

Figure 216 is a graph showing toxicity of TGFRt15-TGFRs in C57BL/6 mice.

Figure 217 is a graph showing antitumor activity of TGFRt15-TGFRs in a

C57BL/6 murine melanoma model.

Figure 218 shows activity of TGFRt15-TGFRs in nine-week old C57BL6/j male

mice, wherein the mice were given 50 ul of bleomycin (2.5 mg/kg, single dose) through

the oropharyngeal route and then were given TGFRt15-TGFRs subcutaneously (3 mg/kg)

on day 17 following bleomycin treatment.

Figure 219 shows fasting plasma glucose levels in db/db mice 4 days post

treatment with TGFRt15-TGFRs or TGFRt15*-TGFRs.

Figures 220A-220C show TGF31-3 levels in db/db mice 4 days post treatment

with TGFRt15-TGFRs or TGFRt15*-TGFRs: TGFß1 (Figure 220A), TGFB2 (Figure

220B), and TGFB3 (Figure 220C).

Figures 221A-E show lymphocyte subsets in db/db mice 4 days post treatment

with TGFRt15-TGFRs or TGFRt15*-TGFRs blood NK cells (Figure 221A), blood

Ki67+ NK cells (Figure 221B), blood granzyme B+ (GzmB ) (Figure 221C), blood CD8+

(Figure 221D), and blood CD8*Ki67+ T cells (Figure 221E).

Figure 222A shows the interaction of TGFRt15*-TGFRs or TGFRt15-TGFRs

with latent TGFB1 (SLC) or with CD39 (control).

Figure 222B shows the interaction of TGFRt15*-TGFRs and TGFRII-Fc with

latent TGFB1.

Figure 223 is a graph showing the clotting time of Innovin in the PT assay.

Figure 224 is a graph showing the clotting time of TGFRt15-TGFRs in the PT

assay.

Figure 225 are graphs showing gene expression of senescence markers PAI-1, IL-

1a, IL6, and IL-1B in kidney and comparing young VS PBS or TGFRt15-TGFRs treated

aged mice with short term VS long term follow-up.

Figure 226 are graphs showing gene expression of senescence markers IL-1a and

IL6 in liver.

Figure 227 shows protein expression of senescence marker PAI-1 in kidney.

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Figure 228 are graphs showing that IL15SA (positive control) or TGFRt15*-

TGFRs + IL15SA mediated an increase in the percentages of CD3*CD8*, CD3*NK1.1 ,

and CD3*CD45+ immune cells in the blood, whereas treatment with TGFRt15*-TGFRs

had little or no effect on the percentage of these cell populations.

Figure 229 are graphs showing that IL15SA (positive control) or TGFRt15*-

TGFRs + IL15SA mediated an increase in the percentages of CD3*CD8*, CD3*NK1.1*,

and CD3*CD45 immune cells in the spleen, whereas treatment with TGFRt15*-TGFRs

had little or no effect on the percentage of these cell populations.

Figure 230A shows gene expression of senescence marker p21, in kidney and

liver tissues, post test article treatment.

Figure 230B shows gene expression of senescence marker PAI1, in kidney and

liver tissues, post study treatment.

Figure 230C shows gene expression of senescence marker IL-1a, in kidney and

liver tissues, post study treatment.

Figure 230D shows gene expression of senescence marker IL-6, in kidney and

liver tissues, post study treatment.

Figure 231A shows CD4+, CD8+, and Treg cell percentages and proliferation

following treatment with the agents shown. Figure 231B shows NK, CD19+, and

monocyte cell percentages and proliferation following treatment with the agents shown.

Figure 232A shows evaluation of gene expression of senescence markers p21 in

lung tissue of mice following chemotherapy and treatment with the agents shown.

Figure 232B shows evaluation of gene expression of senescence marker CD26 in

lung tissue of mice following chemotherapy and treatment with the agents shown.

Figure 232C shows evaluation of gene expression of senescence marker p21 in

liver tissue of mice following chemotherapy and treatment with the agents shown.

Figures 233A-B are graphs showing TGFRt15-TGFRs treatment enhances the

immune cell proliferation, expansion and activation in the peripheral blood of B16F10

tumor bearing mice.

Figure 234 are graphs showing TGFRt15-TGFRs treatment decreases levels of

TGFB in the plasma of B16F10 tumor bearing mice.

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Figure 235 are graphs showing TGFRt15-TGFRs treatment reduces levels of

proinflammatory cytokines in the plasma of B16F10 tumor bearing mice.

Figure 236 shows TGFRt15-TGFRs treatment enhances NK and CD8 expansion

in the spleen of B16F10 tumor bearing mice.

Figures 237A-B show TGFRt15-TGFRs treatment enhances glycolytic activity of

splenocytes in B16F10 tumor bearing mice.

Figures 238A-B show TGFRt15-TGFRs treatment enhances mitochondrial

respiration of splenocytes in B16F10 tumor bearing mice.

Figures 239A-B show TGFRt15-TGFRs treatment enhances NK and CD8

immune cell infiltration (TILs) into tumors of B16F10 tumor bearing mice.

Figure 240 shows histopathological analysis of tumors following TGFRt15-

TGFRs treatment, wherein following TGFRt15-TGFRs+TA99 antibody treatment,

tumors displayed less mitotic and necrotic activity. The mitotic index is correlated to the

dividing cells and presence of necrosis is a measure of more aggressive features and poor

prognosis.

Figure 241 is a graph showing anti-PD-L1 antibody in combination with

TGFRt15-TGFRs+TA99 antibody and chemotherapy in B16F10 melanoma mouse

model.

Figure 242 is a graph showing that anti-tumor efficacy of TGFRt15-TGFRs in

B16F10 melanoma mouse model is dependent on NK and CD8 T cells.

Figures 243A-B are graphs showing gene expression of senescence markers p21,

IL-1a and IL6 in liver and lung tissues of tumor bearing mice following chemotherapy.

Figure 244 is a graph showing induction of gene expression of senescence

markers p21, IL6, H2AX, and NK cell ligands, Raele and ULBP1 by docetaxel treatment

of B16F10 GFP cells.

Figure 245 shows tumor infiltrating lymphocytes/day after 4 days post treatment

in tumor bearing mice.

Figures 246A-B show flow cytometry analysis on tumor cells indicating that mice

which received immunotherapy treatment showed lower number of GFP positive

senescent tumor cells post 4 days and 10 days of treatment as compared to the PBS

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control group (Figure 246A), and tumor cells plated in 24 well plate evaluated by

fluorescence microscopy (Figure 246B).

Figure 247 shows TGFB levels in kidney of mice after inducing kidney injury

with cisplatin and treatment with TGFRt15-TGFRs.

Figures 248A-C show the toxicological effects of repeat dose subcutaneous

administration of TGFRt15-TGFRs in C57BL/6 mice. Changes in body weights are

shown through SD21 (Figure 248A). Spleen weights (Figure 248B) and blood cells

counts and differentials (Figure 248C) are indicated for mice at SD7 after one dose and

SD21 after two doses of TGFRt15-TGFRs.

Figure 249 shows plasma levels of TGF-B isoforms in mice after in vivo treatment

with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFR11***TGFRs (3 mg/kg).

Figures 250A-B show the changes in rates of glycolytic capacity (ECAR) (Figure

250A) and mitochondrial respiratory capacity (OCR) (Figure 250B) in splenocytes of

mice following in vivo treatment with PBS, TGFRt15-TGFRs, TGFR115*-TGFR: or

IL15SA.

Figures 251A-B show the changes in rates of glycolytic capacity (ECAR) (Figure

251A) and mitochondrial respiratory capacity (OCR) (Figure 251B) in mouse splenocytes

following in vitro treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFRs.

Figures 252A-E show the changes in tumor growth and survival of B16F10

melanoma tumors in C57BL/6 mice following in vitro treatment with PBS, TGFRt15-

TGFRs, or TGFRt15*-TGFRs. Tumor volume (Figure 252A) and mouse survival (based

on tumor volume < 4000 mm³) (Figure 252B) were assessed. Mice were intraperitoneally

treated with anti-CD8, anti-NK, or anti-CD8 and anti-NK Abs for 1 week to deplete

immune cells prior to injection with B16F10 melanoma tumor cells as in Figure 252A.

Tumor bearing mice were then treated with PBS or 20 mg/kg TGFRt15-TGFRs on day 1

and 4 post-tumor cell inoculation. Tumor volume of animals (Figure 252C) and mouse

survival (Figure 252D) were assessed. B16F10 tumor bearing mice were treated with

PBS or 20 mg/kg of TGFRt15-TGFRs on day 1 and 7 post-tumor inoculation (Figure

252E). On day 11 post tumor inoculation, tumors were collected and tumor-infiltrating

NK1.1+ cells and CD8+ T cells were quantitated by flow cytometry.

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Figures 253A-B show treatment effects on fasting plasma glucose (Figure 253A)

and insulin (Figure 253B) levels in db/db mice receiving PBS (control) or TGFRt15-

TGFRs. Figure 254A shows the fold change in gene expression levels in pancreas of db/db

mice receiving TGFRt15-TGFRs compared to PBS control.

Figures 254B-D show the average fold change in pancreatic expression levels for

genes of the SASP, Aging and Beta cell indices, respectively, for db/db mice receiving

TGFRt15-TGFRs compared to PBS control.

Figures 255A-B show multispectral imaging of pancreatic tissue sections from

db/db mice treated with PBS (control) (Figure 255A) or TGFRt15-TGFRs (Figure 255B).

A representative pancreatic islet is shown, insulin islet beta cells as OPAL-520,

insulin*p21+ beta cells as OPAL-570 (seen as white cells in gray-scale image) was

reduced in TGRt15-TGFRs treated group (Figure 255B) compared to PBS treated group

(Figure 255A). Figures 255C and 255D show levels of islet insulin+ (Figure 255C) and

islet insulin p21+ (Figure 255D) cells in pancreatic tissue sections from db/db mice

treated with PBS (control) or TGFRt15-TGFRs.

Figures 256A-C show treatment effects on the percentage of blood immune cell

subsets in db/db mice receiving PBS (control) or TGFRt15-TGFRs.

Figure 257 shows the percentage of Ki67 positive immune cells induced in the

blood following subcutaneous treatment of Cynomolgus monkeys with TGFRt15-TGFRs

compared to PBS (vehicle).

Figure 258 shows the extracellular acidification rate (ECAR) representing

glycolytic function of splenocytes isolated from young (6-week-old) and aged (72-week-

old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or

TGFRt15*-TGFRs (3 mg/kg).

Figure 259 shows the oxygen consumption rate (OCR) representing mitochondrial

respiration of splenocytes isolated from young (6-week-old) and aged (72-week-old)

mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-

TGFRs (3 mg/kg).

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Figure 260 shows the percentages of immune cell subsets in the blood of young

(6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS,

TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg).

Figure 261 shows the percentages of immune cell subsets in the spleen of young

(6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS,

TGFRt15-TGFRs or TGFRt15*-TGFRs. Figure 262 shows gene expression levels for IL1-a, IL1-B, IL-6, p21 and PAI-1 in

liver of aged mice after one or two doses of TGFRt15-TGFRs treatment.

Figure 263 shows the inflammation score of liver tissues of aged mice after one or

two doses of TGFRt15-TGFRs treatment.

Figure 264 shows expression levels of IL1-a, IL1-B, IL-6, IL-8, TGF-B, PAI-1,

collagen and fibronectin protein in liver of aged mice after with one or two doses

treatment of TGFRt15-TGFRs.

Figure 265 shows the levels of B-galactosidase in liver tissues of aged mice 4 days

after in vivo treatment with PBS or TGFRt15-TGFRs.

Figure 266 shows the survival curves of 72-week-old C57BL/6 mice following

subcutaneous treatment with PBS or one dose of TGFRt15-TGFRs (3 mg/kg).

Figure 267 shows protein levels of SASP factors in livers of B16F10 tumor-

bearing mice following chemotherapy and TGFRt15-TGFRs + TA99 therapy.

Figures 268A-B show effects of CD8+ T cells (dpCD8) and NK cell (dpNK)

antibody depletion on the levels of TIS B16F10-GFP cells (Figure 268A) and NK and

CD8+ T cells (Figure 268B) in the tumors of mice following chemotherapy and

TGFRt15-TGFRs + TA99 therapy.

Figures 269A-E show the anti-tumor activity and mechanism of action of

TGFRt15-TGFRs + TA99 in combination with immune checkpoint inhibitor in B16F10

tumor-bearing mice. Figure 269A shows an exemplary schematic for treating B16F10

melanoma in a mouse model. Figure 269B shows the change in tumor volume over time

and at day 18 following combination treatments including TGFRt15-TGFRs+TA99+anti-

PD-L1 antibody following doxetaxel as compared to PBS or chemotherapy treatment

alone. Figures 269C and 269D show treatment effects on the percentages of tumor

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infiltrating CD28+CD8 T cells and splenic IFNy+CD8 T cells on day 18. Figure 269E

shows treatment effects on the levels (MFI) of NKG2D of tumor infiltrating CD8+ and

CD8*CD44hi T cells on day 18.

Figures 270A-D show the changes in tumor growth and survival of SW 1990

human pancreatic tumors in C57BL/6 scid mice following in vitro treatment with PBS,

gemcitabine and nab-paclitaxel chemotherapy, TGFRt15-TGFRs, or TGFRt15-

TGFRs+chemotherapy. Figure 270A shows an exemplary schematic for treating SW1990

human pancreatic tumors in a xenograft mouse model. Figure 270B and 270C show the

change in tumor volume over time and at day 38, respectively, following combination

treatments including TGFRt15-TGFRs + chemotherapy as compared to PBS or

chemotherapy treatment alone. Figure 270D shows treatment effects on survival of mice

bearing SW1990 human pancreatic tumors.

DETAILED DESCRIPTION Provided herein are methods of killing or reducing the number of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor. Also provided herein are methods of

decreasing the accumulation of naturally-occurring and/or treatment-induced senescent

cells in a subject that include administering to the subject a therapeutically effective

amount of one or more agent(s) that result(s) in a decrease in the activation of a TGF-B

receptor. Also provided herein are methods of decreasing a level of a marker of

naturally-occurring and/or treatment-induced senescent cells in a subject that include

administering to the subject a therapeutically effective amount of one or more agent(s)

that result(s) in a decrease in the activation of a TGF-B receptor. Also provided herein

are methods of reducing the activity of naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more agent(s) that result(s) in a decrease in the activation of a

TGF-B receptor. Also provided herein are methods of decreasing levels or activity of

SASP factors derived from naturally-occurring and/or treatment-induced senescent cells

WO wo 2021/247604 PCT/US2021/035285

in a subject that include administering to the subject a therapeutically effective amount of

one or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor.

Further provided herein are methods of killing or reducing the number of

naturally-occurring and/or treatment-induced senescent cells in a subject that include

administering to the subject a therapeutically effective amount of one or more common

gamma-chain family cytokine receptor activating agent(s). Also provided herein are

methods of decreasing the accumulation of naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more common gamma-chain family cytokine receptor

activating agent(s). Also provided herein are methods of decreasing a level of a marker

of naturally-occurring and/or treatment-induced senescent cells in a subject that include

administering to the subject a therapeutically effective amount of one or more common

gamma-chain family cytokine receptor activating agent(s). Also provided herein are

methods of reducing the activity of naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more common gamma-chain family cytokine receptor

activating agent(s). Also provided herein are methods of decreasing levels and/or activity

of one or more SASP factor(s) derived from naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more common gamma-chain family cytokine receptor

activating agent(s).

Provided herein are methods of treating an aging-related disease or condition in a

subject in need thereof that include administering to a subject identified as having an

aging-related disease or condition a therapeutically effective amount of one or more

natural killer (NK) cell activating agent(s) and/or a therapeutically effective number of

activated NK cells. Also provided herein are methods of killing or reducing the number

of senescent cells in a subject in need thereof that include administering to the subject a

therapeutically effective amount of one or more NK cell activating agent(s) and/or a

therapeutically effective number of activated NK cells. Also provided herein are

methods of improving the texture and/or appearance of skin and/or hair in a subject in

WO wo 2021/247604 PCT/US2021/035285

need thereof over a period of time that include administering to the subject a

therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s) and/or a therapeutically effective number of activated NK cells. Also provided

herein are methods of assisting in the treatment of obesity in a subject in need thereof

over a period of time that include administering to the subject a therapeutically effective

amount of one or more natural killer (NK) cell activating agent(s) and/or a therapeutically

effective number of activated NK cells.

Activated NK Cells

Some embodiments of any of the methods described herein can include

administering to a subject (e.g., any of the exemplary subjects described herein) a

therapeutically effective number of activated NK cells (e.g., human activated NK cells).

An activated NK cell is an NK cell (e.g., a human NK cell) that has increased expression

levels of two or more (e.g., three, four, five, or six) of CD25, CD69, MTOR-C1,

SREBP1, IFN-y, and a granzyme (e.g., granzyme B), e.g., as compared to a resting NK

cell (e.g., a human resting NK cell). For example, an activated NK cell can have at least

a 10% increase (e.g., at least a 15% increase, at least a 20% increase, at least a 25%

increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least

a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase,

at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80%

increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least

a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160%

increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at

least a 240% increase, at least a 260% increase, at least a 280% increase, or at least a

300% increase) in the expression levels of two of more (e.g., three, four, five, or six) of

CD25, CD69, MTOR-C1, SREBP1, IFN-y, and a granzyme (e.g., granzyme B), e.g., as

compared to a resting NK cell (e.g., a human activated NK cell).

In some embodiments, an activated NK cell can optionally further have at least a

10% increase (e.g., at least a 15% increase, at least a 20% increase, at least a 25%

increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least

WO wo 2021/247604 PCT/US2021/035285

a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase,

at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80%

increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least

a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160%

increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at

least a 240% increase, at least a 260% increase, at least a 280% increase, or at least a

300% increase) in the expression levels of two of more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of CD25, CD59,

CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD16, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C, CCR7, CXCR3, L- Selectin, CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c-Kit,

mTORC1, e.g., as compared to a resting NK cell (e.g., a human activated NK cell).

For example, an activated NK cell (e.g., a human activated NK cell) can have

about a 10% increase to about a 500% increase, about a 10% increase to about a 450%

increase, about a 10% increase to about a 400% increase, about a 10% increase to about a

350% increase, about a 10% increase to about a 300% increase, about a 10% increase to

about a 280% increase, about a 10% increase to about a 260% increase, about a 10%

increase to about a 240% increase, about a 10% increase to about a 220% increase, about

a 10% increase to about a 200% increase, about a 10% increase to about a 180% increase,

about a 10% increase to about a 160% increase, about a 10% increase to about a 140%

increase, about a 10% increase to about a 120% increase, about a 10% increase to about a

100% increase, about a 10% increase to about a 80% increase, about a 10% increase to

about a 60% increase, about a 10% increase to about a 40% increase, about a 10%

increase to about a 20% increase, a 20% increase to about a 500% increase, about a 20%

increase to about a 450% increase, about a 20% increase to about a 400% increase, about

a 20% increase to about a 350% increase, about a 20% increase to about a 300% increase,

about a 20% increase to about a 280% increase, about a 20% increase to about a 260%

increase, about a 20% increase to about a 240% increase, about a 20% increase to about a

220% increase, about a 20% increase to about a 200% increase, about a 20% increase to

about a 180% increase, about a 20% increase to about a 160% increase, about a 20% wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285 increase to about a 140% increase, about a 20% increase to about a 120% increase, about a 20% increase to about a 100% increase, about a 20% increase to about a 80% increase, about a 20% increase to about a 60% increase, about a 20% increase to about a 40% increase, a 40% increase to about a 500% increase, about a 40% increase to about a 450% increase, about a 40% increase to about a 400% increase, about a 40% increase to about a

350% increase, about a 40% increase to about a 300% increase, about a 40% increase to

about a 280% increase, about a 40% increase to about a 260% increase, about a 40%

increase to about a 240% increase, about a 40% increase to about a 220% increase, about

a 40% increase to about a 200% increase, about a 40% increase to about a 180% increase,

about a 40% increase to about a 160% increase, about a 40% increase to about a 140%

increase, about a 40% increase to about a 120% increase, about a 40% increase to about a

100% increase, about a 40% increase to about a 80% increase, about a 40% increase to

about a 60% increase, a 60% increase to about a 500% increase, about a 60% increase to

about a 450% increase, about a 60% increase to about a 400% increase, about a 60%

increase to about a 350% increase, about a 60% increase to about a 300% increase, about

a 60% increase to about a 280% increase, about a 60% increase to about a 260% increase,

about a 60% increase to about a 240% increase, about a 60% increase to about a 220%

increase, about a 60% increase to about a 200% increase, about a 60% increase to about a

180% increase, about a 60% increase to about a 160% increase, about a 60% increase to

about a 140% increase, about a 60% increase to about a 120% increase, about a 60%

increase to about a 100% increase, about a 60% increase to about a 80% increase, a 80%

increase to about a 500% increase, about a 80% increase to about a 450% increase, about

a 80% increase to about a 400% increase, about a 80% increase to about a 350% increase,

about a 80% increase to about a 300% increase, about a 80% increase to about a 280%

increase, about a 80% increase to about a 260% increase, about a 80% increase to about a

240% increase, about a 80% increase to about a 220% increase, about a 80% increase to

about a 200% increase, about a 80% increase to about a 180% increase, about a 80%

increase to about a 160% increase, about a 80% increase to about a 140% increase, about

a 80% increase to about a 120% increase, about a 80% increase to about a 100% increase,

a 100% increase to about a 500% increase, about a 100% increase to about a 450% wo 2021/247604 WO PCT/US2021/035285 increase, about a 100% increase to about a 400% increase, about a 100% increase to about a 350% increase, about a 100% increase to about a 300% increase, about a 100% increase to about a 280% increase, about a 100% increase to about a 260% increase, about a 100% increase to about a 240% increase, about a 100% increase to about a 220% increase, about a 100% increase to about a 200% increase, about a 100% increase to about a 180% increase, about a 100% increase to about a 160% increase, about a 100% increase to about a 140% increase, about a 100% increase to about a 120% increase, a

120% increase to about a 500% increase, about a 120% increase to about a 450%

increase, about a 120% increase to about a 400% increase, about a 120% increase to

about a 350% increase, about a 120% increase to about a 300% increase, about a 120%

increase to about a 280% increase, about a 120% increase to about a 260% increase,

about a 120% increase to about a 240% increase, about a 120% increase to about a 220%

increase, about a 120% increase to about a 200% increase, about a 120% increase to

about a 180% increase, about a 120% increase to about a 160% increase, about a 120%

increase to about a 140% increase, a 140% increase to about a 500% increase, about a

140% increase to about a 450% increase, about a 140% increase to about a 400%

increase, about a 140% increase to about a 350% increase, about a 140% increase to

about a 300% increase, about a 140% increase to about a 280% increase, about a 140%

increase to about a 260% increase, about a 140% increase to about a 240% increase,

about a 140% increase to about a 220% increase, about a 140% increase to about a 200%

increase, about a 140% increase to about a 180% increase, about a 140% increase to

about a 160% increase, a 160% increase to about a 500% increase, about a 160% increase

to about a 450% increase, about a 160% increase to about a 400% increase, about a 160%

increase to about a 350% increase, about a 160% increase to about a 300% increase,

about a 160% increase to about a 280% increase, about a 160% increase to about a 260%

increase, about a 160% increase to about a 240% increase, about a 160% increase to

about a 220% increase, about a 160% increase to about a 200% increase, about a 160%

increase to about a 180% increase, a 180% increase to about a 500% increase, about a

180% increase to about a 450% increase, about a 180% increase to about a 400%

increase, about a 180% increase to about a 350% increase, about a 180% increase to

85 wo 2021/247604 WO PCT/US2021/035285 about a 300% increase, about a 180% increase to about a 280% increase, about a 180% increase to about a 260% increase, about a 180% increase to about a 240% increase, about a 180% increase to about a 220% increase, about a 180% increase to about a 200% increase, a 200% increase to about a 500% increase, about a 200% increase to about a

450% increase, about a 200% increase to about a 400% increase, about a 200% increase

to about a 350% increase, about a 200% increase to about a 300% increase, about a 200%

increase to about a 280% increase, about a 200% increase to about a 260% increase,

about a 200% increase to about a 240% increase, about a 200% increase to about a 220%

increase, a 220% increase to about a 500% increase, about a 220% increase to about a

450% increase, about a 220% increase to about a 400% increase, about a 220% increase

to about a 350% increase, about a 220% increase to about a 300% increase, about a 220%

increase to about a 280% increase, about a 220% increase to about a 260% increase,

about a 220% increase to about a 240% increase, a 240% increase to about a 500%

increase, about a 240% increase to about a 450% increase, about a 240% increase to

about a 400% increase, about a 240% increase to about a 350% increase, about a 240%

increase to about a 300% increase, about a 240% increase to about a 280% increase,

about a 240% increase to about a 260% increase, a 260% increase to about a 500%

increase, about a 260% increase to about a 450% increase, about a 260% increase to

about a 400% increase, about a 260% increase to about a 350% increase, about a 260%

increase to about a 300% increase, about a 260% increase to about a 280% increase, a

280% increase to about a 500% increase, about a 280% increase to about a 450%

increase, about a 280% increase to about a 400% increase, about a 280% increase to

about a 350% increase, about a 280% increase to about a 300% increase, a 300% increase

to about a 500% increase, about a 300% increase to about a 450% increase, about a 300%

increase to about a 400% increase, about a 300% increase to about a 350% increase, a

350% increase to about a 500% increase, about a 350% increase to about a 450%

increase, about a 350% increase to about a 400% increase, a 400% increase to about a

500% increase, about a 400% increase to about a 450% increase, or a 400% increase to

about a 500% increase, in the expression levels of two of more (e.g., three, four, five, or

WO wo 2021/247604 PCT/US2021/035285

six) of CD25, CD69, mTORC1, SREBP1, IFN-y, and a granzyme (e.g., granzyme B),

e.g., as compared to a resting NK cell (e.g., a human resting NK cell).

In some embodiments, an activated NK cell can further have about a 10%

increase to about a 500% increase (e.g., or any of the subranges of this range described

herein) in the expression levels of two of more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of CD25, CD59, CD352,

NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD16, KIR2DS1,

KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C, CCR7, CXCR3, L- Selectin, CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c-Kit,

mTORC1, e.g., as compared to a resting NK cell (e.g., a human activated NK cell).

Non-limiting examples of assays that can be used to determine the expression

level of CD25, CD69, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46,

NKG2D, CD16, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1,

NKG2C, CCR7, CXCR3, L-Selectin, CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1, MYC, SREBP1, IFN-y, and a granzyme (e.g., granzyme

B) include, e.g., immunoblotting, fluorescence-assisted cell sorting, enzyme-linked

immunosorbent assays, and RT-PCR.

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of CD25 are available from Diaclone, Covalab

Biotechnology, and Caltag Medsystems. The protein and cDNA sequences for mature

human CD25 are shown below.

Mature Human CD25 Protein (SEQ ID NO: 1)

elcdddppe iphatfkama ykegtmlnce ckrgfrriks gslymlctgn sshsswdnqc qctssatrnt tkqvtpqpee qkerkttemq spmqpvdqas lpghcreppp weneateriy hfvvgqmvyy qcvqgyralh rgpaesvckm thgktrwtqp qlictgemet sqfpgeekpq aspegrpese tsclvtttdf qiqtemaatm etsiftteyq vavagcvfll isvlllsglt wqrrqrksrr ti

Human CD25 cDNA (SEQ ID NO: 2)

gagctctg tgacgatgac ccgccagaga tcccacacgc cacattcaaa gccatggcct acaaggaagg aaccatgttg aactgtgaat gcaagagagg tttccgcaga ataaaaagcg ggtcactcta tatgctctgt acaggaaact ctagccactc gtcctgggac aaccaatgtc

WO wo 2021/247604 PCT/US2021/035285

aatgcacaag ctctgccact cggaacacaa cgaaacaagt gacacctcaa cctgaagaac agaaagaaag gaaaaccaca gaaatgcaaa gtccaatgca gccagtggad caagcgagco ttccaggtca ctgcagggaa cctccaccat gggaaaatga agccacagag agaatttatc atttcgtggt ggggcagatg gtttattato agtgcgtcca gggatacagg gctctacaca gaggtcctgc tgagagcgtc tgcaaaatga cccacgggaa gacaaggtgg acccagcccc agctcatatg cacaggtgaa atggagacca gtcagtttca aggtgaagag aagcctcagg caagccccga aggccgtcct gagagtgaga cttcctgcct cgtcacaaca acagatttto aaatacagac agaaatggct gcaaccatgg agacgtccat atttacaaca gagtaccagg tagcagtggc cggctgtgtt ttcctgctga tcagcgtcct cctcctgagt gggctcacct ggcagcggag acagaggaag agtagaagaa caatc

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of CD69 are available from RayBiotech, Novus

Biologicals, and Aviscera Bioscience. The protein and cDNA sequences for mature

human CD69 are shown below.

Mature Human CD69 Protein (SEQ ID NO: 3)

mssencfvae nsslhpesgq endatsphfs trhegsfqvp vlcavmnvvf itilialia itiliialia lsvgqyncpg qytfsmpsds hvsscsedwv gyqrkcyfis tvkrswtsaq nacsehgatl avidsekdmn flkryagree hwvglkkepg hpwkwsngke fnnwfnvtgs dkcvflknte vssmeceknl ywicnkpyk

Human CD69 cDNA (SEQ ID NO: 4)

atgagctctg aaaattgttt cgtagcagag aacagctctt tgcatccgga gagtggacaa gaaaatgatg ccaccagtcc ccatttctca acacgtcatg aagggtcctt ccaagttcct gtcctgtgtg ctgtaatgaa tgtggtctto atcaccattt taatcatago tctcattgcc ttatcagtgg gccaatacaa ttgtccagga caatacacat tctcaatgcc atcagacago catgtttctt catgctctga ggactgggtt ggctaccaga ggaaatgcta ctttatttct actgtgaaga ggagctggad ttcagcccaa aatgcttgtt ctgaacatgg tgctactctt gctgtcattg attctgaaaa ggacatgaac tttctaaaac gatacgcagg tagagaggaa cactgggttg gactgaaaaa ggaacctggt cacccatgga agtggtcaaa tggcaaagaa tttaacaact ggttcaacgt tacagggtct gacaagtgtg tttttctgaa aaacacagag gtcagcagca tggaatgtga gaagaattta tactggatat gtaacaaacc ttacaaataa

The protein and cDNA sequences for mature human CD59 are shown below.

Mature Human CD59 Protein (SEQ ID NO: 5)

lqcyncpnptadckt avncssdfda clitkaglqv ynkcwkfehc nfndvttrlr eneltyycck kdlcnfneql en

PCT/US2021/035285

Human CD59 cDNA (SEQ ID NO: 6)

atgggaatco aaggagggto tgtcctgtta gggctgctgc tcgtcctggc tgtcttctgc cattcaggtc atagcctgca gtgctacaac tgtcctaacc caactgctga ctgcaaaaca gccgtcaatt gttcatctga ttttgatgcg tgtctcatta ccaaagctgg gttacaagtg tataacaagt gttggaagtt tgagcattgc aatttcaacg acgtcacaac ccgcttgagg gaaaatgage taacgtacta ctgctgcaag aaggacctgt gtaactttaa cgaacagctt gaaaatggtg ggacatcctt atcagagaaa acagttctta tgctggtgac tccatttctg gcagcagcct ggagccttca tccctaa

The protein and cDNA sequences for mature human CD352 are shown below.

Mature Human CD352 Protein (SEQ ID NO: 7)

qssltplmv ngilgesvtl plefpagekv nfitwlfnet slafivphet kspeihvtnp kqgkrlnftq syslqlsnlk medtgsyraq istktsakls sytlrilrql rniqvtnhsq lfqnmtcelh ltcsvedadd nvsfrwealg ntlssqpnlt vswdprisse qdytciaena vsnlsfsvsa qklcedvkiq ytdtkmilfm vsgicivfgf iillllvlrk rrdslslstq rtqgpaesar nleyvsvspt nntvyasvth snreteiwtp rendtitiys tinhskeskp tfsrataldn VV

Human CD352 cDNA (SEQ ID NO: 8) atgttgtggc tgttccaato gctcctgttt gtcttctgct ttggcccagg gaatgtagtt tcacaaagca gcttaacccc attgatggtg aacgggattc tgggggagto agtaactctt cccctggagt ttcctgcagg agagaaggtc aacttcatca cttggctttt caatgaaaca tctcttgcct tcatagtaca ccatgaaaco aaaagtccag aaatccacgt gactaatccg aaacagggaa agcgactgaa cttcacccag tcctactccc tgcaactcag caacctgaag atggaagaca caggctctta cagagcccag atatccacaa agacctctgo aaagctgtca agttacactc tgaggatatt aagacaactg aggaacatac aagttaccaa tcacagtcag ctatttcaga atatgacctg tgagctccat ctgacttgct ctgtggagga tgcagatgac aatgtctcat tcagatggga ggccttggga aacacacttt caagtcagca aaacctcact gtctcctggg accccaggat ttccagtgaa caggactaca cctgcataga agagaatgct gtcagtaatt tatccttctc tgtctctgca cagaagcttt gcgaagatgt taaaattcaa tatacagata ccaaaatgat tctgtttatg gtttctggga tatgcatagt cttcggtttd atcatactga tgttacttgt tttgaggaaa agaagagatt ccctatcttt gtctactcag cgaacacagg gccccgagtc cgcaaggaac ctagagtatg tttcagtgtd tccaacgaac aacactgtgt atgcttcagt cactcattca aacagggaaa cagaaatctg gacacctaga

gaaaatgata ctatcacaat ttactccaca attaatcatt ccaaagagag taaacccact ttttccaggg caactgccct tgacaatgtc gtgtaa

The protein and cDNA sequences for mature human NKp80 are shown below.

Mature Human NKp80 Protein (SEQ ID NO: 9)

mqdeerymtl nvqskkrssa qtsqltfkdy svtlhwykil lgisgtvngi ltltlislil lvsqgvllkc qkgscsnatq yedtgdlkvn ngtrrnisnk dlcasrsadq tvlcqsewlk wo 2021/247604 WO PCT/US2021/035285 yqgkcywfsn emkswsdsyv yclerkshll iihdglemaf iihdqlemaf iqknlrqlny vwiglnftsl kmtwtwvdgs pidskiffik gpakenscaa ikeskifset cssvfkwicq y

Human NKp80 cDNA (SEQ ID NO: 10) atgcaagatg aagaaagata catgacattg aatgtacagt caaagaaaag gagttctgcc caaacatctc aacttacatt taaagattat tcagtgacgt tgcactggta taaaatctta ctgggaatat ctggaaccgt gaatggtatt ctcactttga ctttgatctc cttgatcctg ttggtactat gccaatcaga atggctcaaa taccaaggga agtgttattg gttctctaat gagatgaaaa gctggagtga cagttatgtg tattgtttgg aaagaaaatc tcatctacta atcatacatg accaacttga aatggctttt atacagaaaa acctaagaca attaaactac gtatggattg ggcttaactt tacctccttg aaaatgacat ggacttgggt ggatggttct ccaatagatt caaagatatt cttcataaag ggaccagcta aagaaaacag ctgtgctgca attaaggaaa gcaaaatttt ctctgaaacc tgcagcagtg ttttcaaatg gatttgtcag tattag

The protein and cDNA sequences for mature human DNAM-1 are shown below.

Mature Human DNAM-1 Protein (SEQ ID NO: 11)

ee vlwhtsvpfa enmslecvyp smgiltqvew fkigtqqdsi aifspthgmv irkpyaervy flnstmasnn mtlffrnase ddvgyyscsl ytypqgtwqk viqvvqsdsf eaavpsnshi vsepgknvtl tcqpqmtwpv qavrwekiqp rqidlltycn lvhgrnftsk fprqivsncs hgrwsvivip dvtvsdsgly rcylqasage netfvmrltv aegktdnqyt lfvaggtvll llfvisitti iviflnrrrr rerrdlftes wdtqkapnny rspistsqpt nqsmddtred iyvnyptfsr rpktrv

Human DNAM-1 cDNA (SEQ ID NO:12) atggattato ctactttact tttggctctt cttcatgtat acagagctct atgtgaagag gtgctttggc atacatcagt tccctttgcc gagaacatgt ctctagaatg tgtgtatcca tcaatgggca tcttaacaca ggtggagtgg ttcaagatcg ggacccagca ggattccata gccattttca gccctactca tggcatggtc ataaggaago cctatgctga gagggtttac tttttgaatt caacgatgga ttccaataac atgactcttt tctttcggaa tgcctctgaa gatgatgttg gctactatto ctgctctctt tacacttacc cacagggaac ttggcagaag gtgatacagg tggttcagtc agatagtttt gaggcagctg tgccatcaaa tagccacatt gtttcggaac ctggaaagaa tgtcacactc acttgtcagc ctcagatgac gtggcctgtg caggcagtga ggtgggaaaa gatccagccc cgtcagatcg acctcttaac ttactgcaac ttggtccatg gcagaaattt cacctccaag ttcccaagac aaatagtgag caactgcago cacggaaggt ggagcgtcat cgtcatcccc gatgtcacag tctcagacto ggggctttac cgctgctact tgcaggccag cgcaggagaa aacgaaacct tcgtgatgag attgactgta gccgagggta aaaccgataa ccaatatacc ctctttgtgg ctggagggad agttttattg ttgttgtttg ttatctcaat taccaccatc attgtcattt tccttaacag aaggagaagg agagagagaa gagatctatt tacagagtcc tgggatacac agaaggcacc caataactat agaagtccca tctctaccag tcaacctacc aatcaatcca tggatgatac aagagaggat atttatgtca actatccaac cttctctcgc agaccaaaga ctagagttta a

WO wo 2021/247604 PCT/US2021/035285

The protein and cDNA sequences for mature human 2B4 are shown below.

Mature Human 2B4 Protein (SEQ ID NO: 13)

gk gcqgsadhvv sisgvplqlq pnsiqtkvds iawkkllpsq ngfhhilkwe ngslpsntsn drfsfivknl sllikaaqqq dsglyclevt sisgkvqtat fqvfvfdkve kprlqgqgki ldrgrcqval sclvsrdgnv syawyrgskl iqtagnltyl deevdingth tytcnvsnpv sweshtlnlt qdcqnahqef rfwpflviiv ilsalflgtl acfcvwrrkr kekqsetspk efltiyedvk dlktrrnheq eqtfpgggst iysmiqsqss aptsgepayt lysliqpsrk sgsrkrnhsp sfnstiyevi gksqpkaqnp arlsrkelen fdvys

Human 2B4 cDNA (SEQ ID NO: 14)

atgctggggc aagtggtcac cctcatactc ctcctgctcc tcaaggtgta tcagggcaaa ggatgccagg gatcagctga ccatgtggtt agcatctcgg gagtgcctct tcagttacaa ccaaacagca tacagacgaa ggttgacage attgcatgga agaagttgct gccctcacaa aatggatttc atcacatatt gaagtgggag aatggctctt tgccttccaa tacttccaat gatagattca gttttatagt caagaacttg agtcttctca tcaaggcago tcagcagcag gacagtggca tctactgcct ggaggtcacc agtatatctg gaaaagttca gacagccacg ttccaggttt ttgtatttga taaagttgag aaaccccgcc tacaggggca ggggaagatc ctggacagag ggagatgcca agtggctctg tcttgcttgg tctccaggga tggcaatgtg tcctatgctt ggtacagagg gagcaagctg atccagacag cagggaacct cacctacctg gacgaggagg ttgacattaa tggcactcao acatatacct gcaatgtcag caatcctgtt agctgggaaa gccacaccct gaatctcact caggactgto agaatgccca tcaggaatto agattttgga cgtttttggt gatcatcgtg attctaagcg cactgttcct tggcaccctt gcctgcttct gtgtgtggag gagaaagagg aaggagaago agtcagagac cagtcccaag gaatttttga caatttacga agatgtcaag gatctgaaaa ccaggagaaa tcacgagcag gagcagactt ttcctggagg ggggagcaco atctactcta tgatccagto ccagtcttct gctcccacgt cacaagaacc tgcatataca ttatattcat taattcagca ttccaggaag tctggatcca ggaagaggaa ccacagccct tccttcaata gcactatcta tgaagtgatt ggaaagagtc aacctaaage ccagaaccct gctcgattga gccgcaaaga gctggagaac tttgatgttt attcctag

The protein and cDNA sequences for mature human NKp30 are shown below.

Mature Human NKp30 Protein (SEQ ID NO: 15) lw vsqppeirtl egssaflpcs fnasqgrlai gsvtwfrdev vpgkevrngt

pefrgrlapl assrflhdhq aelhirdvrg hdasiyvcrv evlglgvgtg ngtrlvveke hpqlgagtvl llragfyavs flsvavgstv yyqgkcltwk gprrqlpavv paplpppcgs sahllppvpg g

Human NKp30 cDNA (SEQ ID NO: 16) atggcctgga tgctgttgct catcttgato atggtccatc caggatcctg tgctctctgg gtgtcccagc cccctgagat tcgtaccctg gaaggatcct ctgccttcct gccctgctco ttcaatgcca gccaagggag actggccatt ggctccgtca cgtggttccg agatgaggtg gttccaggga aggaggtgag gaatggaacc ccagagttca ggggccgcct ggccccactt gcttcttccc gtttcctcca tgaccaccag gctgagctgc acatccggga cgtgcgaggo catgacgcca gcatctacgt gtgcagagtg gaggtgctgg gccttggtgt cgggacaggg aatgggacto ggctggtggt ggagaaagaa catcctcago taggggctgg tacagtcctc ctccttcggg ctggattcta tgctgtcagc tttctctctg tggccgtggg cagcaccgtc tattaccagg qcaaatqcca ctgtcacatg ggaacacact gccactcctc agatgggccc cgaggagtga ttccagagcc cagatgtccc tag

The protein and cDNA sequences for mature human NKp44 are shown below.

Mature Human NKp44 Protein (SEQ ID NO: 17)

qskaqvlqs vagqtltvrc qypptgslye kkgwckeasa lvcirlvtss kprtmawtsr ftiwddpdag fftvtmtdlr eedsghywcr iyrpsdnsvs ksvrfylvvs pasastqtsw tprdlvssqt qtqscvppta gargapesps tipvpsqpqn stlrpgpaap ialvpvfcgl lvakslvlsa llvwwgdiww ktmmelrsld tqkatchlqq vtdlpwtsvs spvereilyh tvartkisdd ddehtl

Human NKp44 cDNA (SEQ ID NO: 18)

atggcctgga gagccctaca cccactgcta ctgctgctgc tgctgttccc aggctctcag gcacaatcca aggctcaggt acttcaaagt gtggcagggc agacgctaac cgtgagatgo cagtacccgc ccacgggcag tctctacgag aagaaaggct ggtgtaagga ggcttcagca cttgtgtgca tcaggttagt caccagctcc aagcccagga cgatggcttg gacctctcga ttcacaatct gggacgacco tgatgctggc ttcttcactg tcaccatgac tgatctgaga gaggaagact caggacatta ctggtgtaga atctaccgcc cttctgacaa ctctgtctct aagtccgtca gattctatct ggtggtatct ccagcctctg cctccacaca gacctcctgg actccccgcg acctggtctc ttcacagacc cagacccaga gctgtgtgcc tcccactgca ggagccagac aagcccctga gtctccatct accatccctg tcccttcaca gccacagaac tccacgctcc gccctggccc tgcagccccc attgccctgg tgcctgtgtt ctgtggactc ctcgtagcca agagcctggt gctgtcagcc ctgctcgtct ggtgggtttt aaggaatcgg cacatgcago atcaagggag gtctctgctg cacccagctc agcccaggcc ccaggcccat agacacttcc cactgagcca cagggcacca ggggggacat atggtggaaa accatga

The protein and cDNA sequences for mature human NKp46 are shown below.

Mature Human NKp46 Protein (SEQ ID NO: 19) qqqt1pkpf iwaephfmvp kekqvticcq gnygaveyql hfegslfavd rpkpperink vqfyipdmns rmaggysciy rvgelwseps nlldlvvtem ydtptlsvhp gpevisgekv tfycrldtat smflllkegr sshvqrgygk vqaefplgpv ttahrgtyro fgsynnhaws fpsepvkllv tgdientsla pedptfpadt wgtyllttet glqkdhalwd htaqnllrmg laflvlvalv wflvedwlsr krtrerasra stwegrrrln tqtl

Human NKp46 cDNA (SEQ ID NO: 20)

atggcctggc gagccctaca cccactgcta ctgctgctgc tgctgttccc aggctctcag gcacaatcca aggctcaggt acttcaaagt gtggcagggo agacgctaac cgtgagatgo wo 2021/247604 WO PCT/US2021/035285 cagtacccgc ccacgggcag tctctacgag aagaaaggct ggtgtaagga ggcttcagca cttgtgtgca tcaggttagt caccagctcc aagcccagga cgatggcttg gacctctcga ttcacaatct gggacgaccc tgatgctggc ttcttcactg tcaccatgac tgatctgaga gaggaagact caggacatta ctggtgtaga atctaccgcc cttctgacaa ctctgtctct aagtccgtca gattctatct ggtggtatct ccagcctctg cctccacaca gacctcctgg actccccgcg acctggtctc ttcacagacc cagacccaga gctgtgtgcc tcccactgca ggagccagac aagcccctga gtctccatct accatccctg tcccttcaca gccacagaac tccacgctcc gccctggccc tgcagccccc attgccctgg tgcctgtgtt ctgtggacto ctcgtagcca agagcctggt gctgtcagca ctgctcgtct ggtgggtttt aaggaatcgg cacatgcage atcaagggag gtctctgctg cacccagcto agcccaggcc ccaggcccat agacacttcc cactgagcca cagggcacca ggggggacat atggtggaaa accatga

The protein and cDNA sequences for mature human NKG2D are shown below.

Mature Human NKG2D Protein (SEQ ID NO: 21)

mgwirgrrsr hswemsefhn ynldlkksdf strwqkqrcp wkskcrena spfffccfia vamgirfiim vaiwsavfln slfngevqip ltesycgpcp knwicyknnc yqffdesknw yesqascmsq nasllkvysk edqdllklvk syhwmglvhi ptngswqwed gsilspnllt iiemqkgdca lyassfkgyi encstpntyi cmqrtv

Human NKG2D cDNA (SEQ ID NO: 22)

atggggtgga ttcgtggtcg gaggtctcga cacagctggg agatgagtga atttcataat tataacttgg atctgaagaa gagtgatttt tcaacacgat ggcaaaagca aagatgtcca gtagtcaaaa gcaaatgtag agaaaatgca tctccatttt ttttctgctg cttcatcgct gtagccatgg gaatccgttt cattattatg gtaacaatat ggagtgctgt attcctaaac tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata gaaaactgtt caactccaaa tacgtacatc tgcatgcaaa ggactgtgta a

The protein and cDNA sequences for mature human CD16a are shown below.

Mature Human CD16a Protein (SEQ ID NO: 23)

maegtlwqil cvssdaqpqt fegvkgadpp tlppgsflpg pvlwwgslar lqteksdevs rkgnwwvtem gggagerlft ssclvglvpl glrislvtcp lqcgimwqll lptallllvs agmrtedlpk avvflepqwy rvlekdsvtl kcqgaysped instqwfhnes lissqassyf idaatvddsg eyrcqtnlst lsdpvqlevh igwlllqapr wvfkeedpih lrchswknta lhkvtylqng kgrkyfhhns dfyipkatlk dsgsyfcrgl fgsknvsset vnititggla vstissffpp gyqvsfclvm vllfavdtgl yfsvktnirs strdwkdhkf kwrkdpqdk

WO wo 2021/247604 PCT/US2021/035285

Human CD16a cDNA (SEQ ID NO: 24)

tggctgagggcacactctggcagattctgtgtgtgtcctcagatgct gccacagacctttgagggagtaaagggggcagacccacccaccttgcctc caggctctttccttcctggtcctgttctatggtggggctcccttgccaga cttcagactgagaagtcagatgaagtttcaagaaaaggaaattggtggg gacagagatgggtggaggggctggggaaaggctgtttacttcctcctgt tagtcggtttggtccctttagggctccggatatctttggtgacttgtcca ctccagtgtggcatcatgtggcagctgctcctcccaactgctctgctact tctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtg tcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctc aagtgccagggagcctactcccctgaggacaattccacacagtggtttca caatgagagcctcatctcaagccaggcctcgagctacttcattgacgct ccacagtcgacgacagtggagagtacaggtgccagacaaacctctccace ctcagtgacccggtgcagctagaagtccatatcggctggctgttgctcca ggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtc cagctggaagaacactgctctgcataaggtcacatatttacagaatgg haaggcaggaagtattttcatcataattctgacttctacattccaaaagc cacactcaaagacagcggctcctacttctgcagggggctttttgggagta aaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttgga gtgtcaaccatctcatcattctttccacctgggtaccaagtctctttct cttggtgatggtactcctttttgcagtggacacaggactatatttctct tgaagacaaacattcgaagctcaacaagagactggaaggaccataaatt aaatggagaaaggaccctcaagacaaatga

The protein and cDNA sequences for mature human CD16b are shown below.

Mature Human CD16b Protein (SEQ ID NO: 25)

mwqlllptal lllvsagmrt edlpkavvfl epqwysvlek dsvtlkcqga yspednstqw fhneslissq assyfidaat vndsgeyrcq tnlstlsdpv qlevhigwll lqaprwvfke edpihlrchs wkntalhkvt ylqngkdrky fhhnsdfhip katlkdsgsy fcrglvgskn vssetvniti tqglavstis sfsppgyqvs fclvmvllfa vdtglyfsvk tni

Human CD16b cDNA (SEQ ID NO: 26)

tgtggcagctgctcctcccaactgctctgctacttctagtttcagctc atgtggcagctgctcctcccaactgctctgctacttctagtttcagctgg patgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaa ggtacagcgtgcttgagaaggacagtgtgactctgaagtgccagggagcc actcccctgaggacaattccacacagtggtttcacaatgagaacctca ctcaagccaggcctcgagctacttcattgacgctgccacagtcaacgaca gtggagagtacaggtgccagacaaacctctccaccctcagtgacccggts agctagaagtccatatcggctggctgttgctccaggcccctcggtggg ttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaad stgctctgcataaggtcacatatttacagaatggcaaagacaggaagtat tttcatcataattctgacttccacattccaaaagccacactcaaagatag cggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcag agactgtgaacatcaccatcactcaaggtttggcagtgtcaaccatctca tcattctctccacctgggtaccaagtctctttctgcttggtgatggtact 94 wo 2021/247604 WO PCT/US2021/035285 cctttttgcagtggacacaggactatatttctctgtgaagacaaacattt cctttttgcagtggacacaggactatatttctctgtgaagacaaacattt ga

The protein and cDNA sequences for mature human KIR2DS1 are shown below.

Human KIR2DS1 Protein (SEQ ID NO: 27)

msltvsmac vgffllqgaw phegvhrkps llahpgrlvk seetvilqcw sdvmfehfll hregmfndtl rligehhdgv skanfsisrm kqdlagtyrc ygsvthspyq vsapsdpldi viiglyekps lsaqpgptvl agesvtlscs srssydmyhl sregeaherr lpagtkvngt fqanfplgpa thggtyrcfg sfrdspyews kssdpllvsv tgnpsnswps ptepssetgn prhlhvligt swkipftil lffllhrwcs dkknaavmdq epagnrtvns edsdeqdhqe vsya

Human KIR2DS1 cDNA (SEQ ID NO: 28)

atgtcgctcacggtcgtcagcatggcgtgtgttgggttcttcttgctgo gggggcctggccacatgagggagtccacagaaaaccttccctcctggcct acccaggtcgcctggtgaaatcagaagagacagtcatcctgcaatgttgo tcagatgtcatgtttgaacacttccttctgcacagagaggggatgtttaa gacactttgcgcctcattggagaacaccatgatggggtctccaaggcca acttctccatcagtcgcatgaagcaagacctggcagggacctacagatgo acggttctgttactcactccccctatcagttgtcagctcccagtgacco tctggacatcgtgatcataggtctatatgagaaaccttctctctcagcco agccgggccccacggttctggcaggagagaatgtgaccttgtcctgcago cccggagctcctatgacatgtaccatctatccagggaaggggaggccc tgaacgtaggctccctgcagggaccaaggtcaacggaacattccaggcca actttcctctgggccctgccacccatggagggacctacagatgcttcggc tctttccgtgactctccatacgagtggtcaaagtcaagtgacccactgct

tgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaa caagctccgaaaccggtaaccccagacacctacatgttctgattgggacc tcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcg ctggtgctccgacaaaaaaaatgctgctgtaatggaccaagagcctgcad ggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggag

gtgtcatacgcataa

The protein and cDNA sequences for mature human KIR2DS2 are shown below.

Human KIR2DS2 Protein (SEQ ID NO: 29) mslmvvsmvc vgffllqgaw phegvhrkps llahpgplvk seetvilqcw sdvrfehfll hregkykdtl hligehhdgv skanfsigpm mqdlagtyrc ygsvthspyq lsapsdpldi vitglyekps lsaqpgptvl agesvtlscs srssydmyhl sregeaherr fsagpkvngt fqadfplgpa thggtyrcfg sfrdspyews nssdpllvsv tgnpsnswps ptepssktgn prhlhvligt svkipftil lffllhrwcs nkknaavmdq epagnrtvns edsdeqdhqe vsya wo 2021/247604 WO PCT/US2021/035285

Human KIR2DS2 cDNA (SEQ ID NO: 30)

atgtcgctcatggtcgtcagcatggcgtgtgttgggttcttcttgcto nggggcctggccacatgagggagtccacagaaaaccttccctcctggc acccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttg tcagatgtcaggtttgagcacttccttctgcacagagaggggaagtataa ggacactttgcacctcattggagagcaccatgatggggtctccaaggcca cttctccatcggtcccatgatgcaagaccttgcagggacctacagatgo tacggttctgttactcactccccctatcagttgtcagctcccagtgacco tctggacatcgtcatcacaggtctatatgagaaaccttctctctcagcco agccgggccccacggttttggcaggagagagcgtgaccttgtcctgcag cccggagctcctatgacatgtaccatctatccagggagggggaggccca tgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggecc actttcctctgggccctgccacccacggaggaacctacagatgcttcggo tctttccgtgactctccctatgagtggtcaaactcgagtgacccactg tgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaal aagctccaaaaccggtaaccccagacacctgcatgttctgattggga cagtggtcaaaatccctttcaccatcctcctcttctttctccttcatc ctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagcctgcag igaacagaacagtgaacagcgaggactctgatgaacaagaccctcaggad gtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcad tcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtaca cggaacttccaaatgctgagtccaga

The protein and cDNA sequences for mature human KIR2DS3 are shown below.

Mature Human KIR2DS3 Protein (SEQ ID NO: 31)

mslmvismac vgffwlqgaw phegfrrkps llahpgrlvk seetvilqcw sdvmfehfll hregtfndtl rligehidgv skanfsigrm rqdlagtyrc ygsvphspyq fsapsdpldi vitglyekps lsaqpgptvl agesvtlscs swssydmyhl stegeaherr fsagpkvngt fqadfplgpa tqggtyrcfg sfhdspyews kssdpllvsv tgnpsnswps ptepssktgn prhlhvligt svklpftil lffllhrwcs dkknasvmdq gpagnrtvnr edsdeqdhqe vsya

Human KIR2DS3 cDNA (SEQ ID NO: 32) atgtcgctcatggtcatcagcatggcatgtgttgggttcttctggctgca atgtcgctcatggtcatcagcatggcatgtgttgggttcttctggctgo gggggcctggccacatgagggattccgcagaaaaccttccctcctggc acccaggtcgcctggtgaaatcagaagagacagtcatcctgcaatgttge tcagatgtcatgtttgagcacttccttctgcacagagaggggacgttt cgacactttgcgcctcattggagagcacattgatggggtctccaaggcc acttctccatcggtcgcatgaggcaagacctggcagggacctacagatgo tacggttctgttcctcactccccctatcagttttcagctcccagtgaccc tctggacatcgtgatcacaggtctatatgagaaaccttctctctcagce agccgggccccacggttctggcaggagagagcgtgaccttgtcctgcago tcctggagctcctatgacatgtaccatctatccacggagggggaggccca tgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggccg wo 2021/247604 WO PCT/US2021/035285 actttcctctgggccctgccacccaaggaggaacctacagatgcttcggo actttcctctgggccctgccacccaaggaggaacctacagatgcttcggc ctttccatgactctccctacgagtggtcaaagtcaagtgacccactgct tgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaa caagctccaaaaccggtaaccccagacacctacacgttctgattgggacc tcagtggtcaaactccctttcaccatcctcctcttctttctccttcatcg :tggtgctccgacaaaaaaaatgcatctgtaatggaccaagggcctgcg ggaacagaacagtgaacagggaggattctgatgaacaggaccatcaggag gtgtcatacgcataa

The protein and cDNA sequences for mature human KIR2DL4 are shown below.

Mature Human KIR2DL4 Protein (SEQ ID NO: 33) hvggqdk pfcsawpsav vpqgghatlr chcrrgfnif tlykkdgvpv pelynrifwn sflispvtpa hagtyrcrgf hphsptewsa psnplvimvt glyekpslta rpgptvrage

nvtlscssqs sfdiyhlsre geahelrlpa vpsingtfqa dfplgpathg etyrcfgsfh gspyewsdps dplpvsvtgn pssswpspte psfktgiarh lhavirysva iilftilpff llhrwcskkk naavmnqepa ghrtvnreds deqdpqevty aqldhciftq rkitgpsqrs krpstdtsvc ielpnaepra lspahehhsq almgssrett alsqtqlass nvpaagi

Human KIR2DL4 cDNA (SEQ ID NO: 34)

tgtccccttcacatgttgtggtcaatgtgtcaactgcacgatecggg cctcaccacatcctctgcaccggtcagtcgagccgagtcactgcgtcct jcagcagaagctgcaccatgtccatgtcacccacggtcatcatcctggca tgtcttgggttcttcttggaccagagtgtgtgggcacacgtgggtggtca jgacaagcccttctgctctgcctggcccagcgctgtggtgcctcaaggad gacacgtgactcttcggtgtcactatcgtcgtgggtttaacatcttcaco :tgtacaagaaagatggggtccctgtccctgagctctacaacagaatat ctggaacagtttcctcattagccctgtgaccccagcacacgcagggacct acagatgtcgaggttttcacccgcactcccccactgagtggtcggcacco

gcaaccccctggtgatcatggtcacaggtctatatgagaaaccttcgc tacagcccggccgggccccacggttcgcgcaggagagaacgtgaccttg cctgcagctcccagagctcctttgacatctaccatctatccagggagggg aagcccatgaacttaggctccctgcagtgcccagcatcaatggaacat caggccgacttccctctgggtcctgccacccacggagagacctacaga cttcggctctttccatggatctccctacgagtggtcagacccgagtga :cactgcctgtttctgtcacaggaaacccttctagtagttggccttcad actgaaccaagcttcaaaactggtatcgccagacacctgcatgctgtga taggtactcagtggccatcatcctctttaccatccttcccttctttct cttcatcgctggtgctccaaaaaaaaagatgctgctgtaatgaaccaagal gcctgcgggacacagaacagtgaacagggaggactctgatgaacaagad ctcaggaggtgacatacgcacagttggatcactgcattttcacacagaga aaaatcactggcccttctcagaggagcaagagaccctcaacagataccad cgtgtgtatagaacttccaaatgctgagcccagagcgttgtctcctgcco atgagcaccacagtcaggccttgatgggatcttctagggagacaacagca wo 2021/247604 WO PCT/US2021/035285 ctgtctcaaacccagcttgccagctctaatgtaccagcagctggaatctg ctgtctcaaacccagcttgccagctctaatgtaccagcagctggaatctg a

The protein and cDNA sequences for mature human KIR2DS4 are shown below.

Mature Human KIR2DS4 Protein (SEQ ID NO: 35) qegvhrkps flalpghlvk seetvilqcw sdvmfehfll hregkfnntl hligehhdgv skanfsigpm mpvlagtyrc yssvphspyq lsapsdpldm viiglyekps lsaqpgptvq agenvslscs siypgrgrpm nvgslqcaas tehsrptflw alpptegptd asalsvtlpt sgqtrvihcl fpsqetlqiv glhplnqapk pvtpdtymf

Human KIR2DS4 cDNA (SEQ ID NO: 36)

atgtcgctcatggtcatcatcatggcgtgtgttgggttcttcttgctgo gggggcctggccacaggagggagtccacagaaaaccttccttcctggccc tcccaggtcacctggtgaaatcagaagagacagtcatcctgcaatgttge cggatgtcatgtttgagcacttccttctgcacagagaggggaagtttaa caacactttgcacctcattggagagcaccatgatggggtttccaaggca acttctccattggtcccatgatgcctgtccttgcaggaacctacagatgo acggttctgttcctcactccccctatcagttgtcagctcccagtgacco tctggacatggtgatcataggtctatatgagaaaccttctctctcagcco agccgggccccacggttcaggcaggagagaatgtgaccttgtcctgcago tccatctatccagggaaggggaggcccatgaacgtaggctccctgcagtg cgcagcatcaacggaacattccaggccgactttcctctgggccctgccao ccacggagggacctacagatgcttcggctctttccgtgacgctccctaco

agtggtcaaactcgagtgatccactgcttgtttccgtcacaggaaaccct caaatagttggccttcacccactgaaccaagctccaaaaccggtaacco cagacacctacatgttctgattgggacctcagtggtcaaaatccctttca catcctcctcttctttctccttcatcgctggtgctccgacaaaaaaaat gctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcga

ggattctgatgaacaagaccatcaggaggtgtcatacgcataa

The protein and cDNA sequences for mature human KIR2DS5 are shown below.

Mature Human KIR2DS5 (SEQ ID NO: 37)

hegfrrkps llahpgplvk seetvilqcw sdvmfehfll hregtfnhtl rligehidgv skgnfsigrm tqdlagtyrc ygsvthspyq lsapsdpldi vitglyekps lsaqpgptvl agesvtlscs srssydmyhl sregeaherr lpagtkvngt fqadfpldpa thggtyrcfg sfrdspyews kssdpllvsv tgntsnswps ptepssktgn prhlhvligt svvlpftil lffllhrwcs nkknasvmdq gpagnrtvnr edsdeqdhqe vsya wo 2021/247604 WO PCT/US2021/035285

Human KIR2DS5 cDNA (SEQ ID NO: 38)

atgtcgctcatggtcatcagcatggcgtgtgttgcgttcttcttgctc jggggcctggccacatgagggattccgcagaaaaccttccctcctgga cccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttg tcagatgtcatgtttgagcacttccttctgcacagagaggggacgtttaa ccacactttgcgcctcattggagagcacattgatggggtctccaagggca cttctccatcggtcgcatgacacaagacctggcagggacctacagatgo tacggttctgttactcactccccctatcagttgtcagcgcccagtgacco tctggacatcgtgatcacaggtctatatgagaaaccttctctctcagcc.

lgccgggccccacggttctggcaggagagagcgtgaccttgtcctgcago tcccggagctcctatgacatgtaccatctatccagggaaggggaggccc tgaacgtaggctccctgcagggcccaaggtcaacagaacattccaggccc actttcctctggaccctgccacccacggagggacctacagatgcttcggo ctttccgtgactctccatacgagtggtcaaagtcaagtgacccactgo tgtttctgtcacaggaaactcttcaaatagttggccttcacccactgaad caagctccgaaaccggtaaccccagacacctacacgttctgattgggacc cagtggtcaaactccctttcaccatcctcctcttctttctccttcatc ctggtgctccaacaaaaaaaatgcatctgtaatggaccaagggcctgcgg ggaacagaacagtgaacagggaggattctgatgaacaggaccatcaggag gtgtcatacgcataa

The protein and cDNA sequences for mature human KIR3DS1 are shown below.

Mature Human KIR3DS1 cDNA (SEQ ID NO: 39) hmggqdkpf lsawpsavvp rgghvtlrch yrhrfnnfml ykedrihvpi fhgrifqegf nmspvttaha gnytcrgshp hsptgwsaps npmvimvtgn hrkpsllahp gplvksgerv ilqcwsdimf ehfflhkegi skdpsrlvgq ihdgvskanf sigsmmrala gtyrcygsvt htpyqlsaps dpldivvtgl yekpslsaqp gpkvqagesv tlscssrssy dmyhlsregg aherrlpavr kvnrtfqadf plgpathggt yrcfgsfrhs pyewsdpsdp llvsvtgnps sswpspteps sksgnlrhlh iligtswki pftillffll hrwcsnkkkc ccngpracre qk

Human KIR3DS1 cDNA (SEQ ID NO: 40)

atgttgctcatggtcgtcagcatggcgtgtgttgggttgttcttggtc atgttgctcatggtcgtcagcatggcgtgtgttgggttgttcttggtoca gagggccggtccacacatgggtggtcaggacaagcccttcctgtctgcc gcccagegctgtggtgcctcgcggaggacacgtgactcttcggtgtca tatcgtcataggtttaacaatttcatgctatacaaagaagacagaatcca egttcccatcttccatggcagaatattccaggagggcttcaacatgaga stgtgaccacagcacatgcagggaactacacatgtcggggttcacaccc. cactcccccactgggtggtcggcacccagcaaccccatggtgatcatgg acaggaaaccacagaaaaccttccctcctggcccacccaggtcccctg jaaatcaggagagagagtcatcctgcaatgttggtcagatatcatgtt: gagcacttctttctgcacaaagagtggatctctaaggacccctcacgcct cgttggacagatccatgatggggtctccaaggccaatttctccatcggtt wo 2021/247604 WO PCT/US2021/035285 ccatgatgcgtgcccttgcagggacctacagatgctacggttctgttact ccatgatgcgtgcccttgcagggacctacagatgctacggttctgttact cacaccccctatcagttgtcagctcccagtgatcccctggacatcgtgg cacaggtctatatgagaaaccttctctctcagcccagccgggccccaage ttcaggcaggagagagcgtgaccttgtcctgtagctcccggagctcctat gacatgtaccatctatccagggaggggggagcccatgaacgtaggctco tgcagtgcgcaaggtcaacagaacattccaggcagatttccctctgggcc ctgccacccacggagggacctacagatgcttcggctctttccgtcactci ccctacgagtggtcagacccgagtgacccactgcttgtttctgtcacagg aacccttcaagtagttggccttcacccacagaaccaagctccaaatct gtaacctcagacacctgcacattctgattgggacctcagtggtcaaaata cctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaa aaaaaatgctgctgtaatggaccaagagcctgcagggaacagaagtga

The protein and cDNA sequences for mature human NKG2C are shown below.

Mature Human NKG2C Protein (SEQ ID NO: 41)

mskqrgtfse vslaqdpkrq qrkpkgnkss isgteqeifq velnlqnpsl nhqgidkiyd cqgllpppek ltaevlgiic ivlmatvlkt ivlipflegn nsspntrtqk arhcghcpee witysnscyy igkerrtwee sllactskns sllsidneee mkflasilps swigvfrnss hhpwvtingl afkhkikdsd naelncavlq vnrlksaqcg ssmiyhckhk l

Human NKG2C cDNA (SEQ ID NO: 42)

atgaataaacaaagaggaaccttctcagaagtgagtctggcccagga aaagcggcagcaaaggaaacctaaaggaataaaagctccatttcaggaa ccgaacaggaaatattccaagtagaattaaatcttcaaaatccttccct aatcatcaagggattgataaaatatatgactgccaaggtttactgccaco tccagagaagctcactgccgaggtcctaggaatcatttgcattgtcctga tggccactgtgttaaaaacaatagttcttattcctttcctggagcagaad aatttttccccgaatacaagaacgcagaaagcacgtcattgtggccattg cctgaggagtggattacatattccaacagttgttattacattggtaago laagaagaacttgggaagagagtttgctggcctgtacttcgaagaactco gtctgctttctatagataatgaagaagaaatgaaatttctggccagcat tttaccttcctcatggattggtgtgtttcgtaacagcagtcatcatccat iggtgacaataaatggtttggctttcaaacataagataaaagactcagat aatgctgaacttaactgtgcagtgctacaagtaaatcgacttaaatca ccagtgtggatcttcaatgatatatcattgtaagcataagctttag

The protein and cDNA sequences for mature human CCR7 are shown below.

Mature Human CCR7 Protein (SEQ ID NO: 43)

qdevtd dyigdnttvd ytlfeslcsk kdvrnfkawf lpimysiicf vgllgnglvv ltyiyfkrlk tmtdtyllnl avadilfllt lpfwaysaak swvfgvhfck lifaiykmsf fsgmllllci sidryvaivq avsahrhrar vllisklscv giwilatvls ipellysdlq

WO wo 2021/247604 PCT/US2021/035285

rssseqamrc slitehveaf itiqvaqmvi gflvpllams fcylviirtl lqarnfernk aikviiavvv vfivfqlpyn gvvlaqtvan fnitsstcel skqlniaydv tyslacvrcc vnpflyafig vkfrndlfkl fkdlgclsqe qlrqwsscrh irrssmsvea ettttfsp

Human CCR7 cDNA (SEQ ID NO: 44)

atggacctggggaaaccaatgaaaagcgtgctggtggtggctctcctt cattttccaggtatgcctgtgtcaagatgaggtcacggacgattacato jagacaacaccacagtggactacactttgttcgagtctttgtgctccaad aaggacgtgcggaactttaaagcctggttcctccctatcatgtactccat

catttgtttcgtgggcctactgggcaatgggctggtcgtgttgacctata actatttcaagaggctcaagaccatgaccgatacctacctgctcaacct gaggtggcagacatcctcttcctcctgacccttcccttctgggcctacal cgcggccaagtcctgggtcttcggtgtccacttttgcaagctcatcttt ccatctacaagatgagcttcttcagtggcatgctcctacttctttgcato agcattgaccgctacgtggccatcgtccaggctgtctcagctcaccgcca ccgtgcccgcgtccttctcatcagcaagctgtcctgtgtgggcatctgga tactagccacagtgctctccatcccagagctcctgtacagtgacctccal aggagcagcagtgagcaagcgatgcgatgctctctcatcacagagcatgt ggaggcctttatcaccatccaggtggcccagatggtgatcggctttctgg tcccctgctggccatgagcttctgttaccttgtcatcatccgcacccto tccaggcacgcaactttgagcgcaacaaggccatcaaggtgatcatcg gtggtcgtggtcttcatagtcttccagctgccctacaatggggtggtc ggcccagacggtggccaacttcaacatcaccagtagcacctgtgagct agtaagcaactcaacatcgcctacgacgtcacctacagcctggcctgcgt ccgctgctgcgtcaaccctttcttgtacgccttcatcggcgtcaagttcc caacgatctcttcaagctcttcaaggacctgggctgcctcagccaggag cagctccggcagtggtcttcctgtcggcacatccggcgctcctccatgag Egtggaggccgagaccaccaccaccttctccccatag

The protein and cDNA sequences for mature human CXCR3 are shown below.

Mature Human CXCR3 Protein (SEQ ID NO: 45)

mvlevsdhqv Indaevaal] enfsssydyg enesdsccts ppcpqdfsln fdraflpaly sllfllgllg ngavaavlls rrtalsstdt fllhlavadt llvltlplwa vdaavqwvfg sglckvagal fninfyagal llacisfdry lnivhatqly rrgpparvtl tclavwglcl lfalpdfifl sahhderlna thcqynfpqv grtalrvlql vagfllpllv maycyahila vllvsrgqrr lramrlvvvv wafalcwtp yhlvvlvdil mdlgalarno gresrvdvak svtsglgymh cclnpllyaf vgvkfrermw mlllrlgcpn qrglqrqpss srrdsswset seasysgl

Human CXCR3 cDNA (SEQ ID NO: 46)

itggagttgaggaagtacggccctggaagactggcggggacagttatagg aggagctgctcagagtaaatcacagactaaatcagactcaatcacaaaad agttcctgccaggcctttacacagccccttcctccccgttcccgccctca wo 2021/247604 WO PCT/US2021/035285 caggtgagtgaccaccaagtgctaaatgacgccgaggttgccgccctcct caggtgagtgaccaccaagtgctaaatgacgccgaggttgccgccctcct ggagaacttcagctcttcctatgactatggagaaaacgagagtgactcg gctgtacctccccgccctgcccacaggacttcagcctgaacttcgaccg gccttcctgccagccctctacagcctcctctttctgctggggctgctggg caacggcgcggtggcagccgtgctgctgagccggcggacagccctgagca caccgacaccttcctgctccacctagctgtagcagacacgctgctggt ctgacactgccgctctgggcagtggacgctgccgtccagtgggtctttgg ctctggcctctgcaaagtggcaggtgccctcttcaacatcaacttctacy aggagccctcctgctggcctgcatcagctttgaccgctacctgaacat gttcatgccacccagctctaccgccgggggcccccggcccgcgtgacco cacctgcctggctgtctgggggctctgcctgcttttcgccctcccagad tcatcttcctgtcggcccaccacgacgagcgcctcaacgccacccactgo aatacaacttcccacaggtgggccgcacggctctgcgggtgctgcago ggtggctggctttctgctgcccctgctggtcatggcctactgctatgcco acatcctggccgtgctgctggtttccaggggccagcggcgcctgcgggcc atgcggctggtggtggtggtcgtggtggcctttgccctctgctggaccco ctatcacctggtggtgctggtggacatcctcatggacctgggcgctttgg cccgcaactgtggccgagaaagcagggtagacgtggccaagtcggtcac tcaggcctgggctacatgcactgctgcctcaacccgctgctctatgcctt tgtaggggtcaagttccgggagcggatgtggatgctgctcttgcgcctgg gctgccccaaccagagagggctccagaggcagccatcgtcttcccgccgg gattcatcctggtctgagacctcagaggcctcctactcgggcttgtga

The protein and cDNA sequences for mature human L-selectin are shown below.

Mature Human L-Selectin Protein (SEQ ID NO: 47)

df lahhgtdcwt yhysekpmnw qrarrfcrdn ytdlvaiqnk aeieylektl pfsrsyywig irkiggiwtw vgtnksltee aenwgdgepn nkknkedcve iyikrnkdag kwnddachkl kaalcytasc qpwscsghge cveiinnytc ncdvgyygpq cqfviqcepl eapelgtmdc thplgnfsfs sqcafscseg tnltgieett cgpfgnwssp eptcqviqce plsapdlgim ncshplasfs ftsactfics egteligkkk ticessgiws npspicqkld ksfsmikegd ynplfipvav mvtafsglaf iiwlarrlkk gkkskrsmnd py

Human L-Selectin cDNA (SEQ ID NO: 48)

atgggctgcagaagaactagagaaggaccaagcaaagccatgatattto atggaaatgtcagagcacccagagggacttatggaacatcttcaagttgt gggggtggacaatgctctgttgtgatttcctggcacatcatggaaccgal tgctggacttaccattattctgaaaaacccatgaactggcaaagggctag hagattctgccgagacaattacacagatttagttgccatacaaaacaag cggaaattgagtatctggagaagactctgcctttcagtcgttcttactad tggataggaatccggaagataggaggaatatggacgtgggtgggaaccaa caaatctcttactgaagaagcagagaactggggagatggtgagcccaaca caagaagaacaaggaggactgcgtggagatctatatcaagagaaacaa gatgcaggcaaatggaacgatgacgcctgccacaaactaaaggcagccct ctgttacacagcttcttgccagccctggtcatgcagtggccatggagaat

WO wo 2021/247604 PCT/US2021/035285

gtgtagaaatcatcaataattacacctgcaactgtgatgtggggtactat gtgtagaaatcatcaataattacacctgcaactgtgatgtggggtactat gggccccagtgtcagtttgtgattcagtgtgagcctttggaggccccaga gctgggtaccatggactgtactcaccctttgggaaacttcagcttcagct cacagtgtgccttcagctgctctgaaggaacaaacttaactgggattgaa gaaaccacctgtggaccatttggaaactggtcatctccagaaccaacct tcaagtgattcagtgtgagcctctatcagcaccagatttggggatcatg. actgtagccatcccctggccagcttcagctttacctctgcatgtacctto atctgctcagaaggaactgagttaattgggaagaagaaaaccatttgtga atcatctggaatctggtcaaatcctagtccaatatgtcaaaaattggac aagtttctcaatgattaaggagggtgattataaccccctcttcattco tggcagtcatggttactgcattctctgggttggcatttatcatttggct ggcaaggagattaaaaaaaggcaagaaatccaagagaagtatgaatgaca catattaa

The protein and cDNA sequences for mature human CXCR1 are shown below.

Mature Human CXCR1 Protein (SEQ ID NO: 49)

msnitdpqmw dfddlnftgm ppadedyspc xletetlnky vviiayalvf llsllgnslv mlvilysrvg rsvtdvylln laladllfal tlpiwaaskv ngwifgtflc kvvsllkevn fysgilllac isvdrylaiv hatrtltqkr hlvkfvclgc wglsmnlslp fflfrqayhp nnsspvcyev lgndtakwrm vlrilphtfg fivplfmmlf cygftlrtlf kahmgqkhra mrvifavvli fllcwlpynl vlladtlmrt qvigescerr nnigraldat eilgflhscl npiiyafigg nfrhgflkil amhglvskef larhrvtsyt sssvnvssnl

Human CXCR1 cDNA (SEQ ID NO: 50)

tgtcaaatattacagatccacagatgtgggattttgatgatctaaat atgtcaaatattacagatccacagatgtgggattttgatgatctaaattt cactggatgccacctgcagatgaagattacagcccctgtatgctagaaz ctgagacactcaacaagtatgttgtgatcatcgcctatgccctagtgtt tgctgagcctgctgggaaactccctggtgatgctggtcatcttataca cagggtcggccgctccgtcactgatgtctacctgctgaacctggccttgg ecgacctactctttgccctgaccttgcccatctgggccgcctccaaggt aatggctggatttttggcacattcctgtgcaaggtggtctcactcctgaa gaagtcaacttctacagtggcatcctgctgttggcctgcatcagtgtgo accgttacctggccattgtccatgccacacgcacactgacccagaagcgt eacttggtcaagtttgtttgtcttggctgctggggactgtctatgaatc itccctgcccttcttccttttccgccaggcttaccatccaaacaattco gtccagtttgctatgaggtcctgggaaatgacacagcaaaatggcggatg tgttgcggatcctgcctcacacctttggcttcatcgtgccgctgtttg atgctgttctgctatggattcaccctgcgtacactgtttaaggcccac ggggcagaagcaccgagccatgagggtcatctttgctgtcgtcctcate Etcctgctttgctggctgccctacaacctggtcctgctggcagacacco catgaggacccaggtgatccaggagagctgtgagcgccgcaacaacatcg gccgggccctggatgccactgagattctgggatttctccatagctgcctc aaccccatcatctacgccttcatcggccaaaattttcgccatggattcc caagatcctggctatgcatggcctggtcagcaaggagttcttggcacgtc atcgtgttacctcctacacttcttcgtctgtcaatgtctcttccaaccto

103 tga

The protein and cDNA sequences for mature human CXCR2 are shown below.

Mature Human CXCR2 Protein (SEQ ID NO: 51)

medfnmesds fedfwkgedl snysysstlp pflldaapce pesleinkyf vviiyalvfl lsllgnslvm lvilysrvgr svtdvyllnl aladllfalt lpiwaaskvn gwifgtflck vvsllkevnf ysgilllaci svdrylaivh atrtltqkry lvkficlsiw glslllalpv llfrrtvyss nvspacyedm gnntanwrml lrilpqsfgf ivpllimlfc ygftlrtlfk ahmgqkhram rvifavvlif llcwlpynlv lladtlmrtq vigetcerrn hidraldate ilgilhscln pliyafiggk frhgllkila ihgliskdsl pkdsrpsfvg sssghtsttl

Human CXCR2 cDNA (SEQ ID NO: 52)

atggaagattttaacatggagagtgacagctttgaagatttctggaaa atggaagattttaacatggagagtgacagctttgaagatttctggaaagg

tgaagatcttagtaattacagttacagctctaccctgcccccttttctad agatgccgccccatgtgaaccagaatccctggaaatcaacaagtattt gtggtcattatctatgccctggtattcctgctgagcctgctgggaaacto cctcgtgatgctggtcatcttatacagcagggtcggccgctccgtcactg tgtctacctgctgaacctagccttggccgacctactctttgccctgac tgcccatctgggccgcctccaaggtgaatggctggatttttggcacat cctgtgcaaggtggtctcactcctgaaggaagtcaacttctatagtggca tcctgctactggcctgcatcagtgtggaccgttacctggccattgtccat gccacacgcacactgacccagaagcgctacttggtcaaattcatatgtct cagcatctggggtctgtccttgctcctggccctgcctgtcttacttttcm gaaggaccgtctactcatccaatgttagcccagectgctatgaggacatg jgcaacaatacagcaaactggcggatgctgttacggatcctgccccagto ctttggcttcatcgtgccactgctgatcatgctgttctgctacggattca ccctgcgtacgctgtttaaggcccacatggggcagaagcaccgggccatg cgggtcatctttgctgtcgtcctcatcttcctgctctgctggctgcccta caacctggtcctgctggcagacaccctcatgaggacccaggtgatccagg agacctgtgagcgccgcaatcacatcgaccgggctctggatgccaccgal attctgggcatccttcacagctgcctcaaccccctcatctacgccttcal tggccagaagtttcgccatggactcctcaagattctagctatacatggct tgatcagcaaggactccctgcccaaagacagcaggccttcctttgttgga acttcttcagggcacacttccactactctctaa

The protein and cDNA sequences for mature human CX3CR1 are shown below.

Mature Human CX3CR1 Protein (SEQ ID NO: 53) mdqfpesvte nfeyddlaea cyigdivvfg tvflsifysv ifaiglvgnl lwfaltnsk kpksvtdiyl Inlalsdllf vatlpfwthy linekglhna mckfttafff igffgsiffi tvisidryla ivlaansmnn rtvqhgvtis lgvwaaailv aapqfmftkq keneclgdyp evlqeiwpvl rnvetnflgf llpllimsyc yfriigtlfs cknhkkakai klillvvivf wo 2021/247604 WO PCT/US2021/035285 flfwtpynvm ifletlklyd ffpscdmrkd lrlalsvtet vafshcclnp liyafagekf rrylyhlygk clavlcgrsv hvdfsssesq rsrhgsvlss nftyhtsdgd allll

Human CX3CR1 cDNA (SEQ ID NO: 54)

atggatcagttccctgaatcagtgacagaaaactttgagtacgatgatt atggatcagttccctgaatcagtgacagaaaactttgagtacgatgattt ggctgaggcctgttatattggggacatcgtggtctttgggactgtgtt gtccatattctactccgtcatctttgccattggcctggtgggaaattt ttggtagtgtttgccctcaccaacagcaagaagcccaagagtgtcaccga catttacctcctgaacctggccttgtctgatctgctgtttgtagccact tgcccttctggactcactatttgataaatgaaaagggcctccacaatgc. atgtgcaaattcactaccgccttcttcttcatcggcttttttggaagcat attcttcatcaccgtcatcagcattgataggtacctggccatcgtcctgo ccgccaactccatgaacaaccggaccgtgcagcatggcgtcaccatcago staggcgtctgggcagcagccattttggtggcagcaccccagttcatgt: cacaaagcagaaagaaaatgaatgccttggtgactaccccgaggtcctcc aggaaatctggcccgtgctccgcaatgtggaaacaaattttcttggctto ctactccccctgctcattatgagttattgctacttcagaatcatccagad jctgttttcctgcaagaaccacaagaaagccaaagccattaaactgatc ttctggtggtcatcgtgtttttcctcttctggacaccctacaacgttatg attttcctggagacgcttaagctctatgacttctttcccagttgtgaca gaggaaggatctgaggctggccctcagtgtgactgagacggttgcattta gccattgttgcctgaatcctctcatctatgcatttgctggggagaagtte gaagatacctttaccacctgtatgggaaatgcctggctgtcctgtgto gcgctcagtccacgttgatttctcctcatctgaatcacaaaggagcagge :ggaagtgttctgagcagcaattttacttaccacacgagtgatggagat gcattgctccttctctga

The protein and cDNA sequences for mature human ChemR23 are shown below.

Mature Human ChemR23 Protein (SEQ ID NO: 55) mrmededynt sisygdeypd yldsivvled lsplearvtr iflvvvysiv cflgilgngl viiiatfkmk ktvnmvwfln lavadflfnv flpihityaa mdyhwvfgta mckisnflli hnmftsvfll tiissdrcis vllpvwsqnh rsvrlaymac mviwvlaffl sspslvfrdt anlhgkiscf nnfslstpgs sswpthsqmd pvgysrhmvv tvtrflcgfl vpvliitacy ltivcklqrn rlaktkkpfk iivtiiitff lcwcpyhtln llelhhtamp gsvfslglpl atalaiansc mnpilyvfmg qdfkkfkval fsrlvnalse dtghssypsh rsftkmssmn ertsmneret gml

Human ChemR23 cDNA (SEQ ID NO: 56) atgagaatggaggatgaagattacaacacttccatcagttacggtgat atgagaatggaggatgaagattacaacacttccatcagttacggtgatga taccctgattatttagactccattgtggttttggaggacttatcccco tggaagccagggtgaccaggatcttcctggtggtggtctacagcatcgto gcttcctcgggattctgggcaatggtctggtgatcatcattgccacct caagatgaagaagacagtgaacatggtctggttcctcaacctggcagtgg cagatttcctgttcaacgtcttcctcccaatccatatcacctatgccgcc wo 2021/247604 WO PCT/US2021/035285 atggactaccactgggttttcgggacagccatgtgcaagatcagcaactt atggactaccactgggttttcgggacagccatgtgcaagatcagcaactt cttctcatccacaacatgttcaccagcgtcttcctgctgaccatcatc jctctgaccgctgcatctctgtgctcctccctgtctggtcccagaaccal cgcagcgttcgcctggcttacatggcctgcatggtcatctgggtcctgga ttcttcttgagttccccatctctcgtcttccgggacacagccaaccte atgggaaaatatcctgcttcaacaacttcagcctgtccacacctgggtc scctcgtggcccactcactcccaaatggaccctgtggggtatagccggca hatggtggtgactgtcacccgcttcctctgtggcttcctggtcccagtcc scatcatcacagcttgctacctcaccatcgtgtgcaaactgcagcgcaa cgcctggccaagaccaagaagcccttcaagattattgtgaccatcatca taccttcttcctctgctggtgcccctaccacacactcaacctcctagago tccaccacactgccatgcctggctctgtcttcagcctgggtttgcccct gccactgcccttgccattgccaacagctgcatgaaccccattctgtatgt tttcatgggtcaggacttcaagaagttcaaggtggccctcttctctcgcc tggtcaatgctctaagtgaagatacaggccactcttcctaccccagccat agaagctttaccaagatgtcatcaatgaatgagaggacttctatgaatga jagggagaccggcatgctttga

The protein and cDNA sequences for mature human CXCR4 are shown below.

Mature Human CXCR4 Protein (SEQ ID NO: 57)

megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiif tgivgnglvi lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh SS

Human CXCR4 cDNA (SEQ ID NO: 58) atgtccattcctttgcctcttttgcagatatacacttcagataactacal atgtccattcctttgcctcttttgcagatatacacttcagataactacac cgaggaaatgggctcaggggactatgactccatgaaggaaccctgttte gtgaagaaaatgctaatttcaataaaatcttcctgcccaccatctactce atcatcttcttaactggcattgtgggcaatggattggtcatcctggtcat gggttaccagaagaaactgagaagcatgacggacaagtacaggctgcaco

agtcagtggccgacctcctctttgtcatcacgcttcccttctgggcagt gatgccgtggcaaactggtactttgggaacttcctatgcaaggcagtcca egtcatctacacagtcaacctctacagcagtgtcctcatcctggccttc cagtctggaccgctacctggccatcgtccacgccaccaacagtcagag ccaaggaagctgttggctgaaaaggtggtctatgttggcgtctggatcc tgccctcctgctgactattcccgacttcatctttgccaacgtcagtgagg agatgacagatatatctgtgaccgcttctaccccaatgacttgtgggt jttgtgttccagtttcagcacatcatggttggccttatcctgcctggta tgtcatcctgtcctgctattgcattatcatctccaagctgtcacactcca gggccaccagaagcgcaaggccctcaagaccacagtcatcctcatccto gctttcttcgcctgttggctgccttactacattgggatcagcatcgact cttcatcctcctggaaatcatcaagcaagggtgtgagtttgagaacactg

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

tgcacaagtggatttccatcaccgaggccctagctttcttccactgttgt ctgaaccccatcctctatgctttccttggagccaaatttaaaacctcte ccagcacgcactcacctctgtgagcagagggtccagcctcaagatcctct ccaaaggaaagcgaggtggacattcatctgtttccactgagtctgagtct tcaagttttcactccagctaa

The protein and cDNA sequences for mature human CCR5 are shown below.

Mature Human CCR5 Protein (SEQ ID NO: 59) mdyqvsspiy dinyytsepo qkinvkqiaa rllpplyslv fifgfvgnml vililinckr lksmtdiyll nlaisdlffl ltvpfwahya aaqwdfgntm cqlltglyfi gffsgiffii lltidrylav vhavfalkar tvtfgvvtsv itwvvavfas lpgiiftrsq keglhytcss hfpysqyqfw knfqtlkivi lglvlpllvm vicysgilkt llrcrnekkr hravrlifti mivyflfwap ynivll1ntf qeffglnncs ssnrldqamq vtetlgmthc cinpiiyafv gekfrnyllv ffqkhiakrf ckccsifqqe aperassvyt rstgeqeisv gl

Human CCR5 cDNA (SEQ ID NO: 60)

atggattatcaagtgtcaagtccaatctatgacatcaattattataca atggattatcaagtgtcaagtccaatctatgacatcaattattatacato gagccctgccaaaaaatcaatgtgaagcaaatcgcagcccgcctcctgo tccgctctactcactggtgttcatctttggttttgtgggcaacatgctc tcatcctcatcctgataaactgcaaaaggctgaagagcatgactgacat ctacctgctcaacctggccatctctgacctgtttttccttcttactgto ccttctgggctcactatgctgccgcccagtgggactttggaaatacaato Egtcaactcttgacagggctctattttataggcttcttctctggaatctt cttcatcatcctcctgacaatcgataggtacctggctgtcgtccatgctg gtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgt atcacttgggtggtggctgtgtttgcgtctctcccaggaatcatctttad sagatctcaaaaagaaggtcttcattacacctgcagctctcattttcca acagtcagtatcaattctggaagaatttccagacattaaagatagtcato htggggctggtcctgccgctgcttgtcatggtcatctgctactcgggaat cctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctc tgaggcttatcttcaccatcatgattgtttattttctcttctgggctccm tacaacattgtccttctcctgaacaccttccaggaattctttggcctga taattgcagtagctctaacaggttggaccaagctatgcaggtgacagaga tcttgggatgacgcactgctgcatcaaccccatcatctatgcctttgt ngggagaagttcagaaactacctcttagtcttcttccaaaagcacattge aaacgcttctgcaaatgctgttctattttccagcaagaggctcccgage gagcaagctcagtttacacccgatccactggggagcaggaaatatctgtg ggcttgtga

The protein and cDNA sequences for mature human S1P5 are shown below.

Mature Human S1P5 Protein (SEQ ID NO: 61)

mesgllrpap vsevivlhyn ytgklrgary qpgaglrada vvclavcafi vlenlavllv lgrhprfhap mflllgsltl sdllagaaya anillsgplt lklspalwfa reggvfvalt wo 2021/247604 WO PCT/US2021/035285 asvlsllaia lersltmarr gpapvssrgr tlamaaaawg vslllgllpa lgwnclgrld acstvlplya kayvlfcvla fvgilaaica lyariycqvr anarrlparp gtagttstra rrkprslall rtlsvvllaf vacwgplfll lllvvacpar tcpvllqadp flglamansl Inpiiytltn rdlrhallrl vccgrhscgr dpsgsqqsas aaeasgglrr clppgldgsf sgsersspqr dgldtsgstg spgaptaart lvsepaad

Human S1P5 cDNA (SEQ ID NO: 62)

atggagtcggggctgctgcggccggcgccggtgagcgaggtcatcgtco atggagtcggggctgctgcggccggcgccggtgagcgaggtcatcgtcct cattacaactacaccggcaagctccgcggtgcgcgctaccagccgggt ccggcctgcgcgccgacgccgtggtgtgcctggcggtgtgcgccttcate gtgctagagaatctagccgtgttgttggtgctcggacgccacccgcgctt cacgctcccatgttcctgctcctgggcagcctcacgttgtcggatctgo tggcaggcgccgcctacgccgccaacatcctactgtcggggccgctcaco ctgaaactgtcccccgcgctctggttcgcacgggagggaggcgtcttcg ggcactcactgcgtccgtgctgagcctcctggccatcgcgctggagcgca cctcaccatggcgcgcagggggcccgcgcccgtctccagtcgggggag cgctggcgatggcagccgcggcctggggcgtgtcgctgctcctcgggct cctgccagcgctgggctggaattgcctgggtcgcctggacgcttgctcca ctgtcttgccgctctacgccaaggcctacgtgctcttctgcgtgctcgcc tcgtgggcatcctggccgctatctgtgcactctacgcgcgcatctactg ccaggtacgcgccaacgcgcggcgcctgccggcacggcccgggactgcgg ggaccacctcgacccgggcgcgtcgcaagccgcgctcgctggccttgctc gcacgctcagcgtggtgctcctggcctttgtggcatgttggggcccco cttcctgctgctgttgctcgacgtggcgtgcccggcgcgcacctgtcctg tactcctgcaggccgatcccttcctgggactggccatggccaactcactt ctgaaccccatcatctacacgctcaccaaccgcgacctgcgccacgcga ectgcgcctggtctgctgcggacgccactcctgcggcagagacccgagtg ctcccagcagtcggcgagcgcggctgaggcttccgggggcctgcgccgo tgcctgcccccgggccttgatgggagcttcagcggctcggagcgctcato

cacccacagccgcccggactctggtatcagaaccggctgcagactga

The protein and cDNA sequences for mature human C-kit are shown below.

Mature Human C-kit Protein (SEQ ID NO: 63)

qpsvs pgepsppsih pgksdlivrv gdeirllctd pgfvkwtfei ldetnenkqn ewitekaeat ntgkytctnk hglsnsiyvf vrdpaklflv drslygkedn dtlvrcpltd pevtnys1kg cqgkplpkdl rfipdpkagi miksvkrayh rlclhcsvdq egksvlsekf ilkvrpafka vpvvsvskas yllregeeft vtctikdvss svystwkren sqtklqekyn swhhgdfnye rqatltissa rvndsgvfmc yanntfgsan vtttlevvdk gfinifpmin ttvfvndgen vdliveyeaf pkpehqqwiy mnrtftdkwe dypksenesn iryvselhlt rlkgteggty tflvsnsdvn aaiafnvyvn tkpeiltydr lvngmlqcva agfpeptidw yfcpgteqrc sasvlpvdvq tlnssgppfg klvvqssids safkhngtve ckayndvgkt sayfnfafkg nnkeqihpht lftplligfv ivagmmciiv miltykylqk pmyevqwkvv eeingnnyvy idptqlpydh kwefprnrls fgktlgagaf gkvveatayg liksdaamtv

WO wo 2021/247604 PCT/US2021/035285

avkmlkpsah lterealmse lkvlsylgnh mnivnllgac tiggptlvit eyccygdlln flrrkrdsfi cskqedhaea alyknllhsk esscsdstne ymdmkpgvsy vvptkadkrr svrigsyier dvtpaimedd elaldledll sfsyqvakgm aflaskncih rdlaarnill thgritkicd fglardiknd snyvvkgnar lpvkwmapes ifncvytfes dvwsygiflw elfslgsspy pgmpvdskfy kmikegfrml spehapaemy dimktcwdad plkrptfkqi vqliekqise stnhiysnla ncspnrqkpv vdhsvrinsv gstasssqpl lvhddv

Human C-kit cDNA (SEQ ID NO: 64)

tgagaggcgctcgcggcgcctgggattttctctgcgttctgctcctad gcttcgcgtccagacaggctcttctcaaccatctgtgagtccaggggal egtctccaccatccatccatccaggaaaatcagacttaatagtccgcgt ggcgacgagattaggctgttatgcactgatccgggctttgtcaaatggad htttgagatcctggatgaaacgaatgagaataagcagaatgaatggatc ggaaaaggcagaagccaccaacaccggcaaatacacgtgcaccaacaa

cacggcttaagcaattccatttatgtgtttgttagagatcctgccaagct tttccttgttgaccgctccttgtatgggaaagaagacaacgacacgctg tccgctgtcctctcacagacccagaagtgaccaattattccctcaagggg tgccaggggaagcctcttcccaaggacttgaggtttattcctgaccccaa ggcgggcatcatgatcaaaagtgtgaaacgcgcctaccatcggctctgt tgcattgttctgtggaccaggagggcaagtcagtgctgtcggaaaaatt atcctgaaagtgaggccagccttcaaagctgtgcctgttgtgtctgtgto caaagcaagctatcttcttagggaaggggaagaattcacagtgacgtgca caataaaagatgtgtctagttctgtgtactcaacgtggaaaagagaaaad agtcagactaaactacaggagaaatataatagctggcatcacggtgactt caattatgaacgtcaggcaacgttgactatcagttcagcgagagttaatg attctggagtgttcatgtgttatgccaataatacttttggatcagcaaat gtcacaacaaccttggaagtagtagataaaggattcattaatatcttcco atgataaacactacagtatttgtaaacgatggagaaaatgtagatttga :tgttgaatatgaagcattccccaaacctgaacaccagcagtggatcta atgaacagaaccttcactgataaatgggaagattatcccaagtctgaga tgaaagtaatatcagatacgtaagtgaacttcatctaacgagattaaaag jcaccgaaggaggcacttacacattcctagtgtccaattctgacgtcaal gctgccatagcatttaatgtttatgtgaatacaaaaccagaaatcctgal ctacgacaggctcgtgaatggcatgctccaatgtgtggcagcaggattco agagcccacaatagattggtatttttgtccaggaactgagcagagate tctgcttctgtactgccagtggatgtgcagacactaaactcatctgggcc ccgtttggaaagctagtggttcagagttctatagattctagtgcatto agcacaatggcacggttgaatgtaaggcttacaacgatgtgggcaagact ctgcctattttaactttgcatttaaaggtaacaacaaagagcaaato tccccacaccctgttcactcctttgctgattggtttcgtaatcgtagct gcatgatgtgcattattgtgatgattctgacctacaaatatttacagaaa cccatgtatgaagtacagtggaaggttgttgaggagataaatggaaaca Etatgtttacatagacccaacacaacttccttatgatcacaaatgggag. ttcccagaaacaggctgagttttgggaaaaccctgggtgctggagcttt ggaaggttgttgaggcaactgcttatggcttaattaagtcagatgcggo catgactgtcgctgtaaagatgctcaagccgagtgcccatttgacagaac gggaagccctcatgtctgaactcaaagtcctgagttaccttggtaatcac tgaatattgtgaatctacttggagcctgcaccattggagggcccaccct atgaatattgtgaatctacttggagcctgcaccattggagggcccaccct gtcattacagaatattgttgctatggtgatcttttgaattttttgaga gaaaacgtgattcatttatttgttcaaagcaggaagatcatgcagaagct gcactttataagaatcttctgcattcaaaggagtcttcctgcagcgatag sactaatgagtacatggacatgaaacctggagtttcttatgttgtccca ccaaggccgacaaaaggagatctgtgagaataggctcatacatagaaaga gatgtgactcccgccatcatggaggatgacgagttggccctagacttag agacttgctgagcttttcttaccaggtggcaaagggcatggctttcctcc cctccaagaattgtattcacagagacttggcagccagaaatatcctcctt actcatggtcggatcacaaagatttgtgattttggtctagccagagacat caagaatgattctaattatgtggttaaaggaaacgctcgactacctgtg.a agtggatggcacctgaaagcattttcaactgtgtatacacgtttgaaagt acgtctggtcctatgggatttttctttgggagctgttctctttaggaad cageccctatcctggaatgccggtcgattctaagttctacaagatgatca aggaaggcttccggatgctcagccctgaacacgcacctgctgaaatgtat jacataatgaagacttgctgggatgcagatcccctaaaaagaccaacatt aagcaaattgttcagctaattgagaagcagatttcagagagcaccaat atatttactccaacttagcaaactgcagccccaaccgacagaagcccgtg gtagaccattctgtgcggatcaattctgtcggcagcaccgcttcctccto ccagcctctgcttgtgcacgacgatgtctga

The protein and cDNA sequences for mature human mTOR are shown below.

Mature Human mTOR Protein (SEQ ID NO: 65) mlgtgpaaat taattssnvs vlqqfasglk srneetraka akelqhyvtm elremsqees trfydqlnhh ifelvsssda nerkggilai asligveggn atrigrfany lrnllpsndp vvmemaskai grlamagdtf taeyvefevk ralewlgadr negrrhaavl vlrelaisvp tfffqqvqpf fdnifvavwd pkqairegav aalraclilt tqrepkemqk pqwyrhtfee aekgfdetla kekgmnrddr ihgallilne lvrissmege rlreemeeit qqqlvhdkyo kdlmgfgtkp rhitpftsfq avqpqqsnal vgllgysshq glmgfgtsps pakstlvesr ccrdlmeekf dqvcqwvlkc rnsknsliqm tilnllprla afrpsaftdt qylqdtmnhv lscvkkeker taafqalgll svavrsefkv ylprvldiir aalppkdfah krqkamqvda tvftcismla ramgpgigqd ikellepmla vglspaltav lydlsrqipq lkkdiqdgll kmlslvlmhk plrhpgmpkg lahqlaspgl ttlpeasdvg sitlalrtlg sfefeghslt qfvrhcadhf Insehkeirm eaartcsrll tpsihlisgh ahvvsqtavq vvadvlskll wgitdpdpd irycvlasld erfdahlaqa enlqalfval ndqvfeirel aictvgrlss mnpafvmpfl rkmliqilte lehsgigrik eqsarmlghl vsnaprlirp ymepilkali 1klkdpdpdp npgvinnvla tigelaqvsg lemrkwvdel fiiimdmlqd ssllakrqva lwtlgqlvas tgyvvepyrk yptllevlln flkteqnqgt rreairvlgl lgaldpykhk vnigmidqr dasavslses kssqdssdys tsemlvnmgn lpldefypav smvalmrifr dqslshhhtm vvqaitfifk slglkcvqfl pqvmptflnv irvcdgaire flfqqlgmlv sfvkshirpy mdeivtlmre fwvmntsiqs tiilliegiv valggefkly lpqliphmlr vfmhdnspgr ivsikllaai qlfganlddy lhlllppivk lfdapeaplp srkaaletvd rltesldftd yasriihpiv rtldqspelr stamdtlssl vfqlgkkyqi fipmvnkvlv rhrinhqryd vlicrivkgy tladeeedpl iyqhrmlrsg qgdalasgpv etgpmkklhv stinlqkawg aarrvskddw lewlrrlsle llkdssspsl rscwalaqay npmardlfna afvscwseln edqqdelirs ielaltsqdi aevtqtl1nl aefmehsdkg plplrddngi vllgeraakc rayakalhyk elefqkgptp aileslisin nklqqpeaaa gvleyamkhf geleiqatwy eklhewedal vaydkkmdtn kddpelmlgr mrclealgew gqlhqqccek wtlvndetqa kmarmaaaaa wglgqwdsme eytcmiprdt hdgafyravl alhqdlfsla qqcidkardl ldaeltamag esysraygam vschmlsele eviqyklvpe rreiirqiww erlqgcqriv edwqkilmvr slvvsphedm rtwlkyaslc gksgrlalah ktlvlllgvd psrqldhplp tvhpqvtyay mknmwksark idafqhmqhf vqtmqqqaqh aiatedqqhk qelhklmard flklgewqln lqginestip kvlqyysaat ehdrswykaw hawavmnfea vlhykhqnqa rdekkklrha sganitnatt aattaatatt tastegsnse seaestensp tpsplqkkvt edlsktllmy tvpavqgffr sislsrgnnl qdtlrvltlw fdyghwpdvn

OL ealvegvkai qidtwlqvip qliaridtpr plvgrlihql ltdigryhpq aliypltvas kstttarhna ankilknmce hsntlvqqam mvseelirva ilwhemwheg leeasrlyfg ernvkgmfev leplhammer gpqtlketsf nqaygrd1me aqewcrkymk sgnvkdltqa wdlyyhvfrr iskqlpqlts lelqyvspkl lmcrdlelav pgtydpnqpi iriqsiaps qvitskqrpr kltlmgsngh efvfllkghe dlrqdervmg lfglvntlla ndptslrknl sigryavipl stnsgligwv phcdtlhali rdyrekkkil lniehrimlr mapdydhltl mqkvevfeha vnntagddla kllwlkspss evwfdrrtny trslavmsmv gyilglgdrh psnlmldrls gkilhidfgd cfevamtrek fpekipfrlt rmltnamevt gldgnyrito htvmevlreh kdsvmavlea fvydpllnwr lmdtntkgnk rsrtrtdsys aggsveildg velgepahkk tgttvpesih sfigdglvkp ealnkkaiqi inrvrdkltg rdfshddtld vptqvellik qatshenlcq cyigwcpfw

Human mTOR cDNA (SEQ ID NO: 66)

atgcttgga accggacctg ccgccgccac caccgctgcc accacatcta gcaatgtgag cgtcctgcag cagtttgcca gtggcctaaa gagccggaat gaggaaacca gggccaaaga gggccaaagc gagatgagto aagaggagtc cgccaaggag ctccagcact atgtcaccat ggaactccga gagatgagtc aagaggagto tactcgctto tatgaccaac tgaaccatca catttttgaa ttggtttcca gctcagatga caatgagagg aaaggtggca tcttggccat agctagcctc ataggagtgg aaggtgggaa tgccacccga attggcagat ttgccaacta tcttcggaac ctcctcccct ccaatgaccc agttgtcatg gaaatggcat ccaaggccat tggccgtctt gccatggcag gggacacttt taccgctgag tacgtggaat ttgaggtgaa gcgagccctg gaatggctgg gtgctgaccg caatgagggc cggagacatg cagctgtcct ggttctccgt gagctggcca tcagcgtccc taccttcttc ttccagcaag tgcaaccctt ctttgacaac atttttgtgg ccgtgtggga ccccaaacag gccatccgtg agggagctgt agccgccctt cgtgcctgtc tgattctcac aacccagcgt gagccgaagg agatgcagaa gcctcagtgg tacaggcaca catttgaaga agcagagaag ggatttgatg agaccttgga caaagagaag ggcatgaato gggatgatcg gatccatgga gccttgttga tccttaacga gctggtccga atcagcagca tggagggaga gcgtctgaga gaagaaatgg aagaaatcac acagcagcag ctggtacacg acaagtactg caaagatctc atgggcttcg gaacaaaacc tcgtcacatt acccccttca ccagtttcca ggctgtacag ccccagcagt caaatgcctt ggtggggctg ctggggtaca gctctcacca aggcctcatg ggatttggga cctcccccag tccagctaag tccaccctgg tggagagccg gtgttgcaga gacttgatgg aggagaaatt tgatcaggtg tgccagtggg tgctgaaatg caggaatage aagaactcgc tgatccaaat gacaatcctt aatttgttga cccgcttgga tgcattccga ccttctgcct tcacagatac ccagtatctc caagatacca tgaaccatgt III cctaagctgt gtcaagaagg agaaggaacg tacagcggcc ttccaagccc tggggctact ttctgtggct gtgaggtctg agtttaaggt ctatttgcct cgcgtgctgg acatcatccg agcggccctg cccccaaagg acttcgccca taagaggcag aaggcaatgc aggtggatga cacagtctto acttgcatca gcatgctggc tcgagcaatg gggccaggca tccagcagga tatcaaggag ctgctggagc ccatgctggc agtgggacta agccctgccc tcactgcagt gctctacgac ctgagccgtc agattccaca gctaaagaag gacattcaag atgggctact gaaaatgctg tccctggtcc ttatgcacaa accccttcgc cacccaggca tgcccaaggg cctggcccat cagctggcct ctcctggcct cacgaccctc cctgaggcca gcgatgtggg cagcatcact cttgccctcc gaacgcttgg cagctttgaa tttgaaggca actctctgac ccaatttgtt cgccactgtg cggatcattt cctgaacagt gagcacaagg agatccgcat ggaggctgcc cgcacctgct cccgcctgct cacaccctcc atccacctca tcagtggcca tgctcatgtg gttagccaga ccgcagtgca agtggtggca gatgtgctta gcaaactgct cgtagttggg ataacagato ctgaccctga cattcgctac tgtgtcttgg cgtccctgga cgagcgcttt gatgcacacc tggcccaggc ggagaacttg caggccttgt ttgtggctct gaatgaccag gtgtttgaga tccgggagct ggccatctgc actgtgggcc gactcagtag catgaaccct gcctttgtca tgcctttcct gcgcaagatg ctcatccaga ttttgacaga gttggagcac agtgggattg gaagaatcaa agagcagagt gcccgcatga gcccgcatgc tggggcacct ggtctccaat gccccccgac tcatccgccc ctacatggag cctattctga aggcattaat tttgaaactg aaagatccag accctgatcc aaacccaggt gtgatcaata atgtcctggc aacaatagga gaattggcad aggttagtgg cctggaaatg aggaaatggg ttgatgaact ttttattato atcatggaca tgctccagga ttcctctttg ttggccaaaa ggcaggtgga tctgtggacc ctgggacagt tggtggccag cactggctat gtagtagage cctacaggaa gtaccctact ttgcttgagg tgctactgaa ttttctgaag actgagcaga accagggtag acgcagagag gccatccgtg tgttagggct tttaggggct ttggatcctt acaagcacaa agtgaacatt ggcatgatag accagtcccg ggatgcctct gctgtcagcc tgtcagaatc caagtcaagt caggattcct ctgactatag cactagtgaa atgctggtca acatgggaaa cttgcctctg gatgagttct acccagctgt gtccatggtg gccctgatgc ggatcttccg agaccagtca ctctctcatc atcacaccat ggttgtccag gccatcacct tcatcttcaa gtccctggga ctcaaatgtg tgcagttcct gccccaggtc atgcccacgt tccttaacgt cattcgagto tgtgatgggg ccatccggga atttttgtta cagcagctgg gaatgttggt gtcctttgtg aagagccaca tcagacctta tatggatgaa atagtcacco tcatgagaga attctgggtc atgaacacct caattcagag cacgatcatt cttctcattg agcaaattgt ggtagctctt gggggtgaat ttaagctcta cctgccccag ctgatcccac acatgctgcg tgtcttcatg catgacaaca gcccaggccg cattgtctct atcaagttac tggctgcaat ccagctgttt ggcgccaacc tggatgacta cctgcattta ctgctgcctc ctattgttaa gttgtttgat gcccctgaag ctccactgcc atctcgaaag gcagcgctag agactgtgga ccgcctgacg gagtccctgg atttcactga ctatgcctcc cggatcatto accctattgt tcgaacactg gaccagagcc cagaactgcg ctccacagcc atggacacgo tgtcttcact tgtttttcag ctggggaaga agtaccaaat tttcattcca atggtgaata aagttctggt gcgacaccga atcaatcatc agcgctatga tgtgctcatc tgcagaattg tcaagggata

WO wo 2021/247604 PCT/US2021/035285

cacacttgct gatgaagagg aggatccttt gatttaccag catcggatga catcggatgc ttaggagtgg ccaaggggat gcattggcta gtggaccagt ggaaacagga cccatgaaga aactgcacgt cagcaccato cagcaccatc aacctccaaa aggcctgggg cgctgccagg agggtctcca aagatgactg gctggaatgg ctgagacggc tgagcctgga gctgctgaag gactcatcat cgccctccct gcgctcctgc tgggccctgg cacaggccta caacccgatg gccagggatc tcttcaatga tgcatttgtg tcctgctggt ctgaactgaa tgaagatcaa caggatgago caggatgagc tcatcagaag catcgagttg gccctcacct cacaagacat cgctgaagtc acacagacco acacagaccc tcttaaactt ggctgaatto ggctgaattc atggaacaca gtgacaaggg ccccctgcca ctgagagatg acaatggcat tgttctgctg ggtgagagag ctgccaagtg ccgagcatat gccaaaacca gccaaagcac tacactacaa agaactggag ttccagaaag gccccacccc tgccattcta gaatctctca tcagcattaa taataagcta cagcagccgg aggcagcggc cggagtgtta gaatatgcca tgaaacactt tggagagctg gagatccagg ctacctggta tgagaaactg cacgagtggg aggatgccct tgtggcctat gacaagaaaa tggacaccaa caaggacgac ccagagctga tgctgggccg catgcgctgc ctcgaggcct tgggggaatg gggtcaactc caccagcagt gctgtgaaaa gtggaccctg gttaatgatg gtggaccctg gttaatgatg agacccaage agacccaagc caagatggcc caagatggcc cggatggctg cggatggctg ctgcagctgc ctgcagctgc atggggttta ggtcagtggg acagcatgga agaatacacc tgtatgatcc ctcgggacac ccatgatggg gcattttata gagctgtgct ggcactgcat caggacctct tctccttggc acaacagtgc attgacaagg ccagggacct gctggatgct gaattaactg cgatggcagg agagagttac agagagttac agtcgggcat agtcgggcat atggggccat atggggccat ggtttcttgc ggtttcttgc cacatgctgt cacatgctgt ccgagctgga ccgagetgga ggaggttatc cagtacaaac ttgtccccga gcgacgagag atcatccgcc agatctggtg ggagagactg cagggctgcc cagggetgcc agcgtatcgt agaggactgg cagaaaatcc ttatggtgcg gtcccttgtg gtcagccctc atgaagacat gagaacctgg ctcaagtatg caagcctgtg cggcaagagt ggcaggctgg ctcttgctca taaaacttta gtgttgctcc tgggagttga tccgtctcgg caacttgace caacttgacc atcctctgcc aacagttcac cctcaggtga cctatgccta

catgaaaaac atgtggaaga gtgcccgcaa gatcgatgcc ttccagcaca tgcagcattt tgtccagacc atgcagcaac aggcccagca tgccatcgct actgaggace actgaggacc agcagcataa gcaggaactg cacaagctca tggcccgatg cttcctgaaa cttggagagt ggcagctgaa tctacaggga tctacagggc atcaatgaga gcacaatccc caaagtgctg cagtactaca gcgccgccao gcgccgccac agagcacgac cgcagctggt acaaggcctg gcatgcgtgg gcagtgatga acttcgaage acttcgaagc

tgtgctacac tacaaacata tacaaacatc agaaccaaga agaaccaagc ccgcgatgag aagaagaaac tgcgtcatga tgcgtcatgc cagcggggcc aacatcacca acgccaccac tgccgccacc acggccgcca ctgccaccac cactgccage cactgccagc accgagggca gcaacagtga gagcgaggcc gagagcaccg agaacagccc caccccatcg caccccateg ccgctgcaga agaaggtcac tgaggatctg tccaaaaccc tcctgatgta cacggtgcct gccgtccagg gcttcttccg ttccatctcc ttgtcacgag gcaacaacct

ccaggataca ctcagagtto ctcagagttc tcaccttatg gtttgattat ggtcactggc cagatgtcaa tgaggcctta gtggaggggg tgaaagccat ccagattgat acctggctac aggttatacc tcagctcatt gcaagaattg atacgcccag acccttggtg ggacgtctca ttcaccagct tctcacagac attggtcggt accaccccca ggccctcatc tacccactga cagtggcttc cagtggctto taagtctacc acgacagccc ggcacaatgo ggcacaatgc agccaacaag attctgaaga acatgtgtga

gcacagcaac accctggtcc agcaggccat gatggtgage gatggtgagc gaggagctga tccgagtgga tccgagtggc catcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggace gtcaaggacc tcacccaage tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag cagctgcctc agctcacatc cttagagctg caatatgttt ccccaaaact tctgatgtgc cgggaccttg aattggctgt gccaggaaca tatgacccca accagccaat cattcgcatt cagtccatag caccgtcttt gcaagtcatc acatccaage acatccaagc agaggccccg agaggecccg gaaattgaca cttatgggca gcaacggaca tgagtttgtt ttccttctaa aaggccatga agatctgcgc caggatgage caggatgagc gtgtgatgca gctcttcggc ctggttaaca cccttctggc caatgaccca acatctcttc ggaaaaacct cagcatccag agatacgctg tcatcccttt atcgaccaac tcgggcctca ttggctgggt tccccactgt gacacactgc acgccctcat ccgggactac agggagaaga agaagatcct tctcaacatc gagcatcgca tcatgttgcg gatggctccg gactatgacc acttgactct gatgcagaag gtggaggtgt ttgagcatgc cgtcaataat acagctgggg acgacctgga acgacctggc caagctgctg tggctgaaaa gccccagctc cgaggtgtgg tttgaccgaa gaaccaatta tacccgttct ttagcggtca tgtcaatggt tgggtatatt ttaggcctgg gagatagaca cccatccaac ctgatgctgg accgtctgag tgggaagato tgggaagatc ctgcacattg actttgggga ctgctttgag gttgctatga cccgagagaa gtttccagag aagattccat ttagactaac aagaatgttg accaatgcta tggaggttac aggcctggat ggcaactaca gaatcacatg ccacacagtg atggaggtga atggaggtgc tgcgagagca caaggacagt gtcatggccg tgctggaago tgctggaagc ctttgtctat gaccccttgc tgaactggag gctgatggac acaaatacca aaggcaacaa gcgatcccga acgaggacgg attcctacto attcctactc tgctggccag tcagtcgaaa ttttggacgg tgtggaactt ggagagccag cccataagaa aacggggacc acagtgccag aatctattca ttctttcatt ggagacggtt tggtgaaacc agaggcccta aataagaaag ctatccagat tattaacagg gttcgagata agctcactgg tcgggacttc tctcatgatg acactttgga tgttccaacg caagttgaga caagttgagc tgctcatcaa acaagcgaca tcccatgaaa acctctgcca gtgctatatt ggctggtgcc ctttctggta a

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of SREBP1 are available from Novus Biologicals and

Abcam. The protein and cDNA sequences for mature human SREBP1 are shown below.

Mature Human SREBP1 Protein (SEQ ID NO: 67)

MDEPPFSEAALEQALGEPCDLDAALLTDIEDMLQLINNQDSDFPGLFDPPYAGSO AGGTDPASPDTSSPGSLSPPPATLSSSLEAFLSGPQAAPSPLSPPQPAPTPLKMYPS AGGTDPASPDTSSPGSLSPPPATLSSSLEAFLSGPQAAPSPLSPPQPAPTPLKMYPS MPAFSPGPGIKEESVPLSILQTPTPQPLPGALLPQSFPAPAPPQFSSTPVLGYPSPI GFSTGSPPGNTQQPLPGLPLASPPGVPPVSLHTQVQSVVPQQLLTVTAAPTAAPV

2021/24764 oM WO 2021/247604 PCT/US2021/035285

TTTVTSQIQQVPVLLQPHFIKADSLLLTAMKTDGATVKAAGLSPLVSGTTVQTGI LPTLVSGGTILATVPLVVDAEKLPINRLAAGSKAPASAQSRGEKRTAHNAIEKRY RSSINDKIIELKDLVVGTEAKLNKSAVLRKAIDYIRFLQHSNQKLKQENLSLRTAV HKSKSLKDLVSACGSGGNTDVLMEGVKTEVEDTLTPPPSDAGSPFQSSPLSLGSI GSGSGGSGSDSEPDSPVFEDSKAKPEQRPSLHSRGMLDRSRLALCTLVFLCLSCN PLASLLGARGLPSPSDTTSVYHSPGRNVLGTESRDGPGWAQWLLPPVVWLLNGL VLVSLVLLFVYGEPVTRPHSGPAVYFWRHRKQADLDLARGDFAQAAQQLWI CRALGRPLPTSHLDLACSLLWNLIRHLLQRLWVGRWLAGRAGGLQQDCALRVD SASARDAALVYHKLHQLHTMGKHTGGHLTATNLALSALNLAECAGDAVSVA TLAEIYVAAALRVKTSLPRALHFLTRFFLSSARQACLAQSGSVPPAMQWLCHPV GHRFFVDGDWSVLSTPWESLYSLAGNPVDPLAQVTQLFREHLLERALNCVTQPN PSPGSADGDKEFSDALGYLQLLNSCSDAAGAPAYSFSISSSMATTTGVDPVAKW WASLTAVVIHWLRRDEEAAERLCPLVEHLPRVLQESERPLPRAALHSFKAARA LGCAKAESGPASLTICEKASGYLQDSLATTPASSSIDKAVQLFLCDLLLVVRTSL WRQQQPPAPAPAAQGTSSRPQASALELRGFQRDLSSLRRLAQSFRPAMRRVFLH, ATARLMAGASPTRTHQLLDRSLRRRAGPGGKGGAVAELEPRPTRREHAEA ASCYLPPGFLSAPGQRVGMLAEAARTLEKLGDRRLLHDCQQMLMRLGGGTTVT SS

Human SREBP1 cDNA (SEQ ID NO: 68)

tggacgagccacccttcagcgaggcggctttggagcaggcgctggg gccgtgcgatctggacgcggcgctgctgaccgacatcgaagacatgctto agcttatcaacaaccaagacagtgacttccctggcctatttgacccaco atgctgggagtggggcagggggcacagaccctgccagccccgatacca ctccccaggcagcttgtctccacctcctgccacattgagctcctctctt hagccttcctgagcgggccgcaggcagcgccctcacccctgtcccctcc cagcctgcacccactccattgaagatgtacccgtccatgcccgctttcto ccctgggcctggtatcaaggaagagtcagtgccactgagcatcctgcad cccccaccccacagcccctgccaggggccctcctgccacagagcttc gecccagccccaccgcagttcagctccacccctgtgttaggctaccco :cctccgggaggcttctctacaggaagccctcccgggaacacccagcag cgctgcctggcctgccactggcttccccgccaggggtcccgcccgtctc ttgcacacccaggtccagagtgtggtcccccagcagctactgacagtcad gctgcccccacggcagcccctgtaacgaccactgtgacctcgcagatc

gcaggtcccggtcctgctgcagccccacttcatcaaggcagactcgct cttctgacagccatgaagacagacggagccactgtgaaggcggcaggtct wo 2021/247604 WO PCT/US2021/035285 cagtcccctggtctctggcaccactgtgcagacagggcctttgccgacco tggtgagtggcggaaccatcttggcaacagtcccactggtcgtagatgcg gagaagctgcctatcaaccggctcgcagctggcagcaaggccccggectc tgcccagagccgtggagagaagcgcacagcccacaacgccattgagaag gctaccgctcctccatcaatgacaaaatcattgagctcaaggatctggtg gtgggcactgaggcaaagctgaataaatctgctgtcttgcgcaaggccat cgactacattcgctttctgcaacacagcaaccagaaactcaagcaggaga acctaagtctgcgcactgctgtccacaaaagcaaatctctgaaggatcto gtgtcggcctgtggcagtggagggaacacagacgtgctcatggagggcgt gaagactgaggtggaggacacactgaccccacccccctcggatgctggct pacctttccagagcagccccttgtcccttggcagcaggggcagtggcag ggtggcagtggcagtgactcggagcctgacagcccagtctttgaggaca caaggcaaagccagagcagcggccgtctctgcacagccggggcatgctgg accgctcccgcctggccctgtgcacgctcgtcttcctctgcctgtcctg aaccccttggcctccttgctgggggcccgggggcttcccagcccctcaga taccaccagcgtctaccatagccctgggcgcaacgtgctgggcaccgaga gcagagatggccctggctgggcccagtggctgctgcccccagtggtctgg ctgctcaatgggctgttggtgctcgtctccttggtgcttctctttgtcta cggtgagccagtcacacggccccactcaggccccgccgtgtacttctgga ggcatcgcaagcaggctgacctggacctggcccggggagactttgcccag gctgcccagcagctgtggctggccctgcgggcactgggccggcccctgcc cacctcccacctggacctggcttgtagcctcctctggaacctcatccgto acctgctgcagcgtctctgggtgggccgctggctggcaggccgggcaggg agacgcagecctggtctaccataagctgcaccagctgcacaccatgggg agcacacaggcgggcacctcactgccaccaacctggcgctgagtgcccto aacctggcagagtgtgcaggggatgccgtgtctgtggcgacgctggccgal gatctatgtggcggctgcattgagagtgaagaccagtctcccacgggcct gcattttctgacacgcttcttcctgagcagtgcccgccaggcctgccte gcacagagtggctcagtgcctcctgccatgcagtggctctgccaccccgt gggccaccgtttcttcgtggatggggactggtccgtgctcagtaccccat gggagagcctgtacagcttggccgggaacccagtggaccccctggcccal gtgactcagctattccgggaacatctcttagagcgagcactgaactgtgt gacccagcccaaccccagccctgggtcagctgatggggacaaggaattct cggatgccctcgggtacctgcagctgctgaacagctgttctgatgctgcg jgggctcctgcctacagcttctccatcagttccagcatggccaccaccal cggcgtagacccggtggccaagtggtgggcctctctgacagctgtggtga tccactggctgcggcgggatgaggaggcggctgagcggctgtgcccgctg gtggagcacctgccccgggtgctgcaggagtctgagagacccctgcccad ggcagctctgcactccttcaaggctgcccgggccctgctgggctgtgcca aggcagagtctggtccagccagcctgaccatctgtgagaaggccagtggg tacctgcaggacagcctggctaccacaccagccagcagctccattgacaa ggccgtgcagctgttcctgtgtgacctgcttcttgtggtgcgcaccagcc tgtggcggcagcagcagcccccggccccggccccagcagcccagggcacc agcagcaggccccaggcttccgcccttgagctgcgtggcttccaacggg cctgagcagcctgaggcggctggcacagagcttccggcccgccatgcgo gggtgttcctacatgaggccacggcccggctgatggcgggggccagccco acacggacacaccagctcctcgaccgcagtctgaggcggcgggcaggcco cggtggcaaaggaggcgcggtggcggagctggagccgcggcccacgcggc gggagcacgcggaggccttgctgctggcctcctgctacctgccccccggc

WO wo 2021/247604 PCT/US2021/035285

ttcctgtcggcgcccgggcagcgcgtgggcatgctggctgaggcggcgcg cacactcgagaagcttggcgatcgccggctgctgcacgactgtcagcaga tgctcatgcgcctgggcggtgggaccactgtcacttccagctag

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of IFN-y are available from R&D Systems, Thermo Fisher

Scientific, Abcam, Enzo Life Sciences, and RayBiotech. The protein and cDNA

sequences for mature human IFN-y are shown below.

Mature Human IFN-y (SEQ ID NO: 69) qdpyvke aenlkkyfna ghsdvadngt lflgilknwk eesdrkimqs qivsfyfklf knfkddqsiq ksvetikedm nvkffnsnkk krddfekltn ysvtdlnvqr kaiheliqvm aelspaaktg krkrsqmlfr g

Human IFN-y cDNA (SEQ ID NO: 70)

caggac ccatatgtaa aagaagcaga aaaccttaag aaatatttta atgcaggtca ttcagatgta gcggataatg gaactctttt cttaggcatt ttgaagaatt ggaaagagga gagtgacaga aaaataatga agagccaaat tgtctccttt tacttcaaac tttttaaaaa ctttaaagat gaccagagca tccaaaagag tgtggagacc atcaaggaag acatgaatgt caagttttto aatagcaaca aaaagaaacg agatgactto gaaaagctga ctaattatto ggtaactgac ttgaatgtcc aacgcaaaga aatacatgaa ctcatccaag tgatggctga actgtcgcca gcagctaaaa cagggaagcg aaaaaggagt cagatgctgt ttcgaggt

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of granzyme B are available from RayBiotech, Thermo

Fisher Scientific, and R&D Systems. The protein and cDNA sequences for mature

human granzyme B are shown below.

Mature Human Granzyme B (SEQ ID NO: 71) iiggheakph srpymaylmi wdqks1krcg gflirddfvl taahcwgssi nvtlgahnik eqeptqgfip vkrpiphpay npknfsndim llqlerkakr travqplrlp snkaqvkpgg tcsvagwgqt aplgkhshtl qevkmtvqed rkcesdlrhy ydstielcvg dpeikktsfk gdsggplvcn kvaqgivsyg rnngmpprac tkvssfvhwi kktmkry

WO wo 2021/247604 PCT/US2021/035285

Human Granzyme B cDNA (SEQ ID NO: 72)

atcatcgggg gacatgaggc caagccccac tcccgcccct acatggctta tcttatgatc tgggatcaga agtctctgaa gaggtgcggt ggcttcctga tacgagacga cttcgtgctg acagctgctc actgttgggg aagctccata aatgtcacct tgggggccca caatatcaaa gaacaggage cgacccagca gtttatccct gtgaaaagac ccatccccca tccagcctat aatcctaaga acttctccaa cgacatcatg ctactgcage ctactgcagc tggagagaaa ggccaagcgg accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacactc acacacacta caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata aagaaaacca tgaaacgcta C

Non-limiting examples of commercial ELISA assays that can be used to

determine the expression level of MYC are available from Invitrogen, LSBio, Biocodon

Technologies, and Elisa Genie. The protein and cDNA sequences for mature human

MYC are shown below.

Human Myc Protein (SEQ ID NO: 329)

mdffrvvenq qppatmplnv sftnrnydld ydsvqpyfyc deeenfyqqq qqselqppap sediwkkfel lptpplspsr rsglcspsyv avtpfslrgd ndggggsfst adqlemvtel lggdmvnqsf icdpddetfi kniiiqdcmw sgfsaaaklv seklasyqaa rkdsgspnpa rghsvcstss lylqdlsaaa secidpsvvf pyplndsssp kscasqdssa fspssdslls stesspqgsp eplvlheetp pttssdseee qedeeeidvv svekrqapgk rsesgspsag ghskpphspl vlkrchvsth qhnyaappst rkdypaakrv kldsvrvlrq isnnrkctsp rssdteenvk rrthnvlerq rrnelkrsff alrdqipele nnekapkvvi lkkatayils vqaeeqklis eedllrkrre qlkhkleqlr nsca

Human Myc cDNA (SEQ ID NO: 330) ctggatt tttttcgggt agtggaaaac cagcageetc ccgcgacgat gcccctcaac gttagcttca ccaacaggaa ctatgaccto gactacgact cggtgcagcc gtatttctac tgcgacgagg aggagaactt ctaccagcag cagcagcaga gcgagctgca gcccccggcg cccagcgagg atatctggaa gaaattcgag ctgctgccca ccccgcccct gtcccctagc

cgccgctccg cgccgetccg ggctctgctc gccctcctac gttgcggtca cacccttctc ccttcgggga

WO wo 2021/247604 PCT/US2021/035285

gacaacgacg gcggtggcgg gagcttctco gagcttctcc acggccgacc agctggagat ggtgaccgag ctgctgggag gagacatggt gaaccagagt ttcatctgcg acccggacga cgagacctto cgagaccttc atcaaaaaca tcatcatcca ggactgtatg tggagcggct tctcggccgc cgccaagctc gtctcagaga agctggcctc ctaccaggct gcgcgcaaag acagcggcag cccgaacccc

gcccgcggcc acagcgtctg ctccacctca agcttgtacc tgcaggatct gagcgccgcc gcctcagagt gcatcgaccc ctcggtggtc ttcccctacc ctctcaacga cagcagctcg cccaaagtcct gcgcctcgca agactccagc cccaagtcct gcgcctcgca agactccage gccttctctc gccttctctc cgtcctcgga cgtcctcgga ttctctgctc ttctctgctc tcctcgacgg agtcctcccc gcagggcage gcagggcagc cccgagcccc tggtgctcca tgaggagaca ccgcccacca ccagcagcga ctctgaggag gaacaagaag atgaggaaga aatcgatgtt gtttctgtgg aaaagaggca ggctcctggc aaaaggtcag agtctggato accttctgct ggaggccaca gcaaacctcc tcacagccca ctggtcctca agaggtgcca cgtctccaca catcagcaca actacgcago gcctccctca actcggaagg actatcctgc tgccaagagg gtcaagttgg acagtgtcag agtcctgaga cagatcagca acaaccgaaa atgcaccago cccaggtcct cggacaccga ggagaatgto ggagaatgtc aagaggcgaa cacacaacgt cttggagcga cttggagcgc cagaggagga acgagctaaa acggagcttt tttgccctgc gtgaccagat cccggagttg gaaaacaatg aaaaggcccc caaggtagtt atccttaaaa aagccacago atacatcctg tccgtccaag cagaggagca aaagctcatt tctgaagagg acttgttgcg gaaacgacga gaacagttga aacacaaact tgaacagcta cggaactctt gtgcgtaa

In some embodiments, activated NK cells (e.g., human activated NK cells) can

show increased (e.g., at least a 10% increase, at least a 20% increase, at least a 30%

increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least

a 70% increase, at least 80% increase, at least a 90% increase, at least a 100% increase, at

least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180%

increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at

least a 260% increase, at least a 280% increase, or at least a 300% increase) ability to kill

senescent cells (e.g., any of the senescent cells described herein) in a subject (e.g., any of

the subjects described herein) or in vitro as compared to resting NK cells (e.g., human

resting NK cells).

In some embodiments, activated NK cells (e.g., human activated NK cells) can

show about a 10% increase to about a 500% increase (or any of the subranges of this

range described herein) ability to kill senescent cells (e.g., any of the senescent cells

WO wo 2021/247604 PCT/US2021/035285

described herein) in a subject (e.g., any of the subjects described herein) or in vivo as

compared to resting NK cells (e.g., human resting NK cells).

In some embodiments, activated NK cells (e.g., human activated NK cells) can

show increased (e.g., at least a 10% increase, at least a 20% increase, at least a 30%

increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least

a 70% increase, at least 80% increase, at least a 90% increase, at least a 100% increase, at

least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180%

increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at

least a 260% increase, at least a 280% increase, or at least a 300% increase) cytotoxic

activity in a contact-cytotoxicity assay in the presence of an antibody that binds

specifically to an antigen present on a senescent or target cell, e.g., as compared to a

resting NK cell (e.g., human resting NK cells).

In some embodiments, activated NK cells (e.g., human activated NK cells) can

show increased (e.g., about a 10% increase to about a 500% increase, or any of the

subranges of this range described herein) cytotoxic activity in a contact-cytotoxicity

assay in the presence of an antibody that binds specifically to an antigen present on a

senescent or target cell, e.g., as compared to a resting NK cell (e.g., human resting NK

cells).

In some embodiments, an activated NK cell can be produced by a method that

includes obtaining a resting NK cell; and contacting the resting NK cell in vitro in a

liquid culture medium including one or more NK cell activating agent(s), where the

contacting results in the generation of the activated NK cells that are subsequently

administered to the subject. In some examples of these methods, the resting NK cell is an

autologous NK cell obtained from the subject. In some examples of these methods, the

resting NK cell is an autologous NK cell obtained from the subject. In some examples of

these methods, the resting NK cell is an haploidentical resting NK cells. In some

examples of these methods, the resting NK cell is an allogeneic resting NK cell. In some

examples of these methods, the resting NK cell is an artificial NK cell. In some examples

of any of these methods, the resting NK cell is a genetically-engineered NK cell carrying

a chimeric antigen receptor or recombinant T cell receptor.

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

In some examples of these methods, the liquid culture medium is a serum-free

liquid culture medium. In some embodiments of any of the methods described herein, the

liquid culture medium is a chemically-defined liquid culture medium. Some examples of

these methods further include isolating the activated NK cells (and optionally further

administering a therapeutically effective amount of the activated NK cells to a subject,

e.g., any of the subjects described herein).

In some embodiments of these methods, the contacting step is performed for a

period of about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2

hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days,

about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7

days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about

4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to

about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6

hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days,

about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6

days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about

3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to

about 18 days, about 12 hours to about 16 days, about 12 hours to about 14 days, about

12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8

days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to

about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12

hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about

1 day to about 16 days, about 1 day to about 15 days, about 1 day to about 14 days, about

1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1

day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day

to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to

about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days

to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2

days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2

days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days,

about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days,

about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days,

about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14

days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about

8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about

5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days to

about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days

to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days, about 6 days

to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6

days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about

6 days to about 7 days, about 7 days to about 18 days, about 7 days to about 16 days,

about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10

days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about

16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to

about 10 days, about 9 days to about 18 days, about 9 days to about 16 days, about 9 days

to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12

days to about 16 days, about 12 days to about 14 days, about 14 days to about 18 days,

about 14 days to about 16 days, or about 16 days to about 18 days.

NK Cell Activating Agents

Provided herein are methods that include the use or administration of one or more

NK cell activating agents. In some embodiments, an NK cell activating agent can be a

protein. In some embodiments, an NK cell activating agent can be a single-chain

chimeric polypeptide (e.g. any of the single-chain chimeric polypeptides described

herein), a multi-chain chimeric polypeptide (e.g. any of the multi-chain chimeric

polypeptides described herein, e.g., the exemplary type A and type B multi-chain

chimeric polypeptides described herein), an antibody, a recombinant cytokine or an

interleukin (e.g. any of the recombinant cytokines or interleukins described herein), and a

soluble interleukin or cytokine receptor (e.g. any of the soluble interleukin or cytokine

WO wo 2021/247604 PCT/US2021/035285

receptors described herein). In some embodiments, the NK cell activating agent can be a

small molecule (e.g., a glycogen synthase kinase-3 (GSK3) inhibitor, e.g., CHIR99021 as

described in Cichocki et al., Cancer Res. 77:5664-5675, 2017) or an aptamer.

In some embodiments of any of the one or more NK cell activating agents

provided herein, at least one of the one or more NK cell activating agent(s) results in

activation of one or more (e.g., two, three, four, five, six, seven, or eight) of: a receptor

for IL-2, a receptor for IL-7, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

18, a receptor for IL-21, a receptor for IL-33, CD16, CD69, CD25, CD59, CD352,

NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1 (e.g., in an immune cell, e.g., a human

immune cell, e.g., a human NK cell) as compared to the level of activation in the absence

of the one or more NK cell activating agent(s).

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-2 is a soluble IL-2 or an agonistic

antibody that binds specifically to an IL-2 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-7 is a soluble IL-7 or an agonistic

antibody that binds specifically to an IL-7 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-12 is a soluble IL-12 or an agonistic

antibody that binds specifically to an IL-12 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-15 is a soluble IL-15 or an agonistic

antibody that binds specifically to an IL-15 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-21 is a soluble IL-21 or an agonistic

antibody that binds specifically to an IL-21 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of a receptor for IL-33 is a soluble IL-33 or an agonistic

antibody that binds specifically to an IL-33 receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of CD16 is an agonistic antibody that binds specifically

to CD16.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of CD69 is an agonistic antibody that binds specifically

to CD69.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of CD25, CD59 is an agonistic antibody that binds

specifically to CD25, CD59.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of CD352 is an agonistic antibody that binds

specifically to CD352.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of NKp80 is an agonistic antibody that binds

specifically to NKp80.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of DNAM-1 is an agonistic antibody that binds

specifically to DNAM-1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of 2B4 is an agonistic antibody that binds specifically to

2B4.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of NKp30 is an agonistic antibody that binds

specifically to NKp30.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of NKp44 is an agonistic antibody that binds

specifically to NKp44.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of NKp46 is an agonistic antibody that binds

specifically to NKp46.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of NKG2D is an agonistic antibody that binds

specifically to NKG2D.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR2DS1 is an agonistic antibody that binds

specifically to KIT2DS1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR2DS2/3 is an agonistic antibody that binds

specifically to KIT2DS2/3.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR2DL4 is an agonistic antibody that binds

specifically to KIT2DL4.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR2DS4 is an agonistic antibody that binds

specifically to KIT2DS4.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR2DS5 is an agonistic antibody that binds

specifically to KIT2DS5.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in activation of KIR3DS1 is an agonistic antibody that binds

specifically to KIT3DS1.

In some embodiments of any of the one or more NK cell activating agents

provided herein, at least one (e.g., two, three, four, or five) of the one or more NK cell

activating agent(s) results in a decrease in the activation of one or more of: PD-1, a TGF-

receptor, TIGIT, CD1, TIM-3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/KLRD1,

KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 (e.g.,

in an immune cell, e.g., a human immune cell, e.g., a human NK cell) as compared to the

level of activation in the absence of the one or more NK cell activating agent(s).

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of a TGF-B receptor is a soluble TGF-B

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

receptor, an antibody that binds specifically to TGF-B, or an antagonistic antibody that

binds specifically to a TGF-B receptor.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of TIGIT is an antagonistic antibody

that binds specifically to TIGIT, a soluble TIGIT, or an antibody that binds specifically to

a ligand of TIGIT.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of CD1 is an antagonistic antibody that

binds specifically to CD1, a soluble CD1, or an antibody that binds specifically to a

ligand of CD1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of TIM-3 is an antagonistic antibody

that binds specifically to TIM-3, a soluble TIM-3, or an antibody that binds specifically

to a ligand of TIM-3.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of Siglec-7 is an antagonistic antibody

that binds specifically to Siglec-7, a soluble Siglec-7, or an antibody that binds

specifically to a ligand of Siglec-7.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of IRP-60 is an antagonistic antibody

that binds specifically to IRP-60, a soluble IRP-60, or an antibody that binds specifically

to a ligand of IRP-60.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of Tactile is an antagonistic antibody

that binds specifically to Tactile, a soluble Tactile, or an antibody that binds specifically

to a ligand of Tactile.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of IL 1R8 is an antagonistic antibody

that binds specifically to IL 1R8, a soluble IL 1R8, or an antibody that binds specifically to

a ligand of IL1R8.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of NKG2A/KLRD1 is an antagonistic

antibody that binds specifically to NKG2A/KLRD1, a soluble NKG2A/KLRD1, or an

antibody that binds specifically to a ligand of NKG2A/KLRD1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of KIR2DL1 is an antagonistic

antibody that binds specifically to KIR2DL1, a soluble KIR2DL1, or an antibody that

binds specifically to a ligand of KIR2DL1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of KIR2DL2/3 is an antagonistic

antibody that binds specifically to KIR2DL2/3, a soluble KIR2DL2/3, or an antibody that

binds specifically to a ligand of KIR2DL2/3.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of KIR2DL5 is an antagonistic

antibody that binds specifically to KIR2DL5, a soluble KIR2DL5, or an antibody that

binds specifically to a ligand of KIR2DL5.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of KIR3DL1 is an antagonistic

antibody that binds specifically to KIR3DL1, a soluble KIR3DL1, or an antibody that

binds specifically to a ligand of KIR3DL1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of KIR3DL2 is an antagonistic

antibody that binds specifically to KIR3DL2, a soluble KIR3DL2, or an antibody that

binds specifically to a ligand of KIR3DL2.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of ILT2/LIR-1 is an antagonistic

antibody that binds specifically to ILT2/LIR-1, a soluble ILT2/LIR-1, or an antibody that

binds specifically to a ligand of ILT2/LIR-1.

In some embodiments, the at least one of the one or more NK cell activating

agent(s) that results in a decrease in the activation of LAG2 is an antagonistic antibody

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

that binds specifically to LAG2, a soluble LAG2, or an antibody that binds specifically to

a ligand of LAG2.

Non-limiting examples of NK cell activating agents are described below and can

be used in any combination.

In some examples, an NK cell activating agents can be a soluble PD-1, a soluble

PD-L1, a soluble TIGIT, a soluble CD1, or a soluble TIM-3. Non-limiting examples of

soluble PD-1, PD-L1, TIGIT, CD1, and TIM-3 are provided below.

Human Soluble PD-1 (SEQ ID NO: 73)

pgwfldspdr pwnpptfspa llvvtegdna tftcsfsnts esfvlnwyrm spsnqtdkla afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt ylcgaislap kaqikeslra elrvterrae vptahpspsp rpagqfqtlv vgvvggllgs lvllvwvlav icsraargti garrtgqplk edpsavpvfs vdygeldfqw rektpeppvp cvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswpl

Human Soluble PD-L1 (SEQ ID NO: 74)

ftvtvpkdlyvv eygsnmtiec kfpvekqldl aalivyweme dkniiqfvhg eedlkvqhss yrqrarl1kd qlslgnaalq itdvklqdag vyrcmisygg adykritvkv napynkingr napynkinqr ilvvdpvtse heltcqaegy pkaeviwtss dhqvlsgktt ttnskreekl fnvtstlrin tttneifyct frrldpeenh lvilgaillo lgvaltfifr lrkgrmmdvk taelvipelp lahppnerth lvilgaillc kcgiqdtnsk kqsdthleet

Human Soluble TIGIT (SEQ ID NO: 75)

mmtgtiett gnisaekggs iilqchlsst taqvtqvnwe qqdqllaicn adlgwhisps fkdrvapgpg lgltlqsltv ndtgeyfciy htypdgtytg riflevless vaehgarfqi pllgamaatl vvictavivv valtrkkkal cgegrgedca rihsvegdlr rksagqeews psapsppgsc vqaeaapagl cgeqrgedca

elhdyfnvls yrslgncsff tetg wo 2021/247604 WO PCT/US2021/035285

Human Soluble CD1A (SEQ ID NO: 76)

nadglkep sfhvt wiasfynhsw kqnlvsgwls dlqthtwdsn sstivflcpw srgnfsneew keletlfrir tirsfegirr yahelqfeyp feiqvtggce 1hsgkvsgsf lqlayqgsdf vsfqnnswlp ypvagnmakh fckvlnqnqh

endithnlls dtcprfilgl ldagkahlqr qvkpeawlsh gpspgpghlq lvchvsgfyp kpvwvmwmrg eqeqqgtqrg dilpsadgtw ylratlevaa geaadlscrv khsslegqdi vlywehhssv gfiilavivp lllliglalw frkrcfc

Human Soluble TIM3 (SEQ ID NO: 77)

seveyraev gqnaylpcfy tpaapgnlvp VCWgkgacpv vcwgkgacpv fecgnvvlrt derdvnywts rywlngdfrk gdvsltienv tladsgiycc riqipgimnd ekfnlklvik pakvtpaptr qrdftaafpr mlttrghgpa etqtlgs1pd etqtlgslpd inltgistla inltqistla nelrdsrlan dlrdsgatir igiyigagic aglalalifg alifkwyshs kekiqnlsli slanlppsgl anavaegirs eeniytieen vyeveepney vyeveepneyycyvssrqqp ycyvssrqqpsqplgcrfam sqplgcrfam

In some embodiments, a soluble PD-1 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 73.

In some embodiments, a soluble PD-L1 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 74.

In some embodiments, a soluble TIGIT protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 75.

In some embodiments, a soluble CD1A protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 76.

In some embodiments, a soluble TIM3 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 77.

Recombinant Antibodies

In some examples, NK activating agent can be: an agonistic antibody that binds

specifically to an IL-2 receptor (see, e.g., those described in Gaulton et al., Clinical

Immunology and Immunopathology 36(1):18-29, 1985), an agonistic antibody that binds

specifically to an IL-7 receptor, an agonistic antibody that binds specifically to IL-12

receptor (see, e.g., those described in Rogge et al., J. Immunol. 162(7): 3926-3932, 1999),

an agonistic antibody that binds specifically to an IL-15 receptor, an agonistic antibody

that binds specifically to an IL-21 receptor (see, e.g., those described in U.S. Patent

Application Publication No. 2006/159655), an agonistic antibody that binds specifically

to an IL-33 receptor (see, e.g., those described in U.S. Patent Application Publication No.

2007/160579), an antagonistic antibody that binds specifically to PD-1 (see, e.g., those

described in U.S. Patent No. 7,521,051), an antibody that binds specifically to PD-L1

(see, e.g., those described in U.S. Patent No. 8,217,149), an antibody that binds

specifically to TGF-B, an antagonistic antibody that binds specifically to TGF-B receptor

(see, e.g., those described in European Patent Application Publication No. 1245676 A1),

an antagonistic antibody that binds specifically to TIGIT (see, e.g., those described in

WO 2017/053748), an antibody that binds specifically to a ligand of TIGIT (see, e.g.,

WO wo 2021/247604 PCT/US2021/035285

those described in WO 2011/127324), an antagonistic antibody that binds specifically to

CD1 (see, e.g., those described in Szalay et al., J. Immunol. 162(12):6955-6958, 1999),

an antibody that binds specifically to a ligand of CD1 (see, e.g., those described in Kain

et al., Immunity 41(4):543-554, 2014), an antagonistic antibody that binds specifically to

TIM-3 (see, e.g., those described in U.S. Patent Application Publication No.

2015/218274), an antibody that binds specifically to a ligand of TIM-3 (see, e.g., those

described in U.S. Patent Application Publication No. 2017/283499), an agonistic

antibody that binds specifically to CD69 (see, e.g., those described in Moretta et al.,

Journal of Experimental Medicine 174:1393, 1991), an agonistic antibody that binds

specifically to CD25, CD59, an agonistic antibody that binds specifically to CD352 (see,

e.g., those described in Yigit et al., Oncotarget 7:26346-26360, 2016), an agonistic

antibody that binds specifically to NKp80 (see, e.g., those described in Peipp et al.,

Oncotarget 6:32075-32088, 2015), an agonistic antibody that binds specifically to

DNAM-1, an agonistic antibody that binds specifically to 2B4 (see, e.g., those described

in Sandusky et al., European J. Immunol. 36:3268-3276, 2006), an agonistic antibody

that binds specifically to NKp30 (see, e.g., those described in Kellner et al.,

OncoImmunology 5:1-12,2016), an agonistic antibody that binds specifically to NKp44,

an agonistic antibody that binds specifically to NKp46 (see, e.g., those described in

Xiong et al., J. Clin. Invest. 123:4264-4272, 2013), an agonistic antibody that binds

specifically to NKG2D (see, e.g., those described in Kellner et al., Oncolmmunology 5:1-

12, 2016), an agonistic antibody that binds specifically to KIR2DS1 (see, e.g., those

described in Xiong et al., J. Clin. Invest. 123:4264-4272, 2013), an agonistic antibody

that binds specifically to KIR2Ds2/3 (see, e.g., those described in Borgerding et al., Exp.

Hematology 38:213-221, 2010), an agonistic antibody that binds specifically to KIR2DL4

(see, e.g., those described in Miah et al., J. Immunol. 180:2922-32, 2008), an agonistic

antibody that binds specifically to KIR2DS4 (see, e.g., those described in Czaja et al.,

Genes and Immunity 15:33-37, 2014), an agonistic antibody that binds specifically to

KIR2DS5 (see, e.g., those described in Czaja et al., Genes and Immunity 15:33-37, 2014),

an agonistic antibody that binds specifically to KIR3DS1 (see, e.g., those described in

Czaja et al., Genes and Immunity 15:33-37, 2014), an antagonistic antibody that binds wo 2021/247604 WO PCT/US2021/035285 specifically to Siglec-7 (see, e.g., those described in Hudak et al., Nature Chemical

Biology 10:69-75, 2014), an antagonistic antibody that binds specifically to IRP60 (see,

e.g., those described in Bachelet et al., J. Biol. Chem. 281:27190-27196, 2006), an

antagonistic antibody that binds specifically to Tactile (see, e.g., those described in

Brooks et al., Eur. J. Cancer 61 (Suppl. 1): S189, 2016), an antagonistic antibody that

binds specifically to IL1R8 (see, e.g., those described in Molgora et al., Frontiers

Immunol. 7:1, 2016), an antagonistic antibody that binds specifically to NKG2A/KLRD1

(see, e.g., those described in Kim et al., Infection Immunity 76:5873-5882, 2008), an

antagonistic antibody that binds specifically to KIR2DL1 (see, e.g., those described in

Weiner et al., Cell 148:1081-1084, 2012), an antagonistic antibody that binds specifically

to KIR2DL2/3 (see, e.g., those described in Weiner et al., Cell 148:1081-1084, 2012), an

antagonistic antibody that binds specifically to KIR2DL5 (see, e.g., those described in US

9,067,997), and an antagonistic antibody that binds specifically KIR3DL1 (see, e.g.,

those described in US 9,067,997), an antagonistic antibody that binds specifically to

KIR3DL2 (see, e.g., those described in US 9,067,997), an antagonistic antibody that

binds specifically to ILT2/LIR-1 (see, e.g., those described in US 8,133,485), and an

antagonistic antibody that binds specifically to LAG-2.

A recombinant antibody that is an NK cell activating agent can be any of

exemplary types of antibodies (e.g., a human or humanized antibody) or any of the

exemplary antibody fragments described herein. A recombinant antibody that is an NK

cell activating agent can include, e.g., any of the antigen-binding domains described

herein.

Recombinant Interleukins or Cytokines

In some examples, NK activating agents can be, e.g., a soluble IL-2, a soluble IL-

7, a soluble IL-12, a soluble IL-15, a soluble IL-21, and a soluble IL-33. Non-limiting

examples of soluble IL-12, IL-15, IL-21, and IL-33. are provided below.

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

Human Soluble IL-2 (SEQ ID NO: 78)

aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr witfcqsiis tlt

Human Soluble IL-7 (SEQ ID NO: 79)

dcdiegkdgkqyesv lmvsidqlld smkeigsncl nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgg vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh

Human Soluble IL-12 subunit alpha (SEQ ID NO: 80)

rnlpvatp dpgmfpclhh sqnllravsn mlqkarqtle fypctseeid heditkdkts tveaclplel tknesclnsr etsfitngsc lasrktsfmm alclssiyed 1kmyqvefkt mnakllmdpk rqifldqnml avidelmqal nfnsetvpqk ssleepdfyk tkiklcillh afriravtid rvmsylnas

Human Soluble IL-12 subunit beta (SEQ ID NO: 81)

iwelkkdv yvveldwypd apgemvvltc dtpeedgitw tldqssevlg sgktltiqvk efgdaggytc efgdagqytc hkggevlshs llllhkkedg 1111hkkedg iwstdilkdq

kepknktflr ceaknysgrf tcwwlttist dltfsvkssr gssdpqgvtc gaatlsaerv rgdnkeyeys vecqedsacp aaeeslpiev mvdavhklky enytssffir diikpdppkn lqlkplknsr qvevsweypd twstphsyfs ltfcvqvqgk skrekkdrvf tdktsatvic rknasisvra qdryysssws

ewasvpcs

Human Soluble IL-15 (SEQ ID NO: 82)

Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin ts

133 wo 2021/247604 WO PCT/US2021/035285

Human Soluble IL-21 (SEQ ID NO: 83)

qgqdrhmi qgqdrhmi rmrqlidivd rmrqlidivdqlknyvndlv qlknyvndlv peflpapedv etncewsafs peflpapedv etncewsafs cfqkaqlksa ntgnneriin vsikklkrkp pstnagrrqk hrltcpscds yekkppkefl erfksllqkm ihqhlssrth gseds

Human Soluble IL-33 (SEQ ID NO: 84)

mkpkmkystn kistakwknt askalcfklg ksqqkakevc pmyfmklrsg lmikkeacyf rrettkrpsl ktgrkhkrhl vlaacqqqst vecfafgisg vqkytralhd ssitgispit eylaslstyn dqsitfaled esyeiyvedl kkdekkdkvl lsyyesqhps nesgdgvdgk mlmvtlsptk dfwlhannke hsvelhkcek plpdqaffvl hnmhsncvsf ecktdpgvfi gvkdnhlali kvdssenlct enilfklset

In some embodiments, a soluble IL-2 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 78.

In some embodiments, a soluble IL-7 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 79.

In some embodiments, a soluble IL-2 protein includes a sequence that is at least

80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to

SEQ ID NO: 80 and a sequence that is at least 80% identical, at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical,

WO wo 2021/247604 PCT/US2021/035285

at least 92% identical, at least 94% identical, at least 96% identical, at least 98%

identical, at least 99% identical, or 100% identical to SEQ ID NO: 81.

In some embodiments, a soluble IL-15 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 82.

In some embodiments, a soluble IL-21 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 83.

In some embodiments, a soluble IL-33 protein can include a sequence that is at

least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical to SEQ ID NO: 84.

Soluble Cytokine or Interleukin Receptors

In some examples of any of the soluble cytokine or interleukin receptors

described herein, the soluble cytokine or interleukin receptors can be a soluble TGF-B

receptor. In some examples, the soluble TGF-B receptor is a soluble TGF-B receptor I

(TGF-BRI) (see, e.g., those described in Docagne et al., Journal of Biological Chemistry

276(49):46243-46250, 2001), a soluble TGF-B receptor II (TGF-BRII) (see, e.g., those

described in Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016), a

soluble TGF-BRIII (see, e.g., those described in Heng et al., Placenta 57:320, 2017). In

some examples, the soluble TGF-B receptor is a receptor "trap" for TGF-B (see, e.g.,

those described in Zwaagstra et al., Mol. Cancer Ther. (7): 1477-1487, 2012, and those

described in De Crescenzo et al. Transforming Growth Factor-B in Cancer Therapy,

Volume II, pp 671-684).

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Additional examples of soluble cytokine or soluble interleukin receptors are

known in the art.

Single Chain Chimeric Polypeptides

Non-limiting examples of NK cell activating agents are single-chain chimeric

polypeptides that include: (i) a first target-binding domain (e.g., any of the target-binding

domains described herein or known in the art), (ii) a soluble tissue factor domain (e.g.,

any of the exemplary soluble tissue factor domains described herein or known in the art),

and (iii) as second target-binding domain (e.g., any of the target-binding domains

described herein or known in the art).

In some examples of any of the single-chain chimeric polypeptides described

herein, the single-chain chimeric polypeptide can have a total length of about 50 amino

acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about

50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino

acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about

950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to

about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino

acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50

amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids,

about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino

acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460

amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to

about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino

acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50

amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids,

about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino

acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240

amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to

about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino

acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about

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100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino

acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about

1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino

acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about

100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino

acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about

650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids

to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100

amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids,

about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420

amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to

about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100

amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids,

about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280

amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to

about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100

amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids,

about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500

amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to

about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150

amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids,

about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800

amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to

about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150

amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids,

about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480

amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to

about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150

amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids,

about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340

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amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to

about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150

amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids,

about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200

amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to

about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200

amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino

acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about

900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids

to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200

amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids,

about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550

amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to

about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200

amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids,

about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380

amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to

about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200

amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids,

about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240

amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to

about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220

amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino

acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about

950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids

to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220

amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids,

about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600

amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to

about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220

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amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids,

about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400

amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to

about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220

amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids,

about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260

amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to

about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240

amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino

acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about

950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids

to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240

amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids,

about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600

amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to

about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240

amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids,

about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400

amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to

about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240

amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids,

about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260

amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to

about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260

amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino

acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about

900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids

to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260

amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids,

about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about

1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino

acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about

300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino

acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about

650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids

to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300

PCT/US2021/035285

amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids,

about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420

amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to

about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300

amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids,

about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500

amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to

about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320

amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids,

about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800

amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to

about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320

amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids,

about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480

amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to

about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320

amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids,

about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340

amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to

about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340

amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino

acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about

900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids

to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340

amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids,

about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550

amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to

about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340

amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids,

about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to

about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360

amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino

acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about

950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids

to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360

amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids,

about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600

amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to

about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360

amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids,

about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400

amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to

about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380

amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino

acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about

950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids

to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380

amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids,

about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600

amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to

about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380

amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids,

about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400

amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to

about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400

amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino

acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about

900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids

to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids,

about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550

amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to

about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400

amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids,

about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500

amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to

about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420

amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids,

about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800

amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to

about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420

amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids,

about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480

amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to

about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440

amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino

acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about

1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino

acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about

440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino

acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about

650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids

to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440

amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids,

about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500

amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to

about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460

amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids,

about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800

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amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to

about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460

amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids,

about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480

amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to

about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480

amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino

acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about

900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids

to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480

amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids,

about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550

amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to

about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500

amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino

acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about

950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids

to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500

amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids,

about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600

amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to

about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550

amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino

acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about

950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids

to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550

amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids,

about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600

amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to

about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600

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amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino

acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about

900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids

to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600

amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids,

about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500

amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to

about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650

amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids,

about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800

amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to

about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700

amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino

acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about

1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino

acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about

700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino

acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about

2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino

acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about

750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino

acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about

800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino

acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about

800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino

acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about

900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids

to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850

amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino

acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about

145

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950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids

to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900

amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino

acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about

950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino

acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about

950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino

acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to

about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000

amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino

acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to

about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000

amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino

acids.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and the soluble tissue factor domain (e.g., any of the

exemplary soluble tissue factor domains described herein) directly abut each other. In

some embodiments of any of the single-chain chimeric polypeptides described herein, the

single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the

exemplary linker sequences described herein or known in the art) between the first target-

binding domain (e.g., any of the exemplary target-binding domains described herein or

known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein). In some embodiments of any of the single-chain

chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the

exemplary soluble tissue factor domains described herein) and the second target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art) directly abut each other. In some embodiments of any of the single-chain

chimeric polypeptides described herein, the single-chain chimeric polypeptide further

comprises a linker sequence (e.g., any of the exemplary linker sequences described herein

PCT/US2021/035285

or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary

soluble tissue factor domains described herein) and the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and the second target-binding domain (e.g., any of

the exemplary target-binding domains described herein or known in the art) directly abut

each other. In some embodiments of any of the single-chain chimeric polypeptides

described herein, the single-chain chimeric polypeptide further comprises a linker

sequence (e.g., any of the exemplary linker sequences described herein or known in the

art) between the first target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art) and the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides described herein,

the second target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and the soluble tissue factor domain (e.g., any of the

exemplary soluble tissue factor domains described herein) directly abut each other. In

some embodiments of any of the single-chain chimeric polypeptides described herein, the

single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the

exemplary linker sequences described herein or known in the art) between the second

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art) and the soluble tissue factor domain (e.g., any of the

exemplary soluble tissue factor domains described herein or known in the art).

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical,

at least 85% identical, at least 90% identical, at least 95% identical, at least 99%

identical, or 100% identical) to

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLA SGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGG

GGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285

RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSA YYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWE KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAG NVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVE NTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQ AVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASG YTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAY EFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGS MTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASG

VPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO: 85).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

nucleic acid that includes a sequence that is at least 70% identical (e.g., at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical) to

AGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGT. AGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCA CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCA

TTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAAC CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCAC AACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCAC< TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC

ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA 148

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CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACO CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGC GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTT AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGT AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTC ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTO AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTC CCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCA TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTO TGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCT TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAA TTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTAC. TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA TGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGG GGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC

GAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCA ATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAG CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATO CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATC TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTC TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA GG (SEQ ID NO: 86).

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical,

at least 85% identical, at least 90% identical, at least 95% identical, at least 99%

identical, or 100% identical) to

149 wo 2021/247604 WO PCT/US2021/035285

MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQ SGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWS KSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWS NPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSC SNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCK ASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS ASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS STAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIV, VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTN TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE IDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGI FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPEL

VKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKE VKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKF KGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGG SGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQI PKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGO SSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGG GTKLETKR (SEQ ID NO: 87). In some embodiments, a single-chain chimeric polypeptide is encoded by a

nucleic acid that includes a sequence that is at least 70% identical (e.g., at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical) to

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCC ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTO CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCO CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAA CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCA TTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAAC

CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTT 150

WO wo 2021/247604 PCT/US2021/035285

AACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAAC TAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAG ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAG CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATT7 GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTT

AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTO AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTO ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTATCO ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC GCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTC ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTC TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTO GTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCT TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAG TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA GGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCG TGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATO GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC GAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTC

CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATC CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATC TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTC TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCAC TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA GG (SEQ ID NO: 88).

wo WO 2021/247604 PCT/US2021/035285

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical,

at least 85% identical, at least 90% identical, at least 95% identical, at least 99%

identical, or 100% identical) to

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYN DYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGR DYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRG TTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSY TTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSY FHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFC FHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCH QYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTV QYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTV

QISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAG QISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVESYPAGNVESTGSAG EPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRD) EPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDV FGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRK FGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRK STDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQ STDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQ QKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQ QKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQW SSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSC SSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSC RASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTDK KASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKS SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID IDNO: SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(SEQ NO: 89).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

nucleic acid that includes a sequence that is at least 70% identical (e.g., at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical) to

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGA GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTG AAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGO AAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGG GTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCT ACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGAC ACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGAC AAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCT AAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTG AGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGG AGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGC CGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGA CGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGA

GGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCA 152

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TGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGC GTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTA ACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTT. GCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGA GATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGG GATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGG AGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCC AGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCGC CTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAG CTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAG CCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGO CCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCG ACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGA ACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGA GAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTAC CGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTAC CCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGA CCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGA ACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATC ACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATC CAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAG CAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAG AGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGO AGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGG CAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGA AGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGA AGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGA AACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACC GAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGG AAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTC TGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGG

TGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAAC GGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCA GCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAAC AGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTAT AAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTAT TACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCT CGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGG AGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGC AGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGC GCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACAC GCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACAC AATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTAT ATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAG ATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAG CCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTC wo WO 2021/247604 PCT/US2021/035285

TTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATO TTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATC ATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 90).

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical,

at least 85% identical, at least 90% identical, at least 95% identical, at least 99%

identical, or 100% identical) to

MKWVTFISLLFLFSSAYSVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWY MKWVTFISLLFLFSSAYSVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWV KQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSA KQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSA LYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTOSPAIMSASL LYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASL GERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSY SLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTN FKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYL FKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYL ARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTV ARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTV EDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKG NYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGI ENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEK VTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYS) VTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSL TISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQL TISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQL QQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY QQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY

TNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWG TNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWG QGTTLTVSS (SEQ ID NO: 91). In some embodiments, a single-chain chimeric polypeptide is encoded by a

nucleic acid that includes a sequence that is at least 70% identical (e.g., at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical) to

ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAG ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAG GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTG CGTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTG LAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATG GGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCC GGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCC

TACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGA 154

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CAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCT CAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCT GAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGG CCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGG CCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGG GGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTA ATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCC CGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTA AACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTT GCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGA AGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGA GATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCG AGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCG AGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGC GAGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCG CCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGA GCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGO GCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGC GACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACO GACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCG ACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTA CCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAG CCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAG AACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCAT CCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGA CCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAG GGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCG AGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGG CAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGA CAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGA AGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGA AGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGA AACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACC GAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGG AAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTC GTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGG TGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAAC GGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCA GCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAAC AGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCT AAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTAT TACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCT CGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTG CGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGG AGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGC 155

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GCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACAC GCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACAC AATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTAT AATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTAT ATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAG ATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAG CCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTC

TTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATC TTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATO ATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 92).

Some embodiments of any of the single-chain chimeric polypeptides described

herein can further include one or more (e.g., two, three, four, five, six, seven, eight, nine,

or ten) additional target-binding domains (e.g., any of the exemplary target-binding

domains described herein or known in the art) at its N- and/or C-terminus.

In some embodiments, the single-chain chimeric polypeptides can include one or

more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

the art) at its N-terminus. In some embodiments, one of the one or more additional

target-binding domains (e.g., any of the exemplary target-binding domains described

herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide can

directly abut the first target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art), the second target-binding domain (e.g.,

any of the exemplary target-binding domains described herein or known in the art), or the

soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains

described herein). In some embodiments, the single-chain chimeric polypeptide further

includes a linker sequence (e.g., any of the exemplary linker sequences described herein

or known in the art) between one of the at least one additional target-binding domains

(e.g., any of the exemplary target-binding domains described herein or known in the art)

at the N-terminus of the single-chain chimeric polypeptide and the first target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art), the second target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art), or the soluble tissue factor domain (e.g.,

any of the exemplary soluble tissue factor domains described herein).

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In some embodiments of any of the single-chain chimeric polypeptides described

herein, the single-chain chimeric polypeptide includes one or more (e.g., two, three, four,

five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the

exemplary target-binding domains described herein or known in the art) at its C-terminus.

In some embodiments, one of the one or more additional target-binding domains (e.g.,

any of the exemplary target-binding domains described herein or known in the art) at the

C-terminus of the single-chain chimeric polypeptide directly abuts the first target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art), the second target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art), or the soluble tissue factor domain (e.g.,

any of the exemplary soluble tissue factor domains described herein or known in the art).

In some embodiments, the single-chain chimeric polypeptide further comprises a linker

sequence (e.g., any of the exemplary linker sequences described herein or known in the

art) between one of the at least one additional target-binding domains (e.g., any of the

exemplary target-binding domains described herein or known in the art) at the C-terminus

of the single-chain chimeric polypeptide and the first target-binding domain (e.g., any of

the exemplary target-binding domains described herein or known in the art), the second

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary

soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the single-chain chimeric polypeptide comprises one or more (e.g., two, three,

four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of

the exemplary target-binding domains described herein or known in the art) at its N-

terminus and its C-terminus. In some embodiments, one of the one or more additional

antigen binding domains (e.g., any of the exemplary target-binding domains described

herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide

directly abuts the first target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art), the second target-binding domain (e.g.,

any of the exemplary target-binding domains described herein or known in the art), or the

WO wo 2021/247604 PCT/US2021/035285

soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains

described herein). In some embodiments, the single-chain chimeric polypeptide further

includes a linker sequence (e.g., any of the exemplary linker sequences described herein

or known in the art) between one of the one or more additional antigen-binding domains

(e.g., any of the exemplary target-binding domains described herein or known in the art)

at the N-terminus and the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor

domains). In some embodiments, one of the one or more additional antigen binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

the art) at the C-terminus directly abuts the first target-binding domain (e.g., any of the

exemplary target-binding domains described herein or known in the art), the second

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary

soluble tissue factor domains). In some embodiments, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linker

sequences described herein or known in the art) between one of the one or more

additional antigen-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) at the C-terminus and the first target-binding

domain(e.g., any of the exemplary target-binding domains described herein or known in

the art), the second target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art), or the soluble tissue factor domain (e.g.,

any of the exemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides described

herein, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain (e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

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described herein or known in the art) bind specifically to the same antigen. In some

embodiments, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the

first target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain (e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) bind specifically to the same epitope. In some

embodiments, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the

first target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain(e.g. any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) include the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art), the second target-binding domain (e.g., any of the

exemplary target-binding domains described herein or known in the art), and the one or

more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

the art) each bind specifically to the same antigen. In some embodiments, the first target-

binding domain (e.g., any of the exemplary target-binding domains described herein or

known in the art), the second target-binding domain(e.g., any of the exemplary target-

binding domains described herein or known in the art), and the one or more (e.g., two,

three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g.,

any of the exemplary target-binding domains described herein or known in the art) each

bind specifically to the same epitope. In some embodiments, the first target-binding

domain, the second target-binding domain, and the one or more (e.g., two, three, four,

five, six, seven, eight, nine, or ten) additional target-binding domains each comprise the

same amino acid sequence.

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In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art), the second target-binding domain(e.g., any of the

exemplary target-binding domains described herein or known in the art), and the one or

more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

the art) bind specifically to different antigens.

In some embodiments of any of the single-chain chimeric polypeptides, one or

more of the first target-binding domain, the second target-binding domain, and the one or

more target-binding domains is an antigen-binding domain (e.g., any of the exemplary

antigen-binding domains described herein or known in the art). In some embodiments of

any of the single-chain chimeric polypeptides described herein, the first target-binding

domain, the second target-binding domain, and the one or more additional target-binding

domains are each an antigen-binding domain (e.g., any of the exemplary antigen-binding

domains described herein or known in the art). In some embodiments, the antigen-

binding domain can include a scFv or a single domain antibody.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain(e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) bind specifically to a target selected from the group

consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1,

VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6,

IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-

2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER,

CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-

BRIII, a ligand of DNAMI, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for

stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a

receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a

receptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble interleukin or cytokine

protein. Non-limiting examples of soluble interleukin proteins and soluble cytokine

proteins include: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, and SCF.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble interleukin or cytokine

receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine

receptors include: a soluble TGF-B receptor II (TGF-BRII), a soluble TGF-BRIII, a

soluble NKG2D, a soluble NKP30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble

CD3, or a soluble CD28.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the target-binding domains described

herein), the second target-binding domain (e.g., any of the target-binding domains

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described herein), and the one or more additional target-binding domains (e.g., any of the

target-binding domains described herein) can each, independently, bind specifically to a

target selected from the group of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAMI, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKP30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a

receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand

(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding

protein, a receptor for CD155, and a receptor for CD122.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble interleukin or cytokine

protein. In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-

2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble interleukin or cytokine

receptor. In some embodiments of any of the multi-chain chimeric polypeptides

described herein, the soluble receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble

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TGF-BRIII, a soluble receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor

for IL-10.

Multi-Chain Chimeric Polypeptides- Type A

Non-limiting examples of NK cell activating agents are multi-chain chimeric

polypeptides that include: (a) a first chimeric polypeptide including: (i) a first target-

binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of

affinity domains; and (b) a second chimeric polypeptide including: (i) a second domain of

a pair of affinity domains; and (ii) a second target-binding domain, where the first

chimeric polypeptide and the second chimeric polypeptide associate through the binding

of the first domain and the second domain of the pair of affinity domains.

In some examples of any of the multi-chain chimeric polypeptides described

herein the total length of first chimeric polypeptide and/or the second chimeric

polypeptide can each independently be about 50 amino acids to about 3000 amino acids,

about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000

amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to

about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino

acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50

amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids,

about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino

acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550

amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to

about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino

acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50

amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids,

about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino

acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300

amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to

about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino

acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50

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amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids,

about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500

amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to

about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100

amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids,

about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800

amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to

about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100

amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids,

about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480

amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to

about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100

amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids,

about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340

amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to

about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100

amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids,

about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200

amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to

about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150

amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino

acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about

950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids

to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150

amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids,

about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600

amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to

about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150

amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids,

about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about

1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino

acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about

200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino

acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about

700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids

to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200

amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids,

about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440

amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to

about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200

amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids,

about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300

amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to

about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200

amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids,

about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000

amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to

about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220

amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids,

about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750

amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to

about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220

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amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids,

about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460

amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to

about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220

amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids,

about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320

amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to

about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220

amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids,

about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000

amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to

about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240

amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids,

about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750

amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to

about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240

amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids,

about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460

amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to

about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240

amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids,

about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320

amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to

about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260

amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino

acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about

1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino

acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about

260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino

acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about

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700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids

to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260

amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids,

about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440

amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to

about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260

amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids,

about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300

amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to

about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280

amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino

acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about

950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids

to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280

amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids,

about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600

amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to

about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280

amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids,

about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400

amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to

about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280

amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids,

about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500

amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to

about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300

amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids,

about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800

amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to

about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300

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amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids,

about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480

amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to

about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300

amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids,

about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340

amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to

about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320

amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino

acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about

950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids

to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320

amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids,

about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600

amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to

about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320

amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids,

about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400

amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to

about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340

amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino

acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about

1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino

acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about

340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino

acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about

700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids

to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340

amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids,

about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440

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amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to

about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340

amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids,

about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000

amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to

about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360

amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids,

about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750

amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to

about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360

amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids,

about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460

amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to

about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360

amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids,

about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000

amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to

about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380

amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids,

about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750

amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to

about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380

amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids,

about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460

amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to

about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400

amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino

acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about

1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino

acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about

400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino

acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about

700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids

to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400

amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids,

about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440

amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to

about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420

amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino

acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about

950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids

to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420

amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids,

about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600

amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to

about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420

amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids,

about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500

amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to

about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440

amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids,

about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800

amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to

about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440

amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids,

about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480

amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to

about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460

amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino

acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about

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950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids

to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460

amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids,

about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600

amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to

about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480

amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino

acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about

1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino

acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about

480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino

acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about

700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids

to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480

amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids,

about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000

amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to

about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500

amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids,

about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750

amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to

about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500

amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids,

about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000

amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to

about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550

amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids,

about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750

amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to

about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about

1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino

acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about

600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino

acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about

700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids

to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650

amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino

acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about

950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids

to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650

amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids,

about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500

amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to

about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700

amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids,

about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800

amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to

about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750

amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino

acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about

950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids

to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800

amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino

acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about

1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino

acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about

800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino

acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about

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2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino

acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about

850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino

acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about

2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino

acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about

950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino

acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about

1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino

acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids,

about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about

1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino

acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids,

about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about

2500 amino acids, or about 2500 amino acids to about 3000 amino acids.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the first target-binding domains

described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein) directly abut each other in the first chimeric

polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides

described herein, the first chimeric polypeptide further comprises a linker sequence (e.g.,

any of the exemplary linker sequences described herein or known in the art) between the

first target-binding domain (e.g., any of the exemplary first target-binding domains

described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor

domains described herein) and the first domain of the pair of affinity domains (e.g., any

of the exemplary first domains of any of the exemplary pairs of affinity domains

described herein) directly abut each other in the first chimeric polypeptide. In some

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embodiments of any of the multi-chain chimeric polypeptides described herein, the first

chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linker sequences described herein or known in the art) between the soluble tissue factor

domain (e.g., any of the exemplary soluble tissue factor domains described herein) and

the first domain of the pair of affinity domains (e.g., any of the exemplary first domains

of any of the exemplary pairs of affinity domains described herein) in the first chimeric

polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the second domain of the pair of affinity domains (e.g., any of the exemplary

second domains of any of the exemplary pairs of affinity domains described herein) and

the second target-binding domain (e.g., any of the exemplary second target-binding

domains described herein) directly abut each other in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein,

the second chimeric polypeptide further comprises a linker sequence (e.g., any of the

exemplary linker sequences described herein or known in the art) between the second

domain of the pair of affinity domains (e.g., any of the exemplary second domains of any

of the exemplary pairs of affinity domains described herein) and the second target-

binding domain (e.g., any of the exemplary second target-binding domains described

herein) in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides, the first

chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven,

eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary target-

binding domains described herein or known in the art), where at least one of the one or

more additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain (e.g., any of the exemplary soluble tissue factor domains described herein or

known in the art) and the first domain of the pair of affinity domains (e.g., any of the

exemplary first domains of any of the exemplary pairs of affinity domains described

herein). In some embodiments, the first chimeric polypeptide can further include a linker

sequence (e.g., any of the exemplary linker sequences described herein or known in the

art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue

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factor domains described herein) and the at least one of the one or more additional target-

binding domain(s) (e.g., any of the exemplary target-binding domains described herein or

known in the art), and/or a linker sequence (e.g., any of the exemplary linker sequences

described herein or known in the art) between the at least one of the one or more

additional target-binding domain(s) (e.g., any of the exemplary target-binding domains

described herein or known in the art) and the first domain of the pair of affinity domains

(e.g., any of the exemplary first domains described herein of any of the exemplary pairs

of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first chimeric polypeptide further includes one or more (e.g., two, three, four,

five, six, seven, eight, nine, or ten) additional target-binding domains at the N-terminal

and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least

one of the one or more additional target-binding domains (e.g., any of the exemplary

target-binding domains described herein or known in the art) directly abuts the first

domain of the pair of affinity domains (e.g., any of the exemplary first domains described

herein of any of the exemplary pairs of affinity domains described herein) in the first

chimeric polypeptide. In some embodiments, the first chimeric polypeptide further

includes a linker sequence (e.g., any of the exemplary linker sequences described herein

or known in the art) between the at least one of the one or more additional target-binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary

first domains described herein of any of the exemplary pairs of affinity domains

described herein). In some embodiments, the at least one of the one or more additional

target-binding domains (e.g., any of the exemplary target-binding domains described

herein or known in the art) directly abuts the first target-binding domain (e.g., any of the

exemplary target-binding domains described herein or known in the art) in the first

chimeric polypeptide. In some embodiments, the first chimeric polypeptide further

comprises a linker sequence (e.g., any of the exemplary linker sequences described herein

or known in the art) between the at least one of the one or more additional target-binding

domains (e.g., any of the exemplary target-binding domains described herein or known in

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the art) and the first target-binding domain (e.g., any of the exemplary target-binding

domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, at least one of the one or more additional target-binding domains (e.g., any of the

exemplary target-binding domains described herein or known in the art) is disposed at the

N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains (e.g., any of the exemplary target-binding

domains described herein or known in the art) is positioned between the soluble tissue

factor domain (e.g., any of the exemplary soluble tissue factor domains described herein

or known in the art) and the first domain of the pair of affinity domains (e.g., any of the

exemplary first domains of any of the exemplary pairs of affinity domains described

herein) in the first chimeric polypeptide. In some embodiments, the at least one

additional target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) of the one or more additional target-binding

domains disposed at the N-terminus directly abuts the first target-binding domain (e.g.,

any of the exemplary target-binding domains described herein or known in the art) or the

first domain of the pair of affinity domains (e.g., any of the exemplary first domains

described herein of any of the exemplary pairs of affinity domains described herein) in

the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide

further comprises a linker sequence (e.g., any of the linker sequences described herein or

known in the art) disposed between the at least one additional target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art)

and the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) or the first domain of the pair of affinity domains

(e.g., any of the exemplary first domains described herein of any of the exemplary pairs

of affinity domains described herein) in the first chimeric polypeptide. In some

embodiments, the at least one additional target-binding domain (e.g., any of the

exemplary target-binding domains described herein or known in the art) of the one or

more additional target-binding domains disposed at the C-terminus directly abuts the first

target-binding domain (e.g., any of the exemplary target-binding domains described

WO wo 2021/247604 PCT/US2021/035285

herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of

the exemplary first domains of any of the exemplary pairs of affinity domains described

herein) in the first chimeric polypeptide. In some embodiments, the first chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linker

sequences described herein or known in the art) disposed between the at least one

additional target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and the first target-binding domain (e.g., any of the

exemplary target-binding domains described herein or known in the art) or the first

domain of the pair of affinity domains (e.g., any of the exemplary first domains described

herein of any of the exemplary pairs of affinity domains described herein) in the first

chimeric polypeptide. In some embodiments, the at least one of the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) positioned between the soluble tissue factor domain

(e.g., any of the exemplary soluble tissue factor domains described herein) and the first

domain of the pair of affinity domains (e.g., any of the first domains described herein or

any of the exemplary pairs of affinity domains described herein), directly abuts the

soluble tissue factor domain and/or the first domain of the pair of affinity domains. In

some embodiments, the first chimeric polypeptide further comprises a linker sequence

(e.g., any of the exemplary linker sequences described herein or known in the art)

disposed (i) between the soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein) and the at least one of the one or more additional

target-binding domains (e.g., any of the exemplary target-binding domains described

herein or known in the art) positioned between the soluble tissue factor domain (e.g., any

of the exemplary soluble tissue factor domains described herein) and the first domain of

the pair of affinity domains (e.g., any of the exemplary first domains of any of the

exemplary pairs of affinity domains described herein), and/or (ii) between the first

domain of the pair of affinity domains and the at least one of the one or more additional

target-binding domains positioned between the soluble tissue factor domain and the first

domain of the pair of affinity domains.

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In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the second chimeric polypeptide further includes one or more (e.g., two, three,

four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of

the exemplary target-binding domains described herein or known in the art) at the N-

terminal end and/or the C-terminal end of the second chimeric polypeptide. In some

embodiments, at least one of the one or more additional target-binding domains (e.g., any

of the exemplary target-binding domains described herein or known in the art) directly

abuts the second domain of the pair of affinity domains (e.g., any of the exemplary

second domains of any of the exemplary pairs of affinity domains described herein) in the

second chimeric polypeptide. In some embodiments, the second chimeric polypeptide

further includes a linker sequence (e.g., any of the exemplary linker sequences described

herein or known in the art) between at least one of the one or more additional target-

binding domains (e.g., any of the exemplary target-binding domains described herein or

known in the art) and the second domain of the pair of affinity domains (e.g., any of the

second domains described herein of any of the exemplary pairs of affinity domains

described herein) in the second chimeric polypeptide. In some embodiments, at least one

of the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) directly abuts the second target-

binding domain (e.g., any of the target-binding domains described herein or known in the

art) in the second chimeric polypeptide. In some embodiments, the second chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linker

sequences described herein or known in the art) between at least one of the one or more

additional target-binding domains (e.g., any of the exemplary target binding domains

described herein or known in the art) and the second target-binding domain (e.g., any of

the exemplary target binding domains described herein or known in the art) in the second

chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or

more, eight or more, nine or more, or ten or more) of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

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bind specifically to the same antigen. In some embodiments, two or more (e.g., three or

more, four or more, five or more, six or more, seven or more, eight or more, nine or

more, or ten or more) of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope. In some embodiments, two or more (e.g., three or more, four or more, five

or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the

first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains include the same amino acid sequence. In some

embodiments, the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains each bind specifically to the same antigen.

In some embodiments, the first target-binding domain, the second target-binding domain,

and the one or more additional target-binding domains each bind specifically to the same

epitope. In some embodiments, the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains each include the same

amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains bind specifically to different antigens. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, one or

more (e.g., two or more, three or more, four or more, five or more, six or more, seven or

more, eight or more, nine or more, or ten or more) of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains is

an antigen-binding domain. In some embodiments, the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains are

each an antigen-binding domain (e.g., a scFv or a single-domain antibody).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain (e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) bind specifically to a target selected from the group

consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1,

VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6,

IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC,

Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER,

CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-

BRIII, a ligand of DNAMI, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D,

a ligand of NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR,

a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor

for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for

stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a

receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a

receptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble interleukin or cytokine

protein. Non-limiting examples of soluble interleukin proteins and soluble cytokine

proteins include: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, and SCF. In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

WO wo 2021/247604 PCT/US2021/035285

binding domains described herein or known in the art) is a soluble interleukin or cytokine

receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine

receptors include: a soluble TGF-B receptor II (TGF-BRII), a soluble TGF-BRIII, a

soluble NKG2D, a soluble NKP30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble

CD3, or a soluble CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the target-binding domains described

herein, the second target-binding domain (e.g., any of the target-binding domains

described herein), and the one or more additional target-binding domains (e.g., any of the

target-binding domains described herein) can each, independently, bind specifically to a

target selected from the group of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a

receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand

(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding

protein, a receptor for CD155, and a receptor for CD122.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain (e.g., any of the target-binding

domains described herein), the second target-binding domain (e.g., any of the target-

binding domains described herein), and the one or more additional binding domains (e.g.,

any of the target-binding described herein) is a soluble interleukin or cytokine protein. In

some embodiments of any of the multi-chain chimeric polypeptides described herein, the

soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-

7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the soluble receptor is a soluble

TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor for TNFa, a

soluble receptor for IL-4, or a soluble receptor for IL-10.

Multi-Chain Chimeric Polypeptides- Type B

Non-limiting examples of NK cell activating agents are multi-chain chimeric

polypeptides that include: (a) a first and second chimeric polypeptide each including: (i) a

first target-binding domain; (ii) a Fc domain; and (iii) a first domain of a pair of affinity

domains; and (b) a third and fourth chimeric polypeptide each including: (i) a second

domain of a pair of affinity domains; and (ii) a second target-binding domain, where the

first and second chimeric polypeptides and the third and fourth chimeric polypeptides

associate through the binding of the first domain and the second domain of the pair of

affinity domains, and the first and second chimeric polypeptides associate through their

Fc domains.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain (e.g., any of the first target-binding domains

described herein) and the Fc domain (e.g., any of the exemplary Fc domains described

herein) directly abut each other in the first and second chimeric polypeptides. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the first

and second chimeric polypeptides further comprise a linker sequence (e.g., any of the

exemplary linker sequences described herein or known in the art) between the first target-

binding domain (e.g., any of the exemplary first target-binding domains described herein)

and the Fc domain (e.g., any of the exemplary Fc domains described herein) in the first

and second chimeric polypeptides.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the Fc domain (e.g., any of the exemplary Fc domains described herein) and the

first domain of the pair of affinity domains (e.g., any of the exemplary first domains of

WO wo 2021/247604 PCT/US2021/035285

any of the exemplary pairs of affinity domains described herein) directly abut each other

in the first and second chimeric polypeptide. In some embodiments of any of the multi-

chain chimeric polypeptides described herein, the first and second chimeric polypeptide

further comprises a linker sequence (e.g., any of the exemplary linker sequences

described herein or known in the art) between the Fc domain (e.g., any of the exemplary

Fc domains described herein) and the first domain of the pair of affinity domains (e.g.,

any of the exemplary first domains of any of the exemplary pairs of affinity domains

described herein) in the first and second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the second domain of the pair of affinity domains (e.g., any of the exemplary

second domains of any of the exemplary pairs of affinity domains described herein) and

the second target-binding domain (e.g., any of the exemplary second target-binding

domains described herein) directly abut each other in the third and fourth chimeric

polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides

described herein, the third and fourth chimeric polypeptide further comprise a linker

sequence (e.g., any of the exemplary linker sequences described herein or known in the

art) between the second domain of the pair of affinity domains (e.g., any of the exemplary

second domains of any of the exemplary pairs of affinity domains described herein) and

the second target-binding domain (e.g., any of the exemplary second target-binding

domains described herein) in the third and fourth chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second target-binding domain bind

specifically to the same antigen. In some embodiments of any of the multi-chain chimeric

polypeptides described herein, the first target-binding domain and the second target-

binding domain bind specifically to the same epitope. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the first target-binding domain and

the second target-binding domain include the same amino acid sequence. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the first

target-binding domain and the second target-binding domain bind specifically to different

antigens. In some embodiments of any of the multi-chain chimeric polypeptides

WO wo 2021/247604 PCT/US2021/035285

described herein, one or both of the first target-binding domain and the second target-

binding domain is an antigen-binding domain (e.g., any of the exemplary second target-

binding domains described herein). In some embodiments of any of the multi-chain

chimeric polypeptides described herein, the first target-binding domain and the second

target-binding domain are each antigen-binding domains (e.g., any of the exemplary

second target-binding domains described herein). In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the antigen-binding domain (e.g.,

any of the exemplary second target-binding domains described herein) includes a scFv or

a single domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art) and the second target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art) bind specifically to a target selected from the group consisting of: CD16a, CD28,

CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-

1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for

IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a

receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a

receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a

receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a

receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a

receptor for CD122, and a receptor for CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain (e.g., any of the exemplary target-

WO wo 2021/247604 PCT/US2021/035285

binding domains described herein or known in the art) and the second target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art) is a soluble interleukin or cytokine protein. Non-limiting examples of soluble

interleukin proteins and soluble cytokine proteins include: IL-1, IL-2, IL-3, IL-7, IL-8,

IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain and the second target-binding

domain (e.g., any of the exemplary target-binding domains described herein or known in

the art) is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble

interleukin receptors and soluble cytokine receptors include: a soluble TGF-B receptor II

(TGF-BRII), a soluble TGF-BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44,

a soluble NKp46, a soluble DNAMI, a scMHCI, a scMHCII, a scTCR, a soluble CD155,

a soluble CD122, a soluble CD3, or a soluble CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second target-binding domain can each,

independently, bind specifically to a target selected from the group of: CD16a, CD33,

CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6,

IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC,

Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-

cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER,

CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-

BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D,

a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR,

a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-

like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a

receptor for a ULP16-binding protein, a receptor for CD155, and a receptor for CD122.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine protein. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine

185

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protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,

IL-17, IL-18, IL-21, PDGF-DD, and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the soluble receptor is a soluble

TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor for TNFa, a

soluble receptor for IL-4, or a soluble receptor for IL-10.

Tissue Factor

Human tissue factor is a 263 amino-acid transmembrane protein containing three

domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1-219); (2) a

22-amino acid transmembrane domain (residues 220-242); and (3) a 21-amino acid

cytoplasmic C-terminal tail (residues 242-263) ((UniProtKB Identifier Number: P13726).

The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one S-

palmitoylation site at Cys245. Deletion or mutation of the cytoplasmic domain was not

found to affect tissue factor coagulation activity. Tissue factor has one S-palmitoylation

site in the intracellular domain of the protein at Cys245. The Cys245 is located at the

amino acid terminus of the intracellular domain and close to the membrane surface. The

tissue factor transmembrane domain is composed of a single-spanning a-helix.

The extracellular domain of tissue factor, composed of two fibronectin type III

domains, is connected to the transmembrane domain through a six-amino acid linker.

This linker provides conformational flexibility to decouple the tissue factor extracellular

domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin

type III module is composed of two overlapping B sheets with the top sheet domain

containing three antiparallel B-strands and the bottom sheet containing four B-strands.

The B-strands are connected by B-loops between strand BA and BB, BC and BD, and BE

and BF, all of which are conserved in conformation in the two modules. There are three

short a-helix segments connecting the B-strands. A unique feature of tissue factor is a 17-

amino acid B-hairpin between strand 310 and strand 311, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12 amino acid loop between B6F and B7G that is not present in the C-terminal domain and is unique to tissue factor. Such a fibronectin type III domain structure is a feature of the immunoglobulin-like family of protein folds and is conserved among a wide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysis once it

binds to tissue to form the active tissue factor-FVIIa complex. The FVIIa, which

circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII),

can also bind directly to tissue factor. The allosteric interaction between tissue factor and

FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of

FVIIa: an approximate 20- to 100-fold increase in the rate of hydrolysis of small,

chromogenic peptidyl substrates, and nearly a million-fold increase in the rate of

activation of the natural macromolecular substrates FIX and FX. In concert with

allosteric activation of the active site of FVIIa upon binding to tissue factor, the

formation of tissue factor-FVIIa complex on phospholipid bilayer (i.e., upon exposure of

phosphatidyl-L-serine on membrane surfaces) increases the rate of FIX or FX activation,

in a Ca2+-dependent manner, an additional 1,000-fold. The roughly million-fold overall

increase in FX activation by tissue factor-FVIIa-phospholipid complex relative to free

FVIIa is a critical regulatory point for the coagulation cascade.

FVII is a ~50 kDa, single-chain polypeptide consisting of 406 amino acid

residues, with an N-terminal "--aarboxyglutamate-rich (GLA) domain, two epidermal

growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain.

FVII is activated to FVIIa by a specific proteolytic cleavage of the Ile-154-Arg152 bond in

the short linker region between the EGF2 and the protease domain. This cleavage results

in the light and heavy chains being held together by a single disulfide bond of Cys13 135

and

Cys262. FVIIa binds phospholipid membrane in a Ca2+-dependent manner through its N-

terminal GLA-domain. Immediately C-terminal to the GLA domain is an aromatic stack

and two EGF domains. The aromatic stack connects the GLA to EGF1 domain which

binds a single Ca2+ ion. Occupancy of this Ca2+ -binding site increases FVIIa amidolytic

activity and tissue factor association. The catalytic triad consist of His 193 , Asp242, and

WO wo 2021/247604 PCT/US2021/035285

Ser344, and binding of a single Ca2+ ion within the FVIIa protease domain is critical for its

catalytic activity. Proteolytic activation of FVII to FVIIa frees the newly formed amino

terminus at Ile 153 to fold back and be inserted into the activation pocket forming a salt

bridge with the carboxylate of Asp343 to generate the oxyanion hole. Formation of this

salt bridge is critical for FVIIa activity. However, oxyanion hole formation does not

occur in free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a

zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to

circulate with a half-life of approximately 90 minutes.

Tissue factor-mediated positioning of the FVIIa active site above the membrane

surface is important for FVIIa towards cognate substrates. Free FVIIa adopts a stable,

extended structure when bound to the membrane with its active site positioned ~80A

above the membrane surface. Upon FVIIa binding to tissue factor, the FVa active site is

repositioned ~6A closer to the membrane. This modulation may aid in a proper

alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA-

domainless FVIIa, it has been shown that the active site was still positioned a similar

distance above the membrane, demonstrating that tissue factor is able to fully support

FVIIa active site positioning even in the absence of FVIIa-membrane interaction.

Additional data showed that tissue factor supported full FVIIa proteolytic activity as long

as the tissue factor extracellular domain was tethered in some way to the membrane

surface. However, raising the active site of FVIIa greater than 80A above the membrane

surface greatly reduced the ability of the tissue factor-FVIIa complex to activate FX but

did not diminish tissue factor-FVIIa amidolytic activity.

Alanine scanning mutagenesis has been used to assess the role of specific amino

acid side chains in the tissue factor extracellular domain for interaction with FVIIa

(Gibbs et al., Biochemistry 33(47): 14003-14010, 1994; Schullek et al., J Biol Chem

269(30): 19399-19403, 1994). Alanine substitution identified a limited number of

residue positions at which alanine replacements cause 5- to 10-fold lower affinity for

FVIIa binding. Most of these residue side chains were found to be well-exposed to

solvent in the crystal structure, concordant with macromolecular ligand interaction. The

FVIIa ligand-binding site is located over an extensive region at the boundary between the

WO wo 2021/247604 PCT/US2021/035285

two modules. In the C-module, residues Arg13 135 and Phe ¹40 located on the protruding B-C

loop provide an independent contact with FVIIa. Leu ¹3 is located at the base of the

fingerlike structure and packed into the cleft between the two modules. This provides

continuity to a major cluster of important binding residues consisting of Lys20, Thr60 ,

Asp58, and Ile22. Thr60 is only partially solvent-exposed and may play a local structural

role rather than making a significant contact with ligand. The binding site extends onto

the concave side of the intermodule angle involving Glu24 and Gln ¹ 10 and potentially the

more distant residue Val207. The binding region extends from Asp58 onto a convex

surface area formed by Lys48, Lys46, Gln ³7. Asp44, and Trp45. Trp45 and Asp44 do not

interact independently with FVIIa, indicating that the mutational effect at the Trp45

position may reflect a structural importance of this side chain for the local packing of the

adjacent Asp44 and Gln³7 side chain. The interactive area further includes two surface-

exposed aromatic residues, Phe76 and Tyr78, which form part of the hydrophobic cluster

in the N-module.

The known physiologic substrates of tissue factor-FVIIa are FVII, FIX, and FX

and certain proteinase-activated receptors. Mutational analysis has identified a number of

residues that, when mutated, support full FVIIa amidolytic activity towards small

peptidyl substrates but are deficient in their ability to support macromolecular substrate

(i.e., FVII, FIX, and FX) activation (Ruf et al., J Biol Chem 267(31): 22206-22210, 1992;

Ruf et al., J Biol Chem 267(9): 6375-6381, 1992; Huang et al., J Biol Chem 271(36):

21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25): 7380-7387, 2000). The

tissue factor loop region at residues 159-165, and residues in or adjacent to this flexible

loop have been shown to be critical for the proteolytic activity of the tissue factor-FVIIa

complex. This defines the proposed substrate-binding exosite region of tissue factor that

is quite distant from the FVIIa active site. A substitution of the glycine residue by a

marginally bulkier residue alanine, significantly impairs tissue factor-FVIIa proteolytic

activity. This suggests that the flexibility afforded by glycine is critical for the loop of

residues 159-165 for tissue factor macromolecular substrate recognition.

The residues Lys165 and Lys 166 have also been demonstrated to be important for

substrate recognition and binding. Mutation of either of these residues to alanine results wo 2021/247604 WO PCT/US2021/035285 in a significant decrease in the tissue factor co-factor function. Lys 165 and Lys16 166 face away from each other, with Lys165 pointing towards FVIIa in most tissue factor-FVIIa structures, and Lys166 pointing into the substrate binding exosite region in the crystal structure. Putative salt bridge formation between Lys165 of and Gla³5 of FVIIa would support the notion that tissue factor interaction with the GLA domain of FVIIa modulates substrate recognition. These results suggest that the C-terminal portion of the tissue factor ectodomain directly interacts with the GLA-domain, the possible adjacent EGF1 domains, of FIX and FX, and that the presence of the FVIIa GLA-domain may modulate these interactions either directly or indirectly.

Soluble Tissue Factor Domain

In some embodiments of any of the polypeptides, compositions, or methods

described herein, the soluble tissue factor domain can be a wildtype tissue factor

polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular

domain. In some examples, the soluble tissue factor domain can be a tissue factor

mutant, wherein a wildtype tissue factor polypeptide lacking the signal sequence, the

transmembrane domain, and the intracellular domain, and has been further modified at

selected amino acids. In some examples, the soluble tissue factor domain can be a

soluble human tissue factor domain. In some examples, the soluble tissue factor domain

can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor

domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble

human tissue factor domains, a mouse soluble tissue factor domain, a rat soluble tissue

factor domain, and mutant soluble tissue factor domains are shown below.

Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO: 93)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGO PTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSG) KTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

Exemplary Nucleic Acid Encoding Soluble Human Tissue Factor Domain (SEQ ID

NO: 94)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACC ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC GTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCA0 CGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGAC TACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTT CGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGA CCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGT

GAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTT GAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTT CTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGO CTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGG AAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTT? TAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGAT CCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGG GCCAAGAAAAGGGCGAGTTCCGGGAG

Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 95)

agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfst dtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgk dlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktn ispgsstvcteqwksflge

Exemplary Soluble Rat Tissue Factor Domain (SEQ ID NO: 96)

Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgt dtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppfti rkflpyrdtkigqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfg indlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktn nkspesitkcteqwksvlge

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 97)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTT DTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNL, wo 2021/247604 WO PCT/US2021/035285

GQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSS GKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEF RE RE

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 98)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTD TECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG PTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E

In some embodiments, a soluble tissue factor domain can include a sequence that

is at least 70% identical, at least 72% identical, at least 74% identical, at least 76%

identical, at least 78% identical, at least 80% identical, at least 82% identical, at least

84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at

least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical,

at least 99% identical, or 100% identical to SEQ ID NO: 93, 95, 96, 97 or 98. In some

embodiments, a soluble tissue factor domain can include a sequence of SEQ ID NO: 93,

95, 96, 97, or 98, with one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, or 20) amino acids removed from its N-terminus and/or one to

twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)

amino acids removed from its C-terminus.

As can be appreciated in the art, one skilled in the art would understand that

mutation of amino acids that are conserved between different mammalian species is more

likely to decrease the activity and/or structural stability of the protein, while mutation of

amino acids that are not conserved between different mammalian species is less likely to

decrease the activity and/or structural stability of the protein.

In some examples of any of the multi-chain chimeric polypeptides described

herein, the soluble tissue factor domain is not capable of binding to Factor VIIa. In some

examples of any of the multi-chain chimeric polypeptides described herein, the soluble

tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi- chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some examples, the soluble tissue factor domain can be a soluble human tissue

factor domain. In some embodiments, the soluble tissue factor domain can be a soluble

mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can

be a soluble rat tissue factor domain.

In some examples, the soluble tissue factor domain does not include one or more

(e.g., two, three, four, five, six, or seven) of: a lysine at an amino acid position that

corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an

isoleucine at an amino acid position that corresponds to amino acid position 22 of mature

wildtype human tissue factor protein; a tryptophan at an amino acid position that

corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an

aspartic acid at an amino acid position that corresponds to amino acid position 58 of

mature wildtype human tissue factor protein; a tyrosine at an amino acid position that

corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an

arginine at an amino acid position that corresponds to amino acid position 135 of mature

wildtype human tissue factor protein; and a phenylalanine at an amino acid position that

corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

In some embodiments, the mutant soluble tissue factor possesses the amino acid sequence

of SEQ ID NO: 97 or SEQ ID NO: 98.

In some examples, the soluble tissue factor domain can be encoded by a nucleic

acid including a sequence that is at least 70% identical, at least 72% identical, at least

74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at

least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical,

at least 90% identical, at least 92% identical, at least 94% identical, at least 96%

identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:

94.

In some embodiments, the soluble tissue factor domain can have a total length of

about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino

acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205

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amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to

about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino

acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20

amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids,

about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino

acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150

amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to

about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino

acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20

amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids,

about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino

acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95

amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to

about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino

acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20

amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids,

about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino

acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215

amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to

about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino

acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30

amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids,

about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino

acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160

amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to

about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino

acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30

amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids,

about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino

acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165

195

PCT/US2021/035285

amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to

about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino

acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50

amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids,

about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino

acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110

amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to

about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino

acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50

amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids,

about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino

acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215

amino acids, about 60 amino acids to about 210 amino acids, about 60 amino acids to

about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino

acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60

amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids,

about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino

acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160

amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to

about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino

acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60

amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids,

about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino

acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105

amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to

about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino

acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60

amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids,

about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino

acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210

WO wo 2021/247604 PCT/US2021/035285

amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to

about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino

acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90

amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids,

about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino

acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155

amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to

about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino

acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90

amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids,

about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino

acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100

amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to

about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100

amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids,

about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190

amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to

about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100

amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids,

about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155

amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to

about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100

amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids,

about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120

amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to

about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino

acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about

110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino

acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about

190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids

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to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110

amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids,

about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155

amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to

about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110

amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids,

about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120

amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to

about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115

amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids,

about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195

amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to

about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115

amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids,

about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160

amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to

about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115

amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids,

about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125

amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to

about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120

amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids,

about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195

amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to

about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120

amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids,

about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160

amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to

about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120

amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to

202

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about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180

amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids,

about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190

amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to

about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185

amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids,

about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195

amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to

about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190

amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids,

about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195

amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to

about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195

amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids,

about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215

amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to

about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205

amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids,

about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215

amino acids, or about 215 amino acids to about 220 amino acids.

Linker Sequences

In some embodiments, the linker sequence can be a flexible linker sequence.

Non-limiting examples of linker sequences that can be used are described in Klein et al.,

Protein Engineering, Design & Selection 27(10):325-330, 2014; Priyanka et al., Protein

Sci. 2(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker

sequence.

In some embodiments of any of the single-chain chimeric polypeptides described

herein can include one, two, three, four, five, six, seven, eight, nine, or ten linker

sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker

WO wo 2021/247604 PCT/US2021/035285

sequences described herein or known in the art). In some embodiments of any of the

single-chain chimeric polypeptides described herein can include one, two, three, four,

five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker

sequences, e.g., any of the exemplary linker sequences described herein or known in the

art).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first chimeric polypeptide can include one, two, three, four, five, six, seven,

eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g.,

any of the exemplary linker sequences described herein or known in the art). In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the

second chimeric polypeptide can include one, two, three, four, five, six, seven, eight,

nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g., any of

the exemplary linker sequences described herein or known in the art).

In some embodiments, a linker sequence can have a total length of 1 amino acid

to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80

amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids,

1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino

acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to

about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 24

amino acids, 1 amino acid to about 22 amino acids, 1 amino acid to about 20 amino acids,

1 amino acid to about 18 amino acids, 1 amino acid to about 16 amino acids, 1 amino

acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to

about 10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 6

amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100

amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about

80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to

about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids

to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino

acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2

amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about

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2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids,

about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino

acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12

amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8

amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4

amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about

90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to

about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids

to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino

acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4

amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about

4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids,

about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino

acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14

amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about

10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about

6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to

about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids

to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino

acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6

amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about

6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids,

about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino

acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18

amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about

14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to

about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to

about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids

to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino

acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8

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amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about

8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids,

about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino

acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20

amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about

16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to

about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids

to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino

acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10

amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids,

about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino

acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30

amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to

about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino

acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10

amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids,

about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino

acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80

amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to

about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino

acids to about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12

amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids,

about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino

acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20

amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to

about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino

acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14

amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids,

about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino

acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40

206 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60

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amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to

about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino

acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22

amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids,

about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino

acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70

amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to

about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino

acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25

amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids,

about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino

acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60

amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to

about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino

acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35

amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids,

about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino

acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45

amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to

about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino

acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40

amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids,

about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino

acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80

amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to

about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino

acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50

amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids,

about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino

acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80

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amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to

about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino

acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80

amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

In some embodiments, the linker is rich in glycine (Gly or G) residues. In some

embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the

linker is rich in glycine and serine residues. In some embodiments, the linker has one or

more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS

pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ

ID NO: 99) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS (SEQ ID NO:

99) sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser

(GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS (SEQ ID NO:

100) sequences In some embodiments, the linker has one or more Gly-Gly-Ser-Gly

(GGSG) (SEQ ID NO: 101) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG

(SEQ ID NO: 101) sequences. In some embodiments, the linker comprises

GGSSRSSSSGGGGSGGGG (SEQ ID NO: 222). In some embodiments, the linker sequence can comprise or consist of

GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker sequence

can be encoded by a nucleic acid comprising or consisting of:

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT( (SEQ ID NO: 103). In some embodiments, the linker sequence can comprise or consist of:

GGGSGGGS (SEQ ID NO: 104),

Target-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain, the second target-binding domain, and/or the

additional one or more target-binding domains can be an antigen-binding domain (e.g.,

any of the exemplary antigen-binding domains described herein or known in the art), a

soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin

proteins or soluble cytokine proteins described herein), and a soluble interleukin or

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cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble

cytokine receptors described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain, the second target-binding domain, and/or the one

or more additional target-binding domains can each independent have a total number of

amino acids of about 5 amino acids to about 1000 amino acids, about 5 amino acids to

about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino

acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5

amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids,

about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino

acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500

amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about

400 amino acids, about 5 amino acids to about 350 amino acids, about 5 amino acids to

about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino

acids to about 260 amino acids, about 5 amino acids to about 240 amino acids, about 5

amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids,

about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino

acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180

amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about

170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to

about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino

acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5

amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids,

about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino

acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115

amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about

105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to

about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids

to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino

acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5

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amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about

5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids,

about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino

acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30

amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about

20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to

about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino

acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10

amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids,

about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino

acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600

amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to

about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino

acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10

amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids,

about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino

acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200

amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to

about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino

acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10

amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids,

about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino

acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145

amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to

about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino

acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10

amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids,

about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino

acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90

amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to

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about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino

acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10

amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids,

about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino

acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35

amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to

about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino

acids to about 15 amino acids, about 15 amino acids to about 1000 amino acids, about 15

amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids,

about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino

acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700

amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to

about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino

acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15

amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids,

about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino

acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240

amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to

about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino

acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15

amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids,

about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino

acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155

amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to

about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino

acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15

amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids,

about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino

acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100

amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to

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about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino

acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20

amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids,

about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino

acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50

amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to

about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino

acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25

amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids,

about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino

acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750

amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to

about 650 amino acids, about 25 amino acids to about 600 amino acids, about 25 amino

acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25

amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids,

about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino

acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260

amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to

about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino

acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25

amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids,

about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino

acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160

amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to

about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino

acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25

amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids,

about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino

acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105

amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to

214 about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to

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about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino

acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30

amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids,

about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino

acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45

amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to

about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino

acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35

amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids,

about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino

acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600

amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to

about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino

acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35

amino acids to about 300 amino acids, about 35 amino acids to about 280 amino acids,

about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino

acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200

amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to

about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino

acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35

amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids,

about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino

acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145

amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to

about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino

acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35

amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids,

about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino

acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90

amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to

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about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino

acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35

amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids,

about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino

acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000

amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to

about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino

acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40

amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids,

about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino

acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450

amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to

about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino

acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40

amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids,

about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino

acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185

amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to

about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino

acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40

amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids,

about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino

acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130

amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to

about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino

acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40

amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids,

about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino

acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75

amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to

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about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino

acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40

amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids,

about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino

acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800

amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to

about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino

acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45

amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids,

about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino

acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280

amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to

about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino

acids to about 200 amino acids, about 45 amino acids to about 195 amino acids, about 45

amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids,

about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino

acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165

amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to

about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino

acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45

amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids,

about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino

acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110

amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to

about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino

acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45

amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids,

about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino

acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55

amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to

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about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino

acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60

amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids,

about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino

acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185

amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to

about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino

acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60

amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids,

about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino

acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130

amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to

about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino

acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60

amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids,

about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino

acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75

amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to

about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino

acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65

amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids,

about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino

acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600

amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to

about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino

acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65

amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids,

about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino

acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200

amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids, about 70 amino acids to about 700 amino acids, about 70 amino acids to about 650 amino acids, about 70 amino acids to about 600 amino acids, about 70 amino acids to about 550 amino acids, about 70 amino acids to about 500 amino acids, about 70 amino acids to about 450 amino acids, about 70 amino acids to about 400 amino acids, about 70 amino acids to about 350 amino acids, about 70 amino acids to about 300 amino acids, about 70 amino acids to about 280 amino acids, about 70 amino acids to about 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to

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about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino

acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70

amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids,

about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino

acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100

amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to

about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino

acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75

amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids,

about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino

acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750

amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to

about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino

acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75

amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids,

about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino

acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260

amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to

about 220 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino

acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75

amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids,

about 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino

acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160

amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to

about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino

acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75

amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids,

about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino

acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105

amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino

acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80

amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids,

about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino

acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750

amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to

about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino

acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80

amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids,

about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino

acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260

amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to

about 220 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino

acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80

amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids,

about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino

acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160

amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to

about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino

acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80

amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids,

about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino

acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105

amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to

about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino

acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85

amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids,

about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino

acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700

amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to

224

PCT/US2021/035285

about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino

acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85

amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids,

about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino

acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240

amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to

about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino

acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85

amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids,

about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino

acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155

amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to

about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino

acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85

amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids,

about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino

acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100

amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to

about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino

acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90

amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids,

about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino

acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600

amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to

about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino

acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90

amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids,

about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino

acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200

amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to

PCT/US2021/035285

about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino

acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90

amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids,

about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino

acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145

amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to

about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino

acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90

amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids,

about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino

acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000

amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to

about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino

acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95

amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids,

about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino

acids, about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450

amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to

about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino

acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95

amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids,

about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino

acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185

amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to

about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino

acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95

amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids,

about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino

acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130

amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to

226

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino

acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95

amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids,

about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900

amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to

about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100

amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids,

about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550

amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to

about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100

amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids,

about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260

amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to

about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100

amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids,

about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180

amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to

about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100

amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids,

about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145

amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to

about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100

amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids,

about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110

amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to

about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105

amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids,

about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750

amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to

about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105

WO wo 2021/247604 PCT/US2021/035285

amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids,

about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400

amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to

about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105

amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids,

about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200

amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to

about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105

amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids,

about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165

amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to

about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105

amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids,

about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130

amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to

about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105

amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids,

about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900

amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to

about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110

amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids,

about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550

amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to

about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110

amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids,

about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260

amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to

about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110

amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids,

about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180

WO wo 2021/247604 PCT/US2021/035285

amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to

about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110

amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids,

about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145

amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to

about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110

amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids,

about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000

amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to

about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115

amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids,

about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650

amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to

about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115

amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids,

about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300

amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to

about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115

amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids,

about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190

amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to

about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115

amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids,

about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155

amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to

about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115

amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids,

about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120

amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to

about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids,

about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700

amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to

about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120

amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids,

about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350

amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to

about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120

amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids,

about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195

amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to

about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120

amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids,

about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160

amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to

about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120

amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids,

about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125

amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to

about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125

amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids,

about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700

amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to

about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125

amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids,

about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350

amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to

about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125

amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids,

about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195

WO wo 2021/247604 PCT/US2021/035285

amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to

about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125

amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids,

about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160

amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to

about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125

amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids,

about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000

amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to

about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130

amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids,

about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650

amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to

about 550 amino acids, about 130 amino acids to about 500 amino acids, about 130

amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids,

about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300

amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to

about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130

amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids,

about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190

amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to

about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130

amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids,

about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155

amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to

about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130

amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids,

about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900

amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to

about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135

WO wo 2021/247604 PCT/US2021/035285

amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids,

about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550

amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to

about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135

amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids,

about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260

amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to

about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135

amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids,

about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180

amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to

about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135

amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids,

about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145

amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to

about 1000 amino acids, about 140 amino acids to about 950 amino acids, about 140

amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids,

about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750

amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to

about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140

amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids,

about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400

amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to

about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140

amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids,

about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200

amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to

about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140

amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids,

about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to

about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140

amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids,

about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900

amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to

about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145

amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids,

about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550

amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to

about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145

amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids,

about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260

amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to

about 220 amino acids, about 145 amino acids to about 200 amino acids, about 145

amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids,

about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180

amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to

about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145

amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids,

about 145 amino acids to about 150 amino acids, about 150 amino acids to about 1000

amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to

about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150

amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids,

about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650

amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to

about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150

amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids,

about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300

amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to

about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids,

about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190

amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to

about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150

amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids,

about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155

amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to

about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155

amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids,

about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700

amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to

about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155

amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids,

about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350

amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to

about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155

amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids,

about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195

amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to

about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155

amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids,

about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160

amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to

about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160

amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids,

about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700

amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to

about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160

amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids,

about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to

about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160

amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids,

about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195

amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to

about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160

amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids,

about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000

amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to

about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165

amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids,

about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650

amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to

about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165

amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids,

about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300

amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to

about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165

amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids,

about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190

amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to

about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165

amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids,

about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900

amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to

about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170

amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids,

about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550

amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to

about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170

PCT/US2021/035285

amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids,

about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260

amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to

about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170

amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids,

about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180

amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to

about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175

amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids,

about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750

amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to

about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175

amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids,

about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400

amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to

about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175

amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids,

about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200

amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to

about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175

amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids,

about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900

amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to

about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180

amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids,

about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550

amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to

about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180

amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids,

about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260

PCT/US2021/035285

amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to

about 220 amino acids, about 180 amino acids to about 200 amino acids, about 180

amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids,

about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000

amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to

about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185

amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids,

about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650

amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to

about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185

amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids,

about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300

amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to

about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185

amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids,

about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190

amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to

about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190

amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids,

about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700

amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to

about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190

amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids,

about 190 amino acids to about 400 amino acids, about 190 amino acids to about 350

amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to

about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190

amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids,

about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195

amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to

about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300

239

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amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids,

about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700

amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to

about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300

amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids,

about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350

amino acids, about 350 amino acids to about 1000 amino acids, about 350 amino acids to

about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350

amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids,

about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700

amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to

about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350

amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids,

about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000

amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to

about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400

amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids,

about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650

amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to

about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400

amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids,

about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900

amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to

about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450

amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids,

about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550

amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to

about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500

amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids,

about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750

240

WO wo 2021/247604 PCT/US2021/035285

amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to

about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500

amino acids to about 550 amino acids, about 550 amino acids to about 1000 amino acids,

about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900

amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to

about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550

amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids,

about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000

amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to

about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600

amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids,

about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650

amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to

about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650

amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids,

about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700

amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to

about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700

amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids,

about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000

amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to

about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750

amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids,

about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900

amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to

about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850

amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids,

about 900 amino acids to about 950 amino acids, or about 950 amino acids to about 1000

amino acids.

PCT/US2021/035285

Any of the target-binding domains described herein can bind to its target with a

dissociation equilibrium constant (KD) of less than 1 X 10-7 M, less than 1 X 10-8 M, less

than 1 X 10-9 M, less than 1 X 10-10 M, less than 1 X 10-11 M, less than 1 X 10-12 M, or less

than 1 X 10-13 M. In some embodiments, the antigen-binding protein construct provided

herein can bind to an identifying antigen with a KD of about 1 X 10-3 M to about 1 X 10-5

M, about 1 X 10-4 M to about 1 X 10-6 M, about 1 X 10-5 M to about 1 X 10-7 M, about 1 X

10-6 M to about 1 X 10-8 M, about 1 x 10-7 M to about 1 X 10-9 M, about 1 X 10-8 M to

about 1 X 10-10 M, or about 1 X 10-9 M to about 1 X 10-11 M (inclusive).

Any of the target-binding domains described herein can bind to its target with a

KD of between about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM

to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to

about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about

950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about

800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about

650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about

500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about

350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about

200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about

90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60

pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30

pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM,

about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2

pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM

to about 15 nM, about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to

about 2 nM, about 2 pM to about 1 nM, about 2 pM to about 950 pM, about 2 pM to

about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM, about 2 pM to

about 750 pM, about 2 pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to

about 600 pM, about 2 pM to about 550 pM, about 2 pM to about 500 pM, about 2 pM to

about 450 pM, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to

about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM to

WO wo 2021/247604 PCT/US2021/035285

about 150 pM, about 2 pM to about 100 pM, about 2 pM to about 90 pM, about 2 pM to

about 80 pM, about 2 pM to about 70 pM, about 2 pM to about 60 pM, about 2 pM to

about 50 pM, about 2 pM to about 40 pM, about 2 pM to about 30 pM, about 2 pM to

about 20 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 2 pM to

about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about

25 nM, about 5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10

nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM to about 1 nM,

about 5 pM to about 950 pM, about 5 pM to about 900 pM, about 5 pM to about 850 pM,

about 5 pM to about 800 pM, about 5 pM to about 750 pM, about 5 pM to about 700 pM,

about 5 pM to about 650 pM, about 5 pM to about 600 pM, about 5 pM to about 550 pM,

about 5 pM to about 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM,

about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM,

about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM,

about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM,

about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM,

about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM,

about 10 pM to about 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM,

about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM to about 5 nM,

about 10 pM to about 2 nM, about 10 pM to about 1 nM, about 10 pM to about 950 pM,

about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800

pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about

650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to

about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10

pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about

10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM,

about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM,

about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM,

about 10 pM to about 30 pM, about 10 pM to about 20 pM, about 15 pM to about 30 nM,

about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM,

about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM,

PCT/US2021/035285

about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900

pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about

750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to

about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15

pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about

15 pM to about 300 pM, about 15 pM to about 250 pM, about 15 pM to about 200 pM,

about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90

pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60

pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30

pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25

nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10

nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1

nM, about 20 pM to about 950 pM, about 20 pM to about 900 pM, about 20 pM to about

850 pM, about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM to

about 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM, about 20

pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM to about 450 pM, about

20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM,

about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150

pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about

80 pM, about 20 pM to about 70 pM, about 20 pM to about 60 pM, about 20 pM to about

50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about

30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM, about 30 pM to about

15 nM, about 30 pM to about 10 nM, about 30 pM to about 5 nM, about 30 pM to about 2

nM, about 30 pM to about 1 nM, about 30 pM to about 950 pM, about 30 pM to about

900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to

about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 30

pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about

30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM,

about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200

pM, about 30 pM to about 150 pM, about 30 pM to about 100 pM, about 30 pM to about

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

90 pM, about 30 pM to about 80 pM, about 30 pM to about 70 pM, about 30 pM to about

60 pM, about 30 pM to about 50 pM, about 30 pM to about 40 pM, about 40 pM to about

30 nM, about 40 pM to about 25 nM, about 40 pM to about 30 nM, about 40 pM to about

15 nM, about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2

nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about

900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to

about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40

pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about

40 pM to about 450 pM, about 40 pM to about 400 pM, about 40 pM to about 350 pM,

about 40 pM to about 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200

pM, about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM to about

90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about

60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about

25 nM, about 50 pM to about 30 nM, about 50 pM to about 15 nM, about 50 pM to about

10 nM, about 50 pM to about 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1

nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about

850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM, about 50 pM to

about 700 pM, about 50 pM to about 650 pM, about 50 pM to about 600 pM, about 50

pM to about 550 pM, about 50 pM to about 500 pM, about 50 pM to about 450 pM, about

50 pM to about 400 pM, about 50 pM to about 350 pM, about 50 pM to about 300 pM,

about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150

pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about

80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about

30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about

15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2

nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about

900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to

about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60

pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about

60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about

90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about

30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about

15 nM, about 70 pM to about 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2

nM, about 70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about

900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about 70 pM to

about 750 pM, about 70 pM to about 700 pM, about 70 pM to about 650 pM, about 70

pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about

70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM,

about 70 pM to about 300 pM, about 70 pM to about 250 pM, about 70 pM to about 200

pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about

90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about

25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80 pM to about

10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM, about 80 pM to about 1

nM, about 80 pM to about 950 pM, about 80 pM to about 900 pM, about 80 pM to about

850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to

about 700 pM, about 80 pM to about 650 pM, about 80 pM to about 600 pM, about 80

pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about

80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM,

about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM to about 150

pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about

30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about

15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2

nM, about 90 pM to about 1 nM, about 90 pM to about 950 pM, about 90 pM to about

900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to

about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90

pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM, about

90 pM to about 450 pM, about 90 pM to about 400 pM, about 90 pM to about 350 pM,

about 90 pM to about 300 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about

100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM,

about 100 pM to about 900 pM, about 100 pM to about 850 pM, about 100 pM to about

800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM

to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about

100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400

pM, about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to

about 250 pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about

150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM,

about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5

nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM, about 150 pM to about

950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM

to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about 700 pM, about

150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 550

pM, about 150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM to

about 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300 pM, about

150 pM to about 250 pM, about 150 pM to about 200 pM, about 200 pM to about 30 nM,

about 200 pM to about 25 nM, about 200 pM to about 30 nM, about 200 pM to about 15

nM, about 200 pM to about 10 nM, about 200 pM to about 5 nM, about 200 pM to about

2 nM, about 200 pM to about 1 nM, about 200 pM to about 950 pM, about 200 pM to

about 900 pM, about 200 pM to about 850 pM, about 200 pM to about 800 pM, about

200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650

pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about 200 pM to

about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM, about

200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250

pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to

about 30 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300

pM to about 5 nM, about 300 pM to about 2 nM, about 300 pM to about 1 nM, about 300

WO wo 2021/247604 PCT/US2021/035285

pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM,

about 300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM to about

700 pM, about 300 pM to about 650 pM, about 300 pM to about 600 pM, about 300 pM

to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about

300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30

nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about

15 nM, about 400 pM to about 10 nM, about 400 pM to about 5 nM, about 400 pM to

about 2 nM, about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pM

to about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800 pM, about

400 pM to about 750 pM, about 400 pM to about 700 pM, about 400 pM to about 650

pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to

about 500 pM, about 500 pM to about 30 nM, about 500 pM to about 25 nM, about 500

pM to about 30 nM, about 500 pM to about 15 nM, about 500 pM to about 10 nM, about

500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about

500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850

pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to

about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about

500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM,

about 600 pM to about 30 nM, about 600 pM to about 15 nM, about 600 pM to about 10

nM, about 600 pM to about 5 nM, about 600 pM to about 2 nM, about 600 pM to about 1

nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to

about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about

600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM to about 30

nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM, about 700 pM to about

15 nM, about 700 pM to about 10 nM, about 700 pM to about 5 nM, about 700 pM to

about 2 nM, about 700 pM to about 1 nM, about 700 pM to about 950 pM, about 700 pM

to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about

700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM,

about 800 pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10

nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pM to about 1

WO wo 2021/247604 PCT/US2021/035285

nM, about 800 pM to about 950 pM, about 800 pM to about 900 pM, about 800 pM to

about 850 pM, about 900 pM to about 30 nM, about 900 pM to about 25 nM, about 900

pM to about 30 nM, about 900 pM to about 15 nM, about 900 pM to about 10 nM, about

900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about

900 pM to about 950 pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about

1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1

nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM

to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to

about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to

about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to

about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to

about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to

about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to

about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to

about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM).

Any of the target-binding domains described herein can bind to its target with a

KD of between about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM

to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to

about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2

nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM,

about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2

nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to

about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6

nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM,

about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4

nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to

about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6

nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM,

about 6 nM to about 7 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9

nM to about 10 nM).

A variety of different methods known in the art can be used to determine the KD

values of any of the antigen-binding protein constructs described herein (e.g., an

electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance,

and a biomolecular binding kinetics assay, etc.).

Antigen-Binding Domains

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, the first target-binding domain and the second target-

binding domain bind specifically to the same antigen. In some embodiments of these

single-chain or multi-chain chimeric polypeptides, the first target-binding domain and the

second target-binding domain bind specifically to the same epitope. In some

embodiments of these single-chain or multi-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain include the same amino acid

sequence.

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, the first target-binding domain and the second target-

binding domain bind specifically to different antigens.

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain. In some embodiments of

any of the single-chain or multi-chain chimeric polypeptides described herein, the first

target-binding domain and the second target-binding domain are each antigen-binding

domains. In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, the antigen-binding domain includes or is a scFv or a

single domain antibody (e.g., a VaHH or a VNAR domain).

In some examples, an antigen-binding domain (e.g., any of the antigen-binding

domains described herein) can bind specifically to any one of CD16a (see, e.g., those

described in U.S. Patent No. 9,035,026), CD28 (see, e.g., those described in U.S. Patent

250 wo 2021/247604 WO PCT/US2021/035285

No. 7,723,482), CD3 (see, e.g., those described in U.S. Patent No. 9,226,962), CD33

(see, e.g., those described in U.S. Patent No. 8,759,494), CD20 (see, e.g., those described

in WO 2014/026054), CD19 (see, e.g., those described in U.S. Patent No. 9,701,758),

CD22 (see, e.g., those described in WO 2003/104425), CD123 (see, e.g., those described

in WO 2014/130635), IL-1R (see, e.g., those described in U.S. Patent No. 8,741,604), IL-

1 (see, e.g., those described in WO 2014/095808), VEGF (see, e.g., those described in

U.S. Patent No. 9,090,684), IL-6R (see, e.g., those described in U.S. Patent No.

7,482,436), IL-4 (see, e.g., those described in U.S. Patent Application Publication No.

2012/0171197), IL-10 (see, e.g., those described in U.S. Patent Application Publication

No. 2016/0340413), PDL-1 (see, e.g., those described in Drees et al., Protein Express.

Purif. 94:60-66, 2014), TIGIT (see, e.g., those described in U.S. Patent Application

Publication No. 2017/0198042), PD-1 (see, e.g., those described in U.S. Patent No.

7,488,802), TIM3 (see, e.g., those described in U.S. Patent No. 8,552,156), CTLA4 (see,

e.g., those described in WO 2012/120125), MICA (see, e.g., those described in WO

2016/154585), MICB (see, e.g., those described in U.S. Patent No. 8,753,640), IL-6 (see,

e.g., those described in Gejima et al., Human Antibodies 11(4):121-129, 2002), IL-8 (see,

e.g., those described in U.S. Patent No. 6,117,980), TNFa (see, e.g., those described in

Geng et al., Immunol. Res. 62(3):377-385, 2015), CD26 (see, e.g., those described in WO

2017/189526), CD36 (see, e.g., those described in U.S. Patent Application Publication

No. 2015/0259429), ULBP2 (see, e.g., those described in U.S. Patent No. 9,273,136),

CD30 (see, e.g., those described in Homach et al., Scand. J. Immunol. 48(5):497-501,

1998), CD200 (see, e.g., those described in U.S. Patent No. 9,085,623), IGF-1R (see, e.g.,

those described in U.S. Patent Application Publication No. 2017/0051063), MUC4AC

(see, e.g., those described in WO 2012/170470), MUC5AC (see, e.g., those described in

U.S. Patent No. 9,238,084), Trop-2 (see, e.g., those described in WO 2013/068946),

CMET (see, e.g., those described in Edwardraja et al., Biotechnol. Bioeng. 106(3):367-

375,2010), EGFR (see, e.g., those described in Akbari et al., Protein Expr. Purif. 127:8-

15, ,2016), HER1 (see, e.g., those described in U.S. Patent Application Publication No.

2013/0274446), HER2 (see, e.g., those described in Cao et al., Biotechnol. Lett.

37(7):1347-1354, 2015), HER3 (see, e.g., those described in U.S. Patent No. 9,505,843),

WO wo 2021/247604 PCT/US2021/035285

PSMA (see, e.g., those described in Parker et al., Protein Expr. Purif. 89(2):136-145,

2013), CEA (see, e.g., those described in WO 1995/015341), B7H3 (see, e.g., those

described in U.S. Patent No. 9,371,395), EPCAM (see, e.g., those described in WO

2014/159531), BCMA (see, e.g., those described in Smith et al., Mol. Ther. 26(6):1447-

1456, 2018), P-cadherin (see, e.g., those described in U.S. Patent No. 7,452,537),

CEACAM5 (see, e.g., those described in U.S. Patent No. 9,617,345), a UL16-binding

protein (see, e.g., those described in WO 2017/083612), HLA-DR (see, e.g., Pistillo et al.,

Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described in WO

2014/007513), TYRO3 (see, e.g., those described in WO 2016/166348), AXL (see, e.g.,

those described in WO 2012/175692), MER (see, e.g., those described in WO

2016/106221), CD122 (see, e.g., those described in U.S. Patent Application Publication

No. 2016/0367664), CD155 (see, e.g., those described in WO 2017/149538), or PDGF-

DD (see, e.g., those described in U.S. Patent No. 9,441,034).

The antigen-binding domains present in any of the single-chain or multi-chain

chimeric polypeptides described herein are each independently selected from the group

consisting of: a VHH domain, a VNAR domain, and a scFv. In some embodiments, any

of the antigen-binding domains described herein is a BiTe, a (scFv)2, a nanobody, a

nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-

scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. Additional examples of antigen-

binding domains that can be used in any of the single-chain or multi-chain chimeric

polypeptide are known in the art.

A VHH domain is a single monomeric variable antibody domain that can be

found in camelids. A VNAR domain is a single monomeric variable antibody domain

that can be found in cartilaginous fish. Non-limiting aspects of VHH domains and VNAR

domains are described in, e.g., Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016;

De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends

Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva

et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol.

Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255,

2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends

252

WO wo 2021/247604 PCT/US2021/035285

Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013;

Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al.,

EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-

1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al.,

Med. Microbiol. Immunol. 198:157-174, 2009.

In some embodiments, each of the antigen-binding domains in the single-chain or

multi-chain chimeric polypeptides described herein are both VHH domains, or at least

one antigen-binding domain is a VHH domain. In some embodiments, each of the

antigen-binding domains in the single-chain or multi-chain chimeric polypeptides

described herein are both VNAR domains, or at least one antigen-binding domain is a

VNAR domain. In some embodiments, each of the antigen-binding domains in the

single-chain or multi-chain chimeric polypeptides described herein are both scFv

domains, or at least one antigen-binding domain is a scFv domain.

In some embodiments, two or more of polypeptides present in the single-chain or

multi-chain chimeric polypeptide can assemble (e.g., non-covalently assemble) to form

any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of

an antibody (e.g., any of the antigen-binding fragments of an antibody described herein),

a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-

in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light

chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a

LUZ-Y, a Fcab, a ka-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-

(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-

IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-

CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-

scFv, a F(ab')2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc,

a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG

conjugate, a Cov-X-Body, and a scFv1-PEG-scFv2. See, e.g., Spiess et al., Mol.

Immunol. 67:95-106, 2015, incorporated in its entirety herewith, for a description of these

elements. Non-limiting examples of an antigen-binding fragment of an antibody include

an Fv fragment, a Fab fragment, a F(ab')2 fragment, and a Fab' fragment. Additional

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examples of an antigen-binding fragment of an antibody is an antigen-binding fragment

of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an

antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1,

IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding

fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized

IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD

(e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding

fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or

an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or

humanized IgM).

An "Fv" fragment includes a non-covalently-linked dimer of one heavy chain

variable domain and one light chain variable domain.

A "Fab" fragment includes, the constant domain of the light chain and the first

constant domain (CH1) of the heavy chain, in addition to the heavy and light chain

variable domains of the Fv fragment.

A "F(ab')2" fragment includes two Fab fragments joined, near the hinge region, by

disulfide bonds.

A "dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and

multispecific binding proteins as described, e.g., in DiGiammarino et al., Methods Mol.

Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent Nos.

7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each

of which is incorporated by reference in its entirety.

DARTS are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799-

801, 2014.

In some embodiments of any of the antigen-binding domains described herein can

bind to an antigen selected from the group consisting of: a protein, a carbohydrate, a

lipid, and a combination thereof.

Additional examples and aspects of antigen-binding domains are known in the art.

Soluble Interleukin or Cytokine Protein

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain can be a soluble interleukin protein or soluble cytokine

protein. In some embodiments, the soluble interleukin or soluble cytokine protein is

selected from the group of: IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-

21, PDGF-DD, and SCF. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10,

IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF are provided below.

Human Soluble IL-3 (SEQ ID NO: 105)

apmtqttplkt swvncsnmid eiithlkqpp lplldfnnln gedgdilmen nlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikd gdwnefrrkl tfylktlena qaqqttlsla if

Human Soluble IL-8 (SEQ ID NO: 106)

egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldp kenwvqrvve kflkraens

Human Soluble IL-10 (SEQ ID NO: 107)

spgqgtqsensc thfpgnlpnm lrdlrdafsr vktffqmkdq ldnlllkesl ledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlr lrlrrchrfl pcenkskave qvknafnklq ekgiykamse fdifinyiea ymtmkirn

Human Soluble IL-17 (SEQ ID NO: 108)

gitiprn pgcpnsedkn fprtvmvnln ihnrntntnp krssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeil vlrrepphcp nsfrlekilv svgctcvtpi vhhva

Human Soluble IL-18 (SEQ ID NO: 109)

yfgklesklsvirn lndqvlfidq gnrplfedmt dsdcrdnapr tifiismykd sqprgmavti svkcekistl scenkiisfk emnppdnikd tksdiiffqr svpghdnkmq fesssyegyf lacekerdlf klilkkedel gdrsimftvq ned

Human Soluble PDGF-DD (SEQ ID NO: 110)

rdtsatpasasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfp nsyprnlllt wrlhsqentr iqlvfdnqfg leeaendicr ydfvevedis ksrtngikit fksddyfvak pgfkiyysll etstiirgrw cghkevppri ksrtnqikit edfqpaaase tnwesvtssi sgvsynspsv tdptliadal dkkiaefdtv edllkyfnpe swqedlenmy ldtpryrgrs yhdrkskvdl drlnddakry sctprnysvn ireelklanv vffprcllvq rcggncgcgt vnwrsctcns gktvkkyhev lqfepghikr rgraktmalv diqldhherc dcicssrppr

Human Soluble SCF (SEQ ID NO: 111)

egicrnrvtnnvkdv tklvanlpkd ymitlkyvpg mdvlpshcwi semvvqlsds ltdlldkfsn iseglsnysi idklvnivdd lvecvkenss kdlkksfksp eprlftpeef frifnrsida fkdfvvaset sdcvvsstls pekdsrvsvt kpfmlppvaa sslrddssss sslrndssss nrkaknppgd sslhwaamal palfsliigf afgalywkkr qpsltraven iqineednei smlqekeref qev

Human Soluble FLT3L (SEQ ID NO: 112)

tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq cafapppscl rfvqtnisrl rwmerlktva gskmqgller vnteihfvtk cafqpppscl 1kpwitrqnf srclelqcqp dsstlpppws prpleatapt lqetseqlva lkpwitrqnf apapplllll apqpplllll 11pvglllla llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl vppvpspqd1 llveh

Additional examples of soluble interleukin proteins and soluble cytokine proteins

are known in the art.

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Soluble Receptor

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is a soluble interleukin receptor or a soluble cytokine

receptor. In some embodiments, the soluble receptor is a soluble TGF-B receptor II

(TGF-B RII) (see, e.g., those described in Yung et al., Am. J. Resp. Crit. Care Med.

194(9):1140-1151, 2016), a soluble TGF-BRIII (see, e.g., those described in Heng et al.,

Placenta 57:320, 2017), a soluble NKG2D (see, e.g., Cosman et al., Immunity 14(2):123-

133,2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:

10.3389/fimmu.2018.01150), a soluble NKp30 (see, e.g., Costa et al., Front. Immunol.,

Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), a soluble NKp44

(see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29,

2018; doi: 10.3389/fimmu.2018.01150), a soluble NKp46 (see, e.g., Mandelboim et al.,

Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May

29, 2018; doi: 10.3389/fimmu.2018.01150) a soluble DNAMI (see, e.g., those described

in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:

10.3389/fimmu.2018.01150), a scMHCI (see, e.g., those described in Washburn et al.,

PLoS One 6(3):e18439, 2011), a scMHCII (see, e.g., those described in Bishwajit et al.,

Cellular Immunol. 170(1):25-33, 1996), a scTCR (see, e.g., those described in Weber et

al., Nature 356(6372):793-796, 1992), a soluble CD155 (see, e.g., those described in

Tahara-Hanaoka et al., Int. Immunol. 16(4):533-538, 2004), or a soluble CD28 (see, e.g.,

Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004).

Additional examples of soluble interleukin receptors and soluble cytokine

receptors are known in the art.

Pairs of Affinity Domains

In some embodiments, a multi-chain chimeric polypeptide includes: 1) a first

chimeric polypeptide that includes a first domain of a pair of affinity domains, and 2) a

second chimeric polypeptide that includes a second domain of a pair of affinity domains

such that the first chimeric polypeptide and the second chimeric polypeptide associate

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through the binding of the first domain and the second domain of the pair of affinity

domains. In some embodiments, the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra) and a soluble IL-15. A sushi domain,

also known as a short consensus repeat or type 1 glycoprotein motif, is a common motif

in protein-protein interaction. Sushi domains have been identified on a number of

protein-binding molecules, including complement components C1r, C1s, factor H, and

C2m, as well as the nonimmunologic molecules factor XIII and B2-glycoprotein. A

typical Sushi domain has approximately 60 amino acid residues and contains four

cysteines (Ranganathan, Pac. Symp Biocomput. 2000:155-67). The first cysteine can

form a disulfide bond with the third cysteine, and the second cysteine can form a

disulfide bridge with the fourth cysteine. In some embodiments in which one member of

the pair of affinity domains is a soluble IL-15, the soluble IL-15 has a D8N or D8A

amino acid substitution. In some embodiments in which one member of the pair of

affinity domains is an alpha chain of human IL-15 receptor (IL-15Ra), the human IL-

15Ra is a mature full-length IL-15Ra. In some embodiments, the pair of affinity

domains is barnase and barnstar. In some embodiments, the pair of affinity domains is a

PKA and an AKAP. In some embodiments, the pair of affinity domains is an

adapter/docking tag module based on mutated RNase I fragments (Rossi, Proc Natl Acad

Sci USA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290, 2008; Rossi et

al., Trends Pharmacol Sci. 33:474-481, 2012) or SNARE modules based on interactions

of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat

Biotechnol. 1486-1492, 2003).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide includes a first domain of a pair of affinity domains and a second chimeric

polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair

of affinity domains, wherein the first domain of the pair of affinity domains and the

second domain of the pair of affinity domains bind to each other with a dissociation

equilibrium constant (KD) of less than 1 X 10-7 M, less than 1 X 10-8 M, less than 1 x 10-9

M, less than 1 X 10-10 M, less than 1 X 10-11 M, less than 1 10-12 M, or less than X 10-13

M. In some embodiments, the first domain of the pair of affinity domains and the second

WO wo 2021/247604 PCT/US2021/035285

domain of the pair of affinity domains bind to each other with a KD of about 1 X 10-4 M to

about 1 X 10-6 M, about 1 10-5 M to about 1 X 10-7 M, about 1 X 10-6 M to about 1 x 10-8

M, about 1 x 10-7 M to about 1 X 10-9 M, about 1 X 10-8 M to about 1 X 10-10 M, about 1 X

10-9 M to about 1 X 10-11 M, about 1 X 10-10 M to about 1 X 10-12 M, about 1 x 10-11 M to

about X 10-13 M, about 1 x 10-4 M to about 1 X 10-5 M, about 1 x 10-5 M to about 1 X 10-

6 M, about 1 x 10-6 M to about 1 X 10-7 M, about 1 x 10-7 M to about 1 X 10-8 M, about 1 X

10-8 M to about 1 x 10-9 M, about 1 x 10-9 M to about 1 X 10-10 M, about 1 X 10-10 M to

about 1 x 10-11 M, about 1 x 10-11 M to about 10-12 M, or about 1 10-12 M to about 1

X 10-13 M (inclusive). Any of a variety of different methods known in the art can be used

to determine the KD value of the binding of the first domain of the pair of affinity

domains and the second domain of the pair of affinity domains (e.g., an electrophoretic

mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular

binding kinetics assay, etc.).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide includes a first domain of a pair of affinity domains and a second chimeric

polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair

of affinity domains, wherein the first domain of the pair of affinity domains, the second

domain of the pair of affinity domains, or both is about 10 to 100 amino acids in length.

For example, a first domain of a pair of affinity domains, a second domain of a pair of

affinity domains, or both can be about 10 to 100 amino acids in length, about 15 to 100

amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids

in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length,

about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to

100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino

acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in

length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length,

about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to

100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino

acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in

length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about

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10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55

amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in

length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about

10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20

amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in

length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about

50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80

amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids

in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length,

about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range

in between. In some embodiments, a first domain of a pair of affinity domains, a second

domain of a pair of affinity domains, or both is about 10, 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the first and/or second domains of a pair of affinity

domains disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5,

6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, SO long as the

function of the first and/or second domains of a pair of affinity domains remains intact.

For example, a sushi domain from an alpha chain of human IL-15 receptor (IL-15Ra) can

include one or more additional amino acids at the N-terminus and/or the C-terminus,

while still retaining the ability to bind to a soluble IL-15. Additionally or alternatively, a

soluble IL-15 can include one or more additional amino acids at the N-terminus and/or

the C-terminus, while still retaining the ability to bind to a sushi domain from an alpha

chain of human IL-15 receptor (IL-15Ra).

A non-limiting example of a sushi domain from an alpha chain of IL-15 receptor

alpha (IL-15Ra) can include a sequence that is at least 70% identical, at least 75%

identical, at least 80% identical, at least 85% identical, at least 90% identical, at least

95% identical, at least 99% identical, or 100% identical to

[TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR (SEQ ID NO: 113). In some embodiments, a sushi domain from an alpha

chain of IL-15Ra can be encoded by a nucleic acid including wo 2021/247604 WO PCT/US2021/035285

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAG CTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGA AGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGT GGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 114). In some embodiments, a soluble IL-15 can include a sequence that is at least 70%

identical, at least 75% identical, at least 80% identical, at least 85% identical, at least

90% identical, at least 95% identical, at least 99% identical, or 100% identical to

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT S (SEQ ID NO: 115). In some embodiments, a soluble IL-15 can be encoded by a

nucleic acid including the sequence of

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTO CATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAA GGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAG

CGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATA ACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGA AGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTO TCCAGATGTTCATCAATACCTCC (SEQ ID NO: 116).

Signal Sequence

In some embodiments, a single-chain chimeric polypeptide comprises a signal

sequence at its N-terminal end. In some embodiments, a multi-chain chimeric

polypeptide includes a first chimeric polypeptide that includes a signal sequence at its N-

terminal end. In some embodiments, a multi-chain chimeric polypeptide includes a

second chimeric polypeptide that includes a signal sequence at its N-terminal end. In

some embodiments, both the first chimeric polypeptide of a multi-chain chimeric

polypeptide and a second chimeric polypeptide of the multi-chain chimeric polypeptide

include a signal sequence. As will be understood by those of ordinary skill in the art, a

signal sequence is an amino acid sequence that is present at the N-terminus of a number

of endogenously produced proteins that directs the protein to the secretory pathway (e.g.,

261 the protein is directed to reside in certain intracellular organelles, to reside in the cell membrane, or to be secreted from the cell). Signal sequences are heterogeneous and differ greatly in their primary amino acid sequences. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region that contains the cleavage site for signal peptidase.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, includes a signal sequence having an amino

acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 117). In some embodiments, a

first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric

polypeptide of the multi-chain chimeric polypeptide, or both, or a single-chain chimeric

polypeptide, includes a signal sequence encoded by the nucleic acid sequence

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 118),

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 119), or

TGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 120).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, includes a signal sequence having an amino

acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 121). In some embodiments, a

first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric

polypeptide of the multi-chain chimeric polypeptide, or both, or a single-chain chimeric

polypeptide, includes a signal sequence having an amino acid sequence

MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 122). In some embodiments, a first chimeric polypeptide of a multi-chain

chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric

polypeptide, or both, or a single-chain chimeric polypeptide, includes a signal sequence

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having an amino acid sequence:

MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMI TAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG(SEQ ID NO: 123). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide,

a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or a

single-chain chimeric polypeptide, includes a signal sequence having an amino acid

sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 124). Those of ordinary skill in the art will be aware of other appropriate signal sequences for use in a

first chimeric polypeptide and/or a second chimeric polypeptide of multi-chain chimeric

polypeptides, or single-chain chimeric polypeptides described herein.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, includes a signal sequence that is about 10

to 100 amino acids in length. For example, a signal sequence can be about 10 to 100

amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids

in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length,

about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to

100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino

acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in

length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length,

about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to

100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino

acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in

length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about

10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60

amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in

length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about

10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25

amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in

length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about

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40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70

amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in

length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about

30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60

amino acids in length, or any range in between. In some embodiments, a signal sequence

is about 10, 15, 20, 25, 30, 35, 40,45,50,55,60,65,70,75,80,85,90,9 95, or 100 amino

acids in length.

In some embodiments, any of the signal sequences disclosed herein can include

one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at

its N-terminus and/or C-terminus, SO long as the function of the signal sequence remains

intact. For example, a signal sequence having the amino acid sequence

MKCLLYLAFLFLGVNC (SEQ ID NO: 125) can include one or more additional amino

acids at the N-terminus or C-terminus, while still retaining the ability to direct the a first

chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric

polypeptide of the multi-chain chimeric polypeptide, or both, or a single-chain chimeric

polypeptide, to the secretory pathway.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, includes a signal sequence that directs the

multi-chain chimeric polypeptide into the extracellular space. Such embodiments are

useful in producing single-chain or multi-chain chimeric polypeptides that are relatively

easy to be isolated and/or purified.

Peptide Tags

In some embodiments, a single-chain chimeric polypeptide includes a peptide tag

(e.g., at the N-terminal end or the C-terminal end of the chimeric polypeptide). In some

embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide

that includes a peptide tag (e.g., at the N-terminal end or the C-terminal end of the first

chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide

includes a second chimeric polypeptide that includes a peptide tag (e.g., at the N-terminal

WO wo 2021/247604 PCT/US2021/035285

end or the C-terminal end of the second chimeric polypeptide). In some embodiments,

both the first chimeric polypeptide of a multi-chain chimeric polypeptide and a second

chimeric polypeptide of the multi-chain chimeric polypeptide include a peptide tag. In

some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a

second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or a single-

chain chimeric polypeptide, includes two or more peptide tags.

Exemplary peptide tags that can be included in a first chimeric polypeptide of a

multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain

chimeric polypeptide, or both, or a single-chain chimeric polypeptide include, without

limitation, AviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 126), a calmodulin-tag

(KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 127), a polyglutamate tag (EEEEEE; SEQ ID NO: 128), an E-tag (GAPVPYPDPLEPR; SEQ ID NO: 129), a

FLAG-tag (DYKDDDDK; SEQ ID NO: 130), an HA-tag, a peptide from hemagglutinin

(YPYDVPDYA; SEQ ID NO: 131), a his-tag (HHHHH (SEQ ID NO: 132); HHHHHH

(SEQ ID NO: 133); HHHHHHH (SEQ ID NO: 134); HHHHHHHH (SEQ ID NO: 135);

HHHHHHHHH (SEQ ID NO: 136); or HHHHHHHHHH (SEQ ID NO: 137)), a myc-tag

(EQKLISEEDL; SEQ ID NO: 138), NE-tag (TKENPRSNQEESYDDNES; SEQ ID NO: 139), S-tag, (KETAAAKFERQHMDS; SEQ ID NO: 140), SBP-tag

(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 141), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 142), Softag 3 (TQDPSRVG; SEQ ID NO:

143), Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 144), Strep-tag (WSHPQFEK; SEQ ID

NO: 145), TC tag (CCPGCC; SEQ ID NO: 146), Ty tag (EVHTNQDPLD; SEQ ID NO:

147), V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 148), VSV-tag (YTDIEMNRLGK;

SEQ ID NO: 149), and Xpress tag (DLYDDDDK; SEQ ID NO: 150). In some

embodiments, tissue factor protein is a peptide tag.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain

chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric

polypeptide, or both, or a single-chain chimeric polypeptide can be used in any of a

variety of applications related to the multi-chain or single-chain chimeric polypeptide,

respectively. For example, a peptide tag can be used in the purification of a multi-chain

WO wo 2021/247604 PCT/US2021/035285

or single-chain chimeric polypeptide. As one non-limiting example, a first chimeric

polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first

chimeric polypeptide), a second chimeric polypeptide of the multi-chain chimeric

polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both, or a

single-chain chimeric polypeptide, can include a myc tag; the multi-chain chimeric

polypeptide that includes the myc-tagged first chimeric polypeptide, the myc-tagged

second chimeric polypeptide, or both, or the myc-tagged single-chain chimeric

polypeptide can be purified using an antibody that recognizes the myc tag(s). One non-

limiting example of an antibody that recognizes a myc tag is 9E10, available from the

non-commercial Developmental Studies Hybridoma Bank. As another non-limiting

example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a

recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of

the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric

polypeptide), or both, or a single-chain chimeric polypeptide, can include a histidine tag;

the multi-chain chimeric polypeptide that includes the histidine-tagged first chimeric

polypeptide, the histidine-tagged second chimeric polypeptide, or both, or the histidine-

tagged single-chain chimeric polypeptide can be purified using a nickel or cobalt chelate.

Those of ordinary skill in the art will be aware of other suitable tags and agents that bind

those tags for use in purifying a single-chain or multi-chain chimeric polypeptide. In

some embodiments, a peptide tag is removed from the first chimeric polypeptide and/or

the second chimeric polypeptide of the multi-chain chimeric polypeptide, or the single-

chain chimeric polypeptide after purification. In some embodiments, a peptide tag is not

removed from the first chimeric polypeptide and/or the second chimeric polypeptide of

the multi-chain chimeric polypeptide, or the single-chain chimeric polypeptide, after

purification.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain

chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric

polypeptide, or both, or a single-chain chimeric polypeptide, can be used, for example, in

immunoprecipitation of the multi-chain chimeric polypeptide or single-chain chimeric

polypeptide, respectively, imaging of the multi-chain chimeric polypeptide or single-

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chain chimeric polypeptide, respectively (e.g., via Western blotting, ELISA, flow

cytometry, and/or immunocytochemistry), and/or solubilization of the multi-chain

chimeric polypeptide or single-chain chimeric polypeptide, respectively.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, includes a peptide tag that is about 10 to

100 amino acids in length. For example, a peptide tag can be about 10 to 100 amino

acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in

length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length,

about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to

100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino

acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in

length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length,

about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to

100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino

acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in

length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about

10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60

amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in

length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about

10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25

amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in

length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about

40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70

amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in

length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about

30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60

amino acids in length, or any range in between. In some embodiments, a peptide tag is

about 10, 15, (20,25,30,35,40,45,50,55,60,65, 70, 75, 80, 85, 90, 95, or 100 amino

acids in length.

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Peptide tags included in a first chimeric polypeptide of a multi-chain chimeric

polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or

both, or a single-chain chimeric polypeptide, can be of any suitable length. For example,

peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino

acids in length. In embodiments in which a single-chain or multi-chain chimeric

polypeptide includes two or more peptide tags, the two or more peptide tags can be of the

same or different lengths. In some embodiments, any of the peptide tags disclosed herein

may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more

amino acids) at the N-terminus and/or C-terminus, SO long as the function of the peptide

tag remains intact. For example, a myc tag having the amino acid sequence

EQKLISEEDL (SEQ ID NO: 138) can include one or more additional amino acids (e.g.,

at the N-terminus and/or the C- terminus of the peptide tag), while still retaining the

ability to be bound by an antibody (e.g., 9E10).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type A

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain and/or the second target-binding domain can

independently bind specifically to CD3 (e.g., human CD3) or CD28 (e.g., human CD28).

In some embodiments, the first target-binding domain binds specifically to CD3 (e.g.,

human CD3) and the second target-binding domain binds specifically to CD28 (e.g.,

human CD28). In some embodiments, the first target-binding domain binds specifically

to CD28 (e.g., human CD28) and the second target-binding domain binds specifically to

CD3 (e.g., human CD3).

In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other. In some

embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble

tissue factor domain and the second target-binding domain directly abut each other. In

some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the soluble tissue factor domain and the second target-binding

domain.

In some embodiments of these single-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is an antigen-

binding domain. In some embodiments of these single-chain chimeric polypeptides, the

first target-binding domain and the second target-binding domain are each an antigen-

binding domain (e.g., any of the exemplary antigen-binding domains described herein).

In some embodiments of these single-chain chimeric polypeptides, the antigen-binding

domain includes a scFv or a single domain antibody.

A non-limiting example of an scFv that binds specifically to CD3 can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKL/

SGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGG GSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 151). In some embodiments, an scFv that binds specifically to CD3 can be encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAA CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAA CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCC

ITCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGO TTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAA0 CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACT AACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACC AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC (SEQ ID NO: 152).

A non-limiting example of an scFv that binds specifically to CD28 can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYN DYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRG TTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSY FHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCH QYHRSPTFGGGTKLETKR (SEQ ID NO: 153).

In some embodiments, an scFv that binds specifically to CD28 can be encoded by

a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84%

identical, at least 86% identical, at least 88% identical, at least 90% identical, at least

92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at

least 99% identical, or 100% identical) to:

GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGA AAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGG GTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTT

ACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAA CTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCG AGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGG ACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGA GGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTAT ATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCA0 CGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTA AACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTT wo 2021/247604 WO PCT/US2021/035285

TTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCT TTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCT GAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGC AGGCGGCACCAAACTGGAGACAAAGAGG(SEQ ID NO: 154). In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and/or the second target-binding domain is a soluble receptor (e.g., a

soluble CD28 receptor or a soluble CD3 receptor). In some embodiments of these single-

chain chimeric polypeptides, the soluble tissue factor domain can be any of the

exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLA SGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGG GGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWER KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAG

NVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR WNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVC VIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKA YTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAY EFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIE MTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASG VPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO: 155).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGA AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTA CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAG

WO wo 2021/247604 PCT/US2021/035285

CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCT, CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTA CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCC AGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAT AATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGA AAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCC GTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGO TTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAA CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACT ACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTA/ AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACC ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA AATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACC

GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC GAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATT GGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACG7 ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCC TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC GCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATT GTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCC TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTO GTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACC TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAA CTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA TGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGG TGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATC GAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCA CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTAC CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAAT TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTC TTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCAC AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA GG (SEQ ID NO: 156).

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, wo 2021/247604 WO PCT/US2021/035285 at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY0 (SGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQW SNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCK ASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS STAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIV DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE LIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPE KPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNE KGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGG

GSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPG SSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGG GTKLETKR (SEQ ID NO: 157).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

TGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTAT CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGA AGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTAT CAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAA CTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCT CTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGC ACAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAA CAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAG CCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTC TCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATO "TGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAA CCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTT TAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACO AGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTG TTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCAC ACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGA CAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGAT CTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACO GAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGG CTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGC AGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAA AGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTC ACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCC TCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCC AGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTG ACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTO TCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAA GAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTC TGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACAC TCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGA

GTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAG TTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACA TGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGG TGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGO GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATO GAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCA CAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGT. CAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAAT TCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTO TTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACC AGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGA GG (SEQ ID NO: 158).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type B

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain and/or the second target-binding domain can

independently bind specifically to an IL-2 receptor (e.g., human IL-2 receptor).

In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other. In some

embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble

tissue factor domain and the second target-binding domain directly abut each other. In

some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the soluble tissue factor domain and the second target-binding

domain.

In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain is a soluble human IL-2 protein. A

non-limiting example of an IL-2 protein that binds specifically to an IL-2 receptor can

include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84%

identical, at least 86% identical, at least 88% identical, at least 90% identical, at least

92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at

least 99% identical, or 100% identical) to:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE, TATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 78). In some embodiments, an IL-2 protein that binds specifically to an IL-2 receptor

can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical,

at least 92% identical, at least 94% identical, at least 96% identical, at least 98%

identical, at least 99% identical, or 100% identical) to:

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTAC TGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAA ACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAA

CTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGC TAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG CAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATG IGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGA TTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 159).

In some embodiments, an IL-2 protein that binds specifically to an IL-2 receptor

can be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTAC TGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAA GCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAG CTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGC TGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGO AACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGT GCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGA CACCTTCTGCCAGTCCATCATCTCCACTTTAACC (SEQ ID NO: 160). In some embodiments of these single-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein.

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSHISTLTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQV YTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTG SAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSL RDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTV

NRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI, NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTO (SEQ ID NO: 161). In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTAC TGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAA

GCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAG 276 wo 2021/247604 WO PCT/US2021/035285

CTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGC TGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGC ACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGT CGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGG ACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACAG TCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCG ATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAG TCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATO TCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTT PAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTT ACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGC CACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG GACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA GTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTC

CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGA' AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCG TGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGe GCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACA ACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATT

CAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAA PAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAA0 TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCA GGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGA AACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTT CTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 162).

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

MKKWVTFISLLFLFSSAYSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVL ELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKST NFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNV VEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDK ENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHI LLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN

WO wo 2021/247604 PCT/US2021/035285

LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI STLT (SEQ ID NO: 163).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CGCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTA CTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCC AGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGA GCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGT CTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAG CAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATG

TGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGA CACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACAG GTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCG AATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCA GTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGAT CTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTG TTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT ATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG GGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGC TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCC CCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGG AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACC GTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAG GGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTAC AACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAAT TACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAA AAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAA GGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGAC GGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGA AACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTT CTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 164).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides- Type C

In some embodiments of any of the single-chain chimeric polypeptides described

herein, the first target-binding domain and/or the second target-binding domain can

independently bind specifically to an IL-15 receptor (e.g., a human IL-15 receptor).

In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other. In some

embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain.

In some embodiments of these single-chain chimeric polypeptides, the soluble

tissue factor domain and the second target-binding domain directly abut each other. In

some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric

polypeptide further includes a linker sequence (e.g., any of the exemplary linkers

described herein) between the soluble tissue factor domain and the second target-binding

domain.

In some embodiments of these single-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain is a soluble human IL-15 protein.

A non-limiting example of an IL-15 protein that binds specifically to an IL-15 receptor

can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least

84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at

least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical,

at least 99% identical, or 100% identical) to:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESC

DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO: 82).

In some embodiments, an IL-15 protein that binds specifically to an IL-15

receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

279

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGA AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGA GCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTG

AAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAG AGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGO CAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGA GTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTG CACATCGTGCAGATGTTCATCAACACTAGC (SEQ ID NO: 165). In some embodiments, an IL-15 protein that binds specifically to an IL-15

receptor can be encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTG AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG

GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA CATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 166). In some embodiments of these single-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein.

In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESO DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLOSFVHIVOMFIN SSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFY7 TDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETN LGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSS

SGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGE FRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLE wo 2021/247604 WO PCT/US2021/035285

SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTS (SEQ ID NO: 167).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGA AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAG GCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTG CAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAG GAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAG CAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGA GTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTG CACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGT TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAA' GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCC GGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTC. CCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAG CTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATAC GAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCA ACATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGA CGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTG TTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGG AGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTG ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT

ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGO TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 168). In some embodiments, a single-chain chimeric polypeptide can include a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92% wo 2021/247604 WO PCT/US2021/035285 identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEED NIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYT VQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGS, GEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRI VFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 169).

In some embodiments, a single-chain chimeric polypeptide is encoded by a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CAACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCA AGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGC GCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTA AGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTA CCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGG AGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCG7 GCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTO GCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAAT GGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGT CGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCT ACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTT GCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATA CGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCC ACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGG ACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGT GTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCC GCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCG AATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGC GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTG

WO wo 2021/247604 PCT/US2021/035285

CACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGT TATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTA ATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCG GCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGC

AATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 170). Exemplary Multi-Chain Chimeric Polypeptides- Type A

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-18 or a receptor of IL-12. In some

examples of these multi-chain chimeric polypeptides, the first target-binding domain and

the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

In some examples of these multi-chain chimeric polypeptides, the first chimeric

polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is an agonistic

antigen-binding domain. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain are

each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is a soluble IL-15 or

a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the

first target-binding domain and the second target-binding domain are each independently

a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain both

bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain bind specifically to the same epitope. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain include the same amino acid sequence

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-12, and the second target-binding

domain binds specifically to a receptor for IL-18. In some embodiments of these multi-

chain chimeric polypeptides, the first target-binding domain binds specifically to a

receptor for IL-18, and the second target-binding domain bind specifically to a receptor

for IL-12.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKN QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:

109).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGAC< AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGA TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA

AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACO CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT (SEQ ID NO: 171).

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some

embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15

includes a sequence of soluble human IL-12B (p40) and a sequence of soluble human IL-

12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble

IL-15 human IL-15 further includes a linker sequence (e.g., any of the exemplary linker

sequences described herein) between the sequence of soluble IL-12B (p40) and the

sequence of soluble human IL-12a (p35). In some examples of these multi-chain

chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID

NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-12B (p40) comprises a sequence that is at least 80% identical (e.g., at

least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical,

at least 90% identical, at least 92% identical, at least 94% identical, at least 96%

identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT QVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFL EAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN

KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDJ SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 81). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12B (p40) is encoded by a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACO CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAA ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA AGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGO AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG

GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGO TGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 172). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12a (p35) includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDL YQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPD

FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS( (SEQ ID NO: 80).

286 wo 2021/247604 WO PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACC CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCA0 ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA

GAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTT GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATC ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA

AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 173).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKI

QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD KQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVO KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEE IDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISD KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 174).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

287 wo 2021/247604 WO PCT/US2021/035285

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACO AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGA TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAAG CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT CTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAG GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACO ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA TGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAAG TTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGC GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAG GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC CTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT TTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCA ACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGO CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA

GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 175).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD

WO wo 2021/247604 PCT/US2021/035285

TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP LETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLY KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECM EKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 176).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGO

CTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACG AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGA ATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAA CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGC ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGG GCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACO ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC GTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATAC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAG CTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACT TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC TTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTA TGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGC' GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAG GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAA GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATO wo 2021/247604 WO PCT/US2021/035285

CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCA CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAN GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 177).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTI QVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTF EAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRG, EYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPP PLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKT EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEE KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTK IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICI

SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 178). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCAC AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAA ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA GCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGA

GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA wo WO 2021/247604 PCT/US2021/035285

CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG AAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGO TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGA GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATO CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG GGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAC CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT TGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT

TTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAAC CAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTA0 AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCC TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 179). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGI MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDG1 VTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGI

STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQG TCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKY NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQ HKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS GSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP

CTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNS, ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEH VVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIP (SEQ ID NO: 180).

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTO ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCC< ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC

CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA GGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA

AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAG GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA ATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCC CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGO AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGO GGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGA CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTAC GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 181).

WO wo 2021/247604 PCT/US2021/035285

Exemplary Multi-Chain Chimeric Polypeptides- Type B

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-21 or to TGF-B. In some examples of

these multi-chain chimeric polypeptides, the first target-binding domain and the soluble

tissue factor domain directly abut each other in the first chimeric polypeptide. In some

examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide

further comprises a linker sequence (e.g., any of the exemplary linkers described herein)

between the first target-binding domain and the soluble tissue factor domain in the first

chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is a soluble IL-21 wo 2021/247604 WO PCT/US2021/035285

(e.g., a soluble human IL-21 polypeptide) or a soluble TGF-B receptor (e.g., a soluble

TGFR3RII receptor). In some embodiments of these multi-chain chimeric polypeptides,

the first target-binding domain and the second target-binding domain are each

independently a soluble IL-21 or a soluble TGF-B receptor (e.g., a soluble TGFR6RII

receptor). In some embodiments of these multi-chain chimeric polypeptides, the first

target-binding domain and the second target-binding domain both bind specifically to a

receptor of IL-21 or to TGF-B. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain bind

specifically to the same epitope. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain

include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-21, and the second target-binding

domain binds specifically to TGF-B. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain binds specifically to TGF-B, and the second

target-binding domain bind specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-21 (e.g., a soluble human IL-21). In some

embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21

includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84%

identical, at least 86% identical, at least 88% identical, at least 90% identical, at least

92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at

least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

294 wo 2021/247604 WO PCT/US2021/035285

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

GAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182). In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain includes a soluble TGF-B receptor (e.g., a soluble TGFR3RII

receptor (e.g., a soluble human TGFR3RII receptor)). In some embodiments of these

multi-chain chimeric polypeptides, the soluble human TGFR6RII includes a first

sequence of soluble human TGFR3RII and a second sequence of soluble human

TGFR3RII. In some embodiments of these multi-chain chimeric polypeptides, the

soluble human TGFR3RII includes a linker disposed between the first sequence of

soluble human TGFR3RII and the second sequence of soluble human TGFR3RII. In

some examples of these multi-chain chimeric polypeptides, the linker includes the

sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183). In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGI FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC< AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT( (SEQ ID NO:

185).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR6RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGAL ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGT? TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCA TCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGA GAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGA AGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGA CAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

186).

296

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human TGFR3RII receptor is encoded by a sequence that is at least 80% identical (e.g., at

least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical,

at least 90% identical, at least 92% identical, at least 94% identical, at least 96%

identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAC AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACO ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTO CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 187). In some embodiments of these multi-chain chimeric polypeptides, the human

TGFßRII receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

297 wo 2021/247604 WO PCT/US2021/035285

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLE KSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNG TVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNV GSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLS LRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRT NRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVI

SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 189).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC< CCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGA CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCO CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAA ACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAAT TGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCA0 CAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGT GATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGG STTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGO CTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCA GCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTO GAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGG ATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCC CCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGT ACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTAC CGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAA GGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATO GAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGA wo 2021/247604 WO PCT/US2021/035285

CGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGO AAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGA TTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGE CCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTT7 TGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 190).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTV STNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKG YLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVI VTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLII DKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN

SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 191). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCA AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCA

TACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATT CTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCA CCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGT wo WO 2021/247604 PCT/US2021/035285

GTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCC GTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCG- GGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAG0 CTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGC CAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCG

CGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCO GGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCT CCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGT GGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGT ACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAA. AGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGA CGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCG ACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCT AGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGA ATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAG

TCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTT CTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 192).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA wo 2021/247604 WO PCT/US2021/035285

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACO TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGG AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACO TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTT7 GAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGT

GTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGO GGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT CTCAAATGTATTAGA (SEQ ID NO: 194).

In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

(WVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRI

CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGS6 GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRE

YICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO: 195). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC

AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGT7 AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCO ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGA GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACG GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCT CAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTG AAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA CGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC CTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTT CGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTC CGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGA0 CGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT CTCAAATGTATTAGA (SEQ ID NO: 196).

Exemplary Multi-Chain Chimeric Polypeptides- Type C

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some

examples of these multi-chain chimeric polypeptides, the first target-binding domain and

the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

In some examples of these multi-chain chimeric polypeptides, the first chimeric

polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

WO wo 2021/247604 PCT/US2021/035285

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is a soluble IL-21

(e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7

polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first

target-binding domain and the second target-binding domain are each independently a

soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain both

bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain bind specifically to the same epitope. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-21, and the second target-binding

domain binds specifically to a receptor for IL-7. In some embodiments of these multi-

chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA CKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLE] PKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTO ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATO AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCO AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGO

ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 197). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG 304 wo WO 2021/247604 PCT/US2021/035285

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTe AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC/ (SEQ ID NO: 182). In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA AGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA

TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGC TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAAC GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA CTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC( (SEQ ID NO: 198). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

WO wo 2021/247604 PCT/US2021/035285

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER KSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNG VQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNV SAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFJ RDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSR VNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHP CKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE0 ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 199).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

AAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC ATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGA PAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACO AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAG ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATA TGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTO TGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACT GTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGAC CTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCT TCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCT GTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAG CCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAG AAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGA TGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTC GGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTG AAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACA TTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAG GGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCG AAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGAC GTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCA AGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAA' TAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTC 306 wo 2021/247604 WO PCT/US2021/035285

CGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCT CGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCT GCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 200).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MGVKVILFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTW KSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDV TYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVN TVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLI DKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIED

[QSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 201). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCA AGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATO AGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGA GATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCC ACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATT AAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAA ACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAA AATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTG TCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTG CAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGG GAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAG GAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCAC

CGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCC TACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATO 307

AGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAA0 AGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAAC AATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGA GAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTT TTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGA

AGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAA GAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAAC CGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAA TTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAT TAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCAC

CCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTAT CTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCT GCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAAT CCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 202) In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 203)

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG CAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGC GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATG. AGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTC ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTC CCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA

CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 204). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

IGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNJ FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN GQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKII MGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 205).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

AATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCG TGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTC AGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTG ATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGT ATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAG CACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATAC GTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTG AGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGA AAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACT

TGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCO CCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTO CAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCC AGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAA CCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 206).

WO wo 2021/247604 PCT/US2021/035285

Exemplary Multi-Chain Chimeric Polypeptides- Type D

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some

examples of these multi-chain chimeric polypeptides, the first target-binding domain and

the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

In some examples of these multi-chain chimeric polypeptides, the first chimeric

polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers

described herein) between the first target-binding domain and the soluble tissue factor

domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is a soluble IL-21

(e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7

polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first

target-binding domain and the second target-binding domain are each independently a

soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain both

bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain bind specifically to the same epitope. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-21, and the second target-binding

domain binds specifically to a receptor for IL-7. In some embodiments of these multi-

chain chimeric polypeptides, the first target-binding domain binds specifically to a

receptor for IL-7, and the second target-binding domain binds specifically to a receptor

for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAG CKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGO CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTAT AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCO

LAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGO ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 197). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC0 GAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEQ DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM "LFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEA PPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to: wo WO 2021/247604 PCT/US2021/035285

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGO GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGA

TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGC TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 198). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKE FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQV

TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS( (SEQ ID NO: 207).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGA GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG/ GGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGA AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA

CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC' GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATO wo 2021/247604 WO PCT/US2021/035285

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT AGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACA GTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACC CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAAT CAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATG GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCC' CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA GAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAA,

GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACAC GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTAC GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC< GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN GQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKI MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEF' LETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL) KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMO QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% wo 2021/247604 WO PCT/US2021/035285 identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTAG GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTC GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGO CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCT TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACA CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTO GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGC GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA CCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTA AGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTT TGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATO AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 210).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to: wo WO 2021/247604 PCT/US2021/035285

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER SKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 211). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG

CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA ACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 212). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED ETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW

VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR( (SEQ ID NO: 213).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

316 least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTO GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACG GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC

CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATO AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCC CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 214).

Exemplary Multi-Chain Chimeric Polypeptides- Type E

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor for IL-18 (e.g., a soluble human IL-18), a receptor for IL-12 (e.g., a soluble human IL-12), or CD16 (e.g., an anti-CD16 scFv). In

some embodiments of these multi-chain chimeric polypeptides described herein, the first

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the second

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the additional

target-binding domain binds specifically to CD16 or a receptor for IL-12.

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

317

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of

the first target-binding domain, the second target-binding domain and the additional

antigen-binding domain is an agonistic antigen-binding domain. In some embodiments

of these multi-chain chimeric polypeptides, the first target-binding domain, the second

target-binding domain, and the additional antigen-binding domain are each agonistic

antigen-binding domains. In some embodiments of these multi-chain chimeric

polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of

the first target-binding domain and the second target-binding domain is a soluble IL-15 or

a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the

first target-binding domain and the second target-binding domain are each independently

a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain both

bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain bind specifically to the same epitope. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain and the second

target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-12, and the second target-binding

domain binds specifically to a receptor for IL-18. In some embodiments of these multi-

chain chimeric polypeptides, the first target-binding domain binds specifically to a

receptor for IL-18, and the second target-binding domain bind specifically to a receptor

for IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first

target-binding domain binds specifically to CD16, and the second target-binding domain

binds specifically to a receptor for IL-18. In some embodiments of these multi-chain

319 chimeric polypeptides, the first target-binding domain binds specifically to a receptor for

IL-18, and the second target-binding domain bind specifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same antigen. In some

embodiments, two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope. In some embodiments, two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKI

QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 109).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACO CCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC

GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT (SEQ ID NO: 171).

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some

embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15

includes a sequence of soluble human IL-12B (p40) and a sequence of soluble human IL-

12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble

IL-15 (e.g., soluble human IL-15) further includes a linker sequence (e.g., any of the

exemplary linker sequences described herein) between the sequence of soluble IL-12B

(p40) and the sequence of soluble human IL-12a (p35). In some examples of these multi-

chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS

(SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-12B (p40) comprises a sequence that is at least 80% identical (e.g., at

least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical,

at least 90% identical, at least 92% identical, at least 94% identical, at least 96%

identical, at least 98% identical, at least 99% identical, or 100% identical) to:

WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT QVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFL CEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT BATVICRKNASISVRAQDRYYSSSWSEWASVPCS( (SEQ ID NO: 81). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12B (p40) is encoded by a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCO ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAG CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG

GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGO GGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 172). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12a (p35) includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKN YQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 80). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-12a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCA ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA AACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTC" GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATC ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG

ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 173).

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain includes an scFv that specifically binds to CD16 (e.g., an anti-

CD16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a light chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP

GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 215).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a light chain variable domain sequence that is at

least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA

GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 216).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a heavy chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW GGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 217).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a heavy chain variable domain sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTO CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAA

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO: 218). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDS YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFISMYKDSQ RGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKN PESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAA LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGT KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNI

LIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 174).

324

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGAC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGA TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAA CCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTO GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAG CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACA CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA

GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATO CGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCA TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCAT CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 175).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least wo 2021/247604 WO PCT/US2021/035285

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM TVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS CFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFT. YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMG EKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE OVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 176).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC ACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC CGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGA' AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA TTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTAG CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATAC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTC PACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACA GTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTAT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT 326 wo 2021/247604 WO PCT/US2021/035285

GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT

TTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATO CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 177).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

VELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGK7 VKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTI CEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVER KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKT IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVG ESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQ

GTLVTVSR (SEQ ID NO: 223).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGAC CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCAC AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAG CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATA ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGG AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAG, GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGA GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAT GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAG TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG "TGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTC ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTA GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGO AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCT TTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT CAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAG AGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCC CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTC CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGA0 GTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTG

GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCG GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGA GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTO TGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTC AGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCC GAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTC CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGA AAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGC" wo 2021/247604 WO PCT/US2021/035285

ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGA ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGT CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC CTGGTGACCGTGTCCAGG (SEQ ID NO: 224). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQ6 TCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE TSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQ

QGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGG SGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLED CTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNS ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHA DIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSE LTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGG GGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVP PGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV

YYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 225). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

WO wo 2021/247604 PCT/US2021/035285

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGT ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG AGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGA0 GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCC. CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG

AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCC GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGO TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCO CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAG AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGO CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA CCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAA. CAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTA AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCO CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGT CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAC

TCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCT GATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCT< GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCG GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTe GTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCC GAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCG GAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCT CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGG/ AGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCG ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAA0 ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTA CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC CTGGTGACCGTGTCCAGG (SEQ ID NO: 226).

WO wo 2021/247604 PCT/US2021/035285

Exemplary Multi-Chain Chimeric Polypeptides- Type F

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor for IL-7 (e.g., a soluble human IL-7), CD16

(e.g., an anti-CD16 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some

embodiments of these multi-chain chimeric polypeptides described herein, the first

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the second

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the additional

target-binding domain binds specifically to CD16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

WO wo 2021/247604 PCT/US2021/035285

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of

the first target-binding domain, the second target-binding domain and the additional

antigen-binding domain is an agonistic antigen-binding domain. In some embodiments

of these multi-chain chimeric polypeptides, the first target-binding domain, the second

target-binding domain, and the additional antigen-binding domain are each agonistic

antigen-binding domains. In some embodiments of these multi-chain chimeric

polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain binds specifically to a receptor IL-7 and the second

target-binding domain binds specifically to CD16 or a receptor for IL-21. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the first

target-binding domain includes a soluble IL-7 protein. In some embodiments of any of

the multi-chain chimeric polypeptides described herein, the soluble IL-7 protein is a

soluble human IL-7. In some embodiments of any of the multi-chain chimeric

polypeptides described herein, the second antigen-binding domain includes a target-

binding domain that binds specifically to CD16. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the second target-binding domain

includes an scFv that binds specifically to CD16. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the second target-binding domain

binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain

chimeric polypeptides described herein, the second target-binding domain includes a

soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides

described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of

any of the multi-chain chimeric polypeptides described herein, the second chimeric

polypeptide further includes an additional target-binding domain that binds specifically to

a receptor for IL-21. In some embodiments of any of the multi-chain chimeric

polypeptides described herein, the additional target-binding domain includes a soluble

IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the

multi-chain chimeric polypeptides described herein, the second chimeric polypeptide

further includes an additional target-binding domain that binds specifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same antigen. In some

embodiments, two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope. In some embodiments, two or more of the first target-binding domain, the wo WO 2021/247604 PCT/US2021/035285 PCT/US2021/035285 second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-7 (e.g., a soluble human IL-7).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical,

at least 84% identical, at least 86% identical, at least 88% identical, at least 90%

identical, at least 92% identical, at least 94% identical, at least 96% identical, at least

98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGO TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG

TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC(SEQ ID NO: 198). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

334

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATO GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCO TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATO AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCO

AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGO ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 197). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCC ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC<

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182) In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain includes an scFv that specifically binds to CD16 (e.g., an anti-

CD16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a light chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGE (SEQ ID NO: 215).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a light chain variable domain sequence that is at

least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGO CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 216).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a heavy chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

VQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW

GGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 217).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a heavy chain variable domain sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC

CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTT ACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 218). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK 337 wo 2021/247604 WO PCT/US2021/035285

DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQV TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTN FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDI KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDT LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO: 207).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC CAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGO GGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATO RAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCA0 ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT GCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAG GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCG CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAAT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATO GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGT CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCC CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT ACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC

338

GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCO GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC

GQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWK; KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFT LETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLY WKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMG PEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE OVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGA GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTO CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC

TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGA CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCA0 AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACA CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTO GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTC wo WO 2021/247604 PCT/US2021/035285

GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA CCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAA CCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTA CGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCO CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA

AGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTT CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC AGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 210).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHC GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ GKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA

YCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRE NSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVD QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKK LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH GSEDS (SEQ ID NO: 232).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG wo WO 2021/247604 PCT/US2021/035285

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTO CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA GTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGO ATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA CAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATO CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATA TCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC ACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGC

CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA CGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAAC CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAAG ACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCC AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGC CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA CGGCTCCGAGGACTCC (SEQ ID NO: 233). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ PGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSR GNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLS LASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRD) SLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVI ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRO

GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQL KSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERF KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 234).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACT CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC AGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTO ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGA GGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGA

GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGG CATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGG ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGG7 CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTO CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG 'GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTAG GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAG CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTC ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGT AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACT GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCA CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCG AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTO CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCC ACGGCTCCGAGGACTCC (SEQ ID NO: 235).

Exemplary Multi-Chain Chimeric Polypeptides- Type G

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGFB (e.g., a human TGFßRII receptor), CD16 (e.g.,

an anti-CD16 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

embodiments of these multi-chain chimeric polypeptides described herein, the first

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the second

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the additional

target-binding domain binds specifically to CD16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

WO wo 2021/247604 PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of

the first target-binding domain, the second target-binding domain and the additional

antigen-binding domain is an agonistic antigen-binding domain. In some embodiments

of these multi-chain chimeric polypeptides, the first target-binding domain, the second

target-binding domain, and the additional antigen-binding domain are each agonistic

antigen-binding domains. In some embodiments of these multi-chain chimeric

polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B, CD16, or a receptor for IL-21. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the first

target-binding domain binds specifically to a TGF-B and the second target-binding

domain binds specifically to CD1 or a receptor of IL-21. In some embodiments of any

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

of the multi-chain chimeric polypeptides described herein, the first target-binding domain

is a soluble TGF-B receptor. In some embodiments of any of the multi-chain chimeric

polypeptides described herein, soluble TGF-B receptor is a soluble TGFßRII receptor. In

some embodiments of any of the multi-chain chimeric polypeptides described herein, the

second target-binding domain binds specifically to CD16. In some embodiments of any

of the multi-chain chimeric polypeptides described herein, the second antigen-binding

domain includes an antigen-binding domain that binds specifically to CD16. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the

second antigen-binding domain includes an scFv that binds specifically to CD16. In

some embodiments of any of the multi-chain chimeric polypeptides described herein, the

second target-binding domain binds specifically to a receptor for IL-21. In some

embodiments of any of the multi-chain chimeric polypeptides described herein, the

second target-binding domain includes a soluble IL-21. In some embodiments of any of

the multi-chain chimeric polypeptides described herein, the second target-binding domain

includes a soluble human IL-21. In some embodiments of any of the multi-chain

chimeric polypeptides described herein, the second chimeric polypeptide further includes

an additional target-binding domain that binds specifically to a receptor for IL-21. In

some embodiments of any of the multi-chain chimeric polypeptides described herein, the

additional target-binding domain includes a soluble IL-21. In some embodiments of any

of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble

human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides

described herein, the second chimeric polypeptide further includes an additional target-

binding domain that binds specifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same antigen. In some

embodiments, two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a TGFßRII receptor (e.g., a soluble human TGFßRII receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human

TGFR3RII includes a first sequence of soluble human TGFR3RII and a second sequence

of soluble human TGFR3RII. In some embodiments of these multi-chain chimeric

polypeptides, the soluble human TGFR3RII includes a linker disposed between the first

sequence of soluble human TGFR3RII and the second sequence of soluble human

TGFR3RII. In some examples of these multi-chain chimeric polypeptides, the linker

includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE, KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

346 identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAC AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACO

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO: 185)

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGAT ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT

TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT TGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNN

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK 347

NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human TGFßRII receptor is encoded by a sequence that is at least 80% identical (e.g., at

least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical,

at least 90% identical, at least 92% identical, at least 94% identical, at least 96%

identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA CTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACO ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO: 187).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of

soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA

GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATO AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCO AAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC( (SEQ ID NO: 197). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTO LACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCO CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182). In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a light chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRI GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 215).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGO CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 216).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a heavy chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY

WGQGTLVTVSR (SEQ ID NO: 217).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a heavy chain variable domain sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTO CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAA0

TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA CAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAC GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO: 218).

wo 2021/247604 WO PCT/US2021/035285

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNN VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRI INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQIS7 WWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLY SPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLI YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPV

ECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG. CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTO CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTO

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAG GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC wo 2021/247604 WO PCT/US2021/035285

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG AACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTC GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCA CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGO CACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATAC AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCA CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT CGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAA GCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTG ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG

AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGO ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGT `CTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTT `CATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGG TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC( (SEQ ID NO: 237). In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEW KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFS GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV

RRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSV QAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238).

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACT CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC ICTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGA AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCA ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGT

GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACG2 ACAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGT AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCT AGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTG CCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCC GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAG CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC ACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCC GTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAN TAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACG ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGO

TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 239). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHG GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ PGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT YYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYIO WSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIV QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERINVSIKK

LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH GSEDS (SEQ ID NO: 232).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGT GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCO CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAG TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC

TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATG CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGO CTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCC wo WO 2021/247604 PCT/US2021/035285

GCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA CGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAAC

CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA ACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGG AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGO CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA CGGCTCCGAGGACTCC (SEQ ID NO: 233). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQO KPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSS GNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSG AASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNA

KNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQ GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQJ KSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERE KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 234).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGT AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACA GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGG, GGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGA GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGO CATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGC ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGT TCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTO CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG CTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACAT GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATA CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCT ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTO AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAAC GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA

CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGC GAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTG CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCO ACGGCTCCGAGGACTCC (SEQ ID NO: 235).

Exemplary Multi-Chain Chimeric Polypeptides- Type H

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7. In some examples of these multi-

chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor

domain directly abut each other in the first chimeric polypeptide. In some examples of

these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises

a linker sequence (e.g., any of the exemplary linkers described herein) between the first

target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain each independently bind

specifically to a receptor for IL-7. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain bind

specifically to the same epitope. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain and the second target-binding domain

include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain include a soluble IL-7 (e.g., a

soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides,

the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least

82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at

least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical,

at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ

PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO: 79).

357 wo WO 2021/247604 PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATO GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEO FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK

DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVO TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNI LIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDT LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO: 207).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to: wo WO 2021/247604 PCT/US2021/035285

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATO GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTO GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT RAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCA CCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACA GTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACC CCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG

CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAAT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAG CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGT CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAAT GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCC' CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA GAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACAC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTO (SEQ ID NO: 208).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNO GQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKI MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWK KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP

WO wo 2021/247604 PCT/US2021/035285

YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTG CAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAA AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGA GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCT TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCT GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTC GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCA TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA CCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAA TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA CGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCO TCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGO AAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGAT AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC

360

AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 210).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ

PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 203).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGO TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT

GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAN CAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGG0 GAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA

GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTC CCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA

CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 204). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

361

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC

TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 250).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT

GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAG PAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCAC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATATTACATGO CCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCO TCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGG CACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA0 TGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 251).

Exemplary Multi-Chain Chimeric Polypeptides- Type I

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B. In some examples of these multi-chain

chimeric polypeptides, the first target-binding domain and the soluble tissue factor

domain directly abut each other in the first chimeric polypeptide. In some examples of

these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises

a linker sequence (e.g., any of the exemplary linkers described herein) between the first

WO wo 2021/247604 PCT/US2021/035285

target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain and the second target-binding domain each independently bind

specifically to TGF-B. In some embodiments of these multi-chain chimeric polypeptides,

the first target-binding domain and the second target-binding domain bind specifically to

the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the

first target-binding domain and the second target-binding domain include the same amino

acid sequence

In some embodiments of these multi-chain chimeric polypeptides, the first

target-binding domain and the second target-binding domain is a soluble TGF-B receptor

(e.g., a soluble TGFßRII receptor, e.g., a soluble human TGFBRII). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR6RII includes a first sequence of soluble human TGFR3RII and a second sequence of soluble human TGFR3RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR3RII includes a linker disposed between the first sequence of soluble human TGFR3RII and the second sequence of soluble human TGFR3RIL In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI KPOEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184). In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA AACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT 364 wo 2021/247604 WO PCT/US2021/035285

GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO: 185). In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT

CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least wo 2021/247604 WO PCT/US2021/035285

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAG ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATO CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCO CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 187).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVOKSVNNDM VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKS GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLY PEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKI YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPV ECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK LLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFI QSFVHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

WO wo 2021/247604 PCT/US2021/035285

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG, CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG

AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG AGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC CCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGA TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG AACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTO GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGO TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCA CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATA GCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA CTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTG ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGG TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO: 237).

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF; CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILI AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGS GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI, TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEV EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV RNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSV QAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVT ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCO ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGA GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACC ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTC GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA

CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC wo 2021/247604 WO PCT/US2021/035285

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCG GCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATO AACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCC GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAG CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAG TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGG GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGG AGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTG ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA

TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGT' TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 239). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWE INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NUFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

369 wo WO 2021/247604 PCT/US2021/035285 least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC< AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACO

CAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGT CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC AGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTG AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA0 GAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGO GGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGG GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCAC CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 257). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF DNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFIL AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGS GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRER YICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO: 195). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ITGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTAC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCG. AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCO ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACG GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG

AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACA. CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC< TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGO TGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAG GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCA0 CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 259).

Exemplary Multi-Chain Chimeric Polypeptides- Type J

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7, a receptor of IL-21, or a receptor of

CD137L. In some embodiments of these multi-chain chimeric polypeptides described

herein, the second chimeric polypeptide further includes the additional target-binding

domain. In some embodiments of these multi-chain chimeric polypeptides described

herein, the additional target-binding domain binds specifically to a receptor for IL-21

(e.g., a soluble IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a

soluble CD137L, e.g., a soluble human CD137L).

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In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

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In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments, the second chimeric polypeptide can include an additional

target-binding domain. In some embodiments, the additional target-binding domain and

the

In some embodiments of these multi-chain chimeric polypeptides, one or more of

the first target-binding domain, the second target-binding domain and the additional

target-binding domain is an agonistic antigen-binding domain. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain, the second

target-binding domain, and the additional target-binding domain are each agonistic

antigen-binding domains. In some embodiments of these multi-chain chimeric

polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-7, and the second target-binding

domain binds specifically to a receptor for IL-21 or a receptor for CD137L. In some

embodiments, the additional target-binding domain binds specifically to a receptor for IL-

21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain is a soluble IL-7 (e.g., a soluble human IL-7). In some embodiments of

these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ

PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCO TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT( (SEQ ID NO: 227).

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain or the additional target-binding domain binds specifically to a

receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the

second target-binding domain or the additional target-binding domain is a soluble IL-21

(e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). 374

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO: 182). In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain binds specifically to a receptor for CD137L. In some

embodiments of these multi-chain chimeric polypeptides, the second chimeric

polypeptide further comprises an additional target-binding domain that binds specifically

to a receptor for CD137L. In some embodiments of these multi-chain chimeric

polypeptides, the second target-binding domain and/or the additional target-binding

domain is a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGI SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 260).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATG

GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGG4 GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGO CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGC7

CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAAT CCAGCCGGACTCCCTTCACCGAGGTCGGAAG (SEQ ID NO: 261). In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

PAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKED WVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVD] PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEI (SEQ ID NO: 262).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGO TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTO ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA

376 wo 2021/247604 WO PCT/US2021/035285

GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 263). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGN LFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEI DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDL

KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 207).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGA AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACO

GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCO CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT wo 2021/247604 WO PCT/US2021/035285

TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATG TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT ACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGO CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC TCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAAT GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT

GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNI EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL IGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWK CFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPER

YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC wo WO 2021/247604 PCT/US2021/035285 PCT/US2021/035285

CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGO CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG- AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCAC CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAG TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACA CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTO GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCT GAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACO TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAA

GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTC GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA PCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGG CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAA GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATO AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTO C (SEQ ID NO: 210).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

QDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLE FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGI KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELS DDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDT LVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVD

LPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF LPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGLPSPRSE (SEQ ID NO: 268)

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC ACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGC CCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGC CGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAAT GTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCA GCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGC GTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGO ACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCA CCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGC GCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGG GCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCC

GGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA( (SEQ ID NO: 269).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

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MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGS GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLA PPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 270). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

TGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA0

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATO GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACG PAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATO AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTAC CGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCAG GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGG TCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCC CCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAA TGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGC. GGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTG GTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCG TGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTG0 CCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCC ACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGC GCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAG GGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTT CGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO: 271).

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAG LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLL LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA GVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSE, NSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEJ (SEQ ID NO: 272).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGA GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCO

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG AGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGA ACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTG0 CCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCAT GTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCT GGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTA CAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTC

TTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCG TTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGC

CTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGG GTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAC GCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO: 273). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIV VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGG GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG

AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQL TQGATVLGLFRVTPEI (SEQ ID NO: 274).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGT GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAC GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATO AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGA AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGG TCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCA TGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGC TGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCT

WO wo 2021/247604 PCT/US2021/035285

ACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATG ACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATG TCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTC CGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCC CCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCG

GCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGG GCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO: 275).

Exemplary Multi-Chain Chimeric Polypeptides- Type K

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-7 or TGF-B. In some examples of

these multi-chain chimeric polypeptides, the first target-binding domain and the soluble

tissue factor domain directly abut each other in the first chimeric polypeptide. In some

examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide

further comprises a linker sequence (e.g., any of the exemplary linkers described herein)

between the first target-binding domain and the soluble tissue factor domain in the first

chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to a receptor for IL-7, and the second target-binding

domain binds specifically to TGF-B. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain binds specifically to TGF-B, and the second

target-binding domain binds specifically to a receptor for IL-7.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain includes a soluble IL-7 protein (e.g., a soluble human IL-7 protein). In

some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7

protein includes a sequence that is at least 80% identical (e.g., at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical,

at least 92% identical, at least 94% identical, at least 96% identical, at least 98%

identical, at least 99% identical, or 100% identical) to:

EDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEG FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:

79).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC

AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227).

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain comprises a target-binding domain that binds specifically to TGF-

B. In some embodiments of these multi-chain chimeric polypeptides, the second target-

binding domain is a soluble TGF-B receptor (e.g., a soluble TGFßRII receptor, e.g., a

soluble human TGFßRII receptor). In some embodiments of these multi-chain chimeric

polypeptides, the soluble human TGFR3RII includes a first sequence of soluble human

TGFR3RII and a second sequence of soluble human TGFR3RII. In some embodiments

of these multi-chain chimeric polypeptides, the soluble human TGFR3RII includes a

linker disposed between the first sequence of soluble human TGFR3RII and the second

sequence of soluble human TGFR3RII. In some examples of these multi-chain chimeric

polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO:

102).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT( (SEQ ID NO: 185). In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG

AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

387 wo 2021/247604 WO PCT/US2021/035285

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVOKSVNN VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW,

INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCAT GAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTO ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGAT CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG ACGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGT

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 187). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKE FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD

VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTK VNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLI DVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILA NNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 207). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGO TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCT AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAG CATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA0 ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGO AGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAA CCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAA AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGC ATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCG0 TTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGC TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGC AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGG TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGG GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGT7 TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCO TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208).

WO wo 2021/247604 PCT/US2021/035285

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKD MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK CFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWK SSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQ GEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGG TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTC AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGG GCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGA. CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACC ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGC AGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAA GACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACO AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTG GATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGT

GTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG 390

WO wo 2021/247604 PCT/US2021/035285

AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGG. TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGA AAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGAC<

GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGO GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTA TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATO ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCC TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNND VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA CATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTO ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAA ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC wo 2021/247604 WO PCT/US2021/035285

CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG

TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG G (SEQ ID NO: 257).

In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILI AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK] GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSREE

ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO: 195). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTT ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGAT CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTG

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACA. TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCC ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAG AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG G (SEQ ID NO: 259).

Exemplary Multi-Chain Chimeric Polypeptides- Type L

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B, a receptor of IL-21, or a receptor of CD137L.

In some embodiments of these multi-chain chimeric polypeptides described herein, the

second chimeric polypeptide further includes the additional target-binding domain. In

some embodiments of these multi-chain chimeric polypeptides described herein, the

additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble

IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L,

e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

WO wo 2021/247604 PCT/US2021/035285

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described wo 2021/247604 WO PCT/US2021/035285 herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of

the first target-binding domain, the second target-binding domain and the additional

target-binding domain is an agonistic antigen-binding domain. In some embodiments of

these multi-chain chimeric polypeptides, the first target-binding domain, the second

target-binding domain, and the additional target-binding domain are each agonistic

antigen-binding domains. In some embodiments of these multi-chain chimeric

polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to TGF-B and the second target-binding domain binds

specifically to a receptor for IL-21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain is a soluble TGF-B receptor (e.g., a soluble TGFßRII receptor, e.g., a

soluble human TGFßRII receptor). In some embodiments of these multi-chain chimeric

polypeptides, the soluble human TGFR6RII includes a first sequence of soluble human

TGFR6RII and a second sequence of soluble human TGFR3RII. In some embodiments

of these multi-chain chimeric polypeptides, the soluble human TGFR3RII includes a

linker disposed between the first sequence of soluble human TGFR3RII and the second

sequence of soluble human TGFR3RII. In some examples of these multi-chain chimeric

polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO:

102).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE" FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185). In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGT? TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGA AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT

CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA wo 2021/247604 WO PCT/US2021/035285

GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC, IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT CGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC

TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 187).

wo 2021/247604 WO PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain or the additional target-binding domain binds specifically to a

receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the

second target-binding domain or the additional target-binding domain includes a soluble

IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). In some embodiments of these multi-chain chimeric polypeptides, the soluble

human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182). In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain or the additional target-binding domain binds specifically to a

receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides,

the second target-binding domain and/or the additional target-binding domain includes a

soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical,

at least 92% identical, at least 94% identical, at least 96% identical, at least 98%

identical, at least 99% identical, or 100% identical) to:

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGJ SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAP ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTO GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 260).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGO GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC

CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 261). In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at

least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%

identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEI (SEQ ID NO: 262).

399

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%,

at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%

identical) to:

ATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA

CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATO (SEQ ID NO: 263). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD KSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENS FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECN GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE

LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSE VHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

400 wo 2021/247604 WO PCT/US2021/035285

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATC' GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAG

TCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGA GAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGA AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGT< TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGO TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACA/ PACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTG AGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCA GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGA TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGO AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG CGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAAT CACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAG AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA CGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATAT ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT

AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATC< GCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGA GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 237).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% wo 2021/247604 WO PCT/US2021/035285 identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

KWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF] AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGS GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA PSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATL SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESO CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC AATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAA ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGAT CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGT TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC CCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC 402 wo 2021/247604 WO PCT/US2021/035285

ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCO TAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAAT? TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAG AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATC ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACO ACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTC. CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG GCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCO

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCO AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATG CATCAATACCTCC (SEQ ID NO: 239).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGE KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELS DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK

LVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGLPSPRSE (SEQ ID NO: 268).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to: wo 2021/247604 WO PCT/US2021/035285

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG CCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC< CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCA CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAAC. CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG CGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCC CTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATG

TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT GTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGT GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGG TGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACT GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCG CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC TGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 269)

In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT PSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIV KSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGG GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPG

LAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 270).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCO GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCA CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG ACGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCO

CCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT GTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGG9 GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGC GGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCA0

GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCO CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC CTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO:

271).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAG LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER wo 2021/247604 WO PCT/US2021/035285

FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSG KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLD RQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAG YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARD

AFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO: 272).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGO AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGO GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAG CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCC GCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTT GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAA GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG

CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTT GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATO (SEQ ID NO: 273). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

406 wo 2021/247604 WO PCT/US2021/035285 least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

KWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPE VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRL CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGO GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEI (SEQ ID NO: 274).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG CCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG CCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC< CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGO GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGC CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCC GGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATG GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAA GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTT CTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCT CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC( (SEQ ID NO: 275).

407

WO wo 2021/247604 PCT/US2021/035285

Exemplary Multi-Chain Chimeric Polypeptides- Type M

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B or a receptor of IL-21. In some embodiments

of these multi-chain chimeric polypeptides described herein, the second chimeric

polypeptide further includes the additional target-binding domain. In some embodiments

of these multi-chain chimeric polypeptides described herein, the additional target-binding

domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble

human IL-21) or a TGF-B (e.g., a soluble TGF-B receptor, e.g., a soluble TGFßRII

receptor).

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to TGF-B, and the second target-binding domain binds

specifically to TGF-B or a receptor for IL-21. In some embodiments of these multi-chain

chimeric polypeptides, the first target-binding domain is a soluble TGF-B receptor (e.g., a

soluble TGFßRII receptor, e.g., a soluble human TGFßRII receptor). In some

embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR3RII

includes a first sequence of soluble human TGFR3RII and a second sequence of soluble

human TGFR3RII. In some embodiments of these multi-chain chimeric polypeptides,

the soluble human TGFR6RII includes a linker disposed between the first sequence of

soluble human TGFR3RII and the second sequence of soluble human TGFR3RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102) In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAC AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC< ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:

185).

410

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT

CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE7 FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

WO wo 2021/247604 PCT/US2021/035285

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACO ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA

AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACO TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO: 187).

In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain binds specifically to a receptor for IL-21. In some embodiments of

these multi-chain chimeric polypeptides, the second target-binding domain includes a

soluble IL-21 (e.g., a human soluble IL-21). In some embodiments of these multi-chain

chimeric polypeptides, the soluble IL-21 includes a sequence that is at least 80% identical

(e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88%

identical, at least 90% identical, at least 92% identical, at least 94% identical, at least

96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-

21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at

least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical,

at least 92% identical, at least 94% identical, at least 96% identical, at least 98%

identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCO CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO

AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC wo 2021/247604 WO PCT/US2021/035285

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO: 182). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN IDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD KSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENS TPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTI YYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSE VHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

CCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAG ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATO GGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGA AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTO TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG wo WO 2021/247604 PCT/US2021/035285

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTC CCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC CTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACO TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTG AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAA GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGA ACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT CACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAA. AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAC

CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATO ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGO ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGC AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 237).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST DNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEW PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA TIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE

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SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG KECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAG ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC TTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAA CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAA ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG GCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTO CTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATAG ACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCC TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTO GGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAA ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGT ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATO ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC wo 2021/247604 WO PCT/US2021/035285

ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 239).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC] KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVOKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI FSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTE VLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPA EDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHR LTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 300). In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

TCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTO ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATO GGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGA ACGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG wo 2021/247604 WO PCT/US2021/035285

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC TAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA ACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGO

CATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTA AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTC AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCC< GCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC PACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGC

CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 301). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI

SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKE GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIROGODRHMIRMRQLIDIV PQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVS KLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSR2

HGSEDS (SEQ ID NO: 302).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA

WO wo 2021/247604 PCT/US2021/035285

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA CATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACT ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC

CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTO AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCC GCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTO GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGA. GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC< CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 303).

Exemplary Multi-Chain Chimeric Polypeptides- Type N

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B or CD16. In some embodiments of these multi-

chain chimeric polypeptides described herein, the second chimeric polypeptide further

includes the additional target-binding domain. In some embodiments of these multi-

chain chimeric polypeptides described herein, the additional target-binding domain binds

specifically to CD16 (e.g., an anti-CD16 scFv) or a TGF-B (e.g., a soluble TGF-B

receptor, e.g., a soluble TGFBRII receptor).

WO wo 2021/247604 PCT/US2021/035285

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to TGF-B, and the second target-binding domain binds

specifically to TGF-B or CD16. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain is a soluble TGF-B receptor (e.g., a soluble

TGFßRII receptor, e.g., a soluble human TGFBRII receptor). In some embodiments of

these multi-chain chimeric polypeptides, the soluble human TGFR6RII includes a first

sequence of soluble human TGFR3RII and a second sequence of soluble human

TGFR3RII. In some embodiments of these multi-chain chimeric polypeptides, the

soluble human TGFR3RII includes a linker disposed between the first sequence of

soluble human TGFR3RII and the second sequence of soluble human TGFR3RII. In

some examples of these multi-chain chimeric polypeptides, the linker includes the

sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

420 wo 2021/247604 WO PCT/US2021/035285

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE7 FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG

AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT( (SEQ ID NO: 185).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR6RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGAT ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT `CCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAL

TGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT wo 2021/247604 WO PCT/US2021/035285

CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCA AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR EDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAG CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGG AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA0 CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 187).

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In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain binds specifically to CD16. In some embodiments of these multi-

chain chimeric polypeptides, the second target-binding domain includes an anti-CD16

scFv. In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a light chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 215).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a light chain variable domain sequence that is at

least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGO CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAG

TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 216).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 includes a heavy chain variable domain that includes a

sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical,

at least 86% identical, at least 88% identical, at least 90% identical, at least 92%

identical, at least 94% identical, at least 96% identical, at least 98% identical, at least

99% identical, or 100% identical) to: wo 2021/247604 WO PCT/US2021/035285

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW GGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 217).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that

binds specifically to CD16 is encoded by a heavy chain variable domain sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTO CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA

TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAC GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 218).

In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC

PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE7 ICSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD

WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF

VHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

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identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGA ACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAG AATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTT ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAG ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAA GCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTT CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTO AATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT ACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGA AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC ACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG GTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCO CGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGC' AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATO AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 237).

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In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKI GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSY GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYT DVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC CACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTT ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAG ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATO CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGT TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTO CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC 426 wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285

TAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA ACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC CTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACO TAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAG CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTO AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT ACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGA AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC CACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTO TGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATA

CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCO ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 239).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQ

QKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDS BGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLR CAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO: 308).

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In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG

GCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTT CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC AAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATG TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA ACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGG. GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO: 309).

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In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNG SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVR ITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTIT GAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEV

LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQG TLVTVSR (SEQ ID NO: 310).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC. CACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATO GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAA

CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGT TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTO CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC 429

CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGO TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCC ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAA0

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTO AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCO GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTO CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGA

GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO: 311).

Exemplary Multi-Chain Chimeric Polypeptides- Type O

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each

independently bind specifically to TGF-B or a receptor of CD137L. In some

embodiments of these multi-chain chimeric polypeptides described herein, the second

chimeric polypeptide further includes the additional target-binding domain. In some

embodiments of these multi-chain chimeric polypeptides described herein, the additional

target-binding domain binds specifically to a receptor to TGF- (e.g., a soluble TGF-B

receptor, e.g., a soluble TGFßRII receptor) or CD137L.

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

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linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

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any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to TGF-B, and the second target-binding domain binds

specifically to CD137L. In some embodiments of these multi-chain chimeric

polypeptides, the first target-binding domain or the additional target-binding domain is a

soluble TGF-B receptor (e.g., a soluble TGFßRII receptor, e.g., a soluble human TGFBRII

receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human TGFR3RII includes a first sequence of soluble human TGFR6RII and a second

sequence of soluble human TGFR3RII. In some embodiments of these multi-chain

chimeric polypeptides, the soluble human TGFR3RII includes a linker disposed between

the first sequence of soluble human TGFR3RII and the second sequence of soluble

human TGFR3RII. In some examples of these multi-chain chimeric polypeptides, the

linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR6RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGI FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC. TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO:

185).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGT7 TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT

CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAC AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGA0 AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO:

186).

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In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTO AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAC AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG

CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAG CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 187). In some embodiments of these multi-chain chimeric polypeptides, the second

target-binding domain includes a soluble CD137L protein (e.g., a soluble human CD137L

protein). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

EGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGI

YKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 260).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%,

at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%

identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTG GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGO CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC

AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATO CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 261). In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at

least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%

identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFE

VTPEI (SEQ ID NO: 262).

In some embodiments of these multi-chain chimeric polypeptides, a soluble

human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%,

at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%

identical) to: wo 2021/247604 WO PCT/US2021/035285

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAG,

TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTG0 ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 263). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI FSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSC WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVEC

GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA AACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT

GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTO wo 2021/247604 WO PCT/US2021/035285

ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGO CCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC CTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCO TAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAG CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTG AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAAT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAG AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCC GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT CACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAA ACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTT CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCO CGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCG ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG

GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 237).

In some embodiments, a first chimeric polypeptide can include a sequence that is

at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG

WO wo 2021/247604 PCT/US2021/035285

GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE KPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP IVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLY NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238). In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

TGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTAC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAG CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAG ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC GAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGA AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC CCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTO CTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCO TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT ACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG wo 2021/247604 WO PCT/US2021/035285

GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATC GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGT ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCO ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGC AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC

AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGA GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 239).

In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKI IDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC LNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLE

GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFG FQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRS E (SEQ ID NO: 316).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA

CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA wo 2021/247604 WO PCT/US2021/035285

ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGO CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC TCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGG GAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGA AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCO CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGT TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCT CACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGO TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCC ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC

AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCT GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACT GTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCA TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAAC CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 317). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFS CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGS GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY

WO wo 2021/247604 PCT/US2021/035285

ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSRE ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSRE GPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVL GLFRVTPEIPAGLPSPRSE (SEQ ID NO: 318).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC. CACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA

CGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGA0 ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAG ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGAT CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAG CTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGO CGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGT CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCO CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTG TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA ACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGC ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTC AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCC GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTAG ATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGO TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACT

WO wo 2021/247604 PCT/US2021/035285

CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 319).

Exemplary Multi-Chain Chimeric Polypeptides- Type P

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, the first target-binding domain and the second targeting-binding domain each bind

specifically to TGF-B. In some embodiments of these multi-chain chimeric polypeptides

described herein, the first chimeric polypeptide further includes the additional target-

binding domain. A non-limiting example of this type of multi-chain chimeric

polypeptide is shown in Figures 209 and 210.

In some examples of these multi-chain chimeric polypeptides, the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the

first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary

linkers described herein) between the first target-binding domain and the soluble tissue

factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain and the first domain of the pair of affinity domains directly abut

each other in the first chimeric polypeptide. In some embodiments of these multi-chain

chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence

(e.g., any of the exemplary linkers described herein) between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second

domain of the pair of affinity domains and the second target-binding domain directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

WO wo 2021/247604 PCT/US2021/035285

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second domain of the pair of affinity domains directly abut

each other in the second chimeric polypeptide. In some embodiments of these multi-

chain chimeric polypeptides, the second chimeric polypeptide further includes a linker

sequence (e.g., any of the exemplary linkers described herein) between the second

domain of the pair of affinity domains and the additional target-binding domain in the

second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional

target-binding domain and the second target-binding domain directly abut each other in

the second chimeric polypeptide. In some embodiments of these multi-chain chimeric

polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g.,

any of the exemplary linkers described herein) between the second target-binding domain

and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble

tissue factor domain can be any of the exemplary soluble tissue factor domains described

herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of

affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target-

binding domain binds specifically to TGF-B, and the second target-binding domain binds

specifically to TGF-B. In some embodiments of these multi-chain chimeric polypeptides,

the first target-binding domain and/or the second target-binding domain is a soluble TGF-

receptor (e.g., a soluble TGFßRII receptor, e.g., a soluble human TGFßRII receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

human TGFR6RII includes a first sequence of soluble human TGFR6RII and a second

sequence of soluble human TGFR3RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR3RII includes a linker disposed between the first sequence of soluble human TGFR3RII and the second sequence of soluble human TGFR3RII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE" FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 183).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor comprises a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPOEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA CTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACO AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG

ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO: 185).

In some embodiments of these multi-chain chimeric polypeptides, the second

sequence of soluble human TGFR3RII receptor is encoded by a sequence that is at least

80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical,

at least 88% identical, at least 90% identical, at least 92% identical, at least 94%

identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100%

identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGAT ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA

GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor includes a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET

FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK INDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble

TGF-B receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82%

identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at

least 98% identical, at least 99% identical, or 100% identical) to: wo 2021/247604 WO PCT/US2021/035285

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA CTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGA GAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCAC CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGC CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA ACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAG

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAC CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 187). In some embodiments, the first chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

IVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP0

PSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDN VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD KSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSP

FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSE VHIVQMFINTS (SEQ ID NO: 238).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

446

WO wo 2021/247604 PCT/US2021/035285

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACA ACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATT CACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTO CTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGA AACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGAC TTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAA AGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATG AACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCG GATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGC AAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAA ATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA AGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCA

GGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAA ACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC CGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGC GAGGAATACAATACCAGCAACCCCGACTCAGGCACTACAAATACTGTGGCAG CATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGA CCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGA ACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGA GATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCO ACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCT. CCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAG AACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAA TTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGA ACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTG GCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAG AAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAG AAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGG 447 wo WO 2021/247604 PCT/US2021/035285

AAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTO AGAGAAAACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTA "TCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCC TTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCC AGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTC AGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTC

ACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATO ACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATC CTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCA CTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCA AAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTT AAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTT TGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ TGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID ID NO: NO: 239). 239). In some embodiments, a first chimeric polypeptide can include a soluble IL-15

including a D8N amino acid substitution and have a sequence that is at least 80%

identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at

least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical,

at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

(Signal peptide)

MGVKVLFALICIAVAEA (Single chain Human TGF-beta Receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE) MCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM

DNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR, IDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IFSEEYNTSNPD (Tissue factor)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTT) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD

TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNI, PTIOSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGK TAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (IL-15D8N)

NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT S

(SEQ ID NO: 238).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC (Single chain Human TGF-beta Receptor II homodimer)

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACA ACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTT TCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCA TCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGA GAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGAC TTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAA, AGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGA AACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCG GATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA ATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACC

AGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCA GAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAA ACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACG CCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCT CCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCT TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGC GAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACT TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTA ATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACA TGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACA

ACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAG 449 wo WO 2021/247604 PCT/US2021/035285

ACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCO GTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTO GAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACA AAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAA

ACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTA TTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAAT GAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGC GAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGO AGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAG TGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA (Human IL-15D8N)

AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAAT AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAAT ATATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGC CTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGC AAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGA AAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGA GTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGO GTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCA

AACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAAT AACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAAT GTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACA TATTGTCCAAATGTTCATCAACACTTCT TATTGTCCAAATGTTCATCAACACTTCT (SEQ (SEQ ID ID NO: NO: 244). 244). In some embodiments, the second chimeric polypeptide can include a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPOEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKI IDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR (SEQ ID NO: 240).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

450 wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285 least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACO TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA TTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACC AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC AGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGO GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTO CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 241). In some embodiments, a second chimeric polypeptide can include a sequence that

is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86%

identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG

GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR( (SEQ ID NO: 242).

In some embodiments, a second chimeric polypeptide is encoded by a sequence

that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at

least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,

or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGA AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC

GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAG GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAA ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGT GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACA CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCT AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA0

GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC< TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAC CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAG GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA GTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCA0

CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 243).

WO wo 2021/247604 PCT/US2021/035285

Methods of Treating an Aging-Related Disease or Condition

Provided herein are methods of treating an aging-related disease or condition (e.g.

any of the exemplary types of aging-related disease or condition described herein or

known in the art) in a subject in need thereof that include administering to a subject

identified as having an aging-related disease or condition (e.g. any of the exemplary types

of aging-related disease or condition described herein or known in the art) a

therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s) (e.g. any of the natural killer (NK) cell activating agent(s) described herein or

known in the art).

Provided herein are methods of treating an aging-related disease or condition (e.g.

any of the exemplary types of aging-related disease or condition described herein or

known in the art) in a subject in need thereof that include administering to a subject

identified as having an aging-related disease or condition (e.g. any of the exemplary types

of aging-related disease or condition described herein or known in the art) a

therapeutically effective amount of activated NK cells (e.g. any of the activated NK cells

described herein or known in the art).

Some embodiments of these methods further include: obtaining a resting NK cell;

and contacting the resting NK cell in vitro in a liquid culture medium including one or

more NK cell activating agent(s), where the contacting results in the generation of the

activated NK cells that are subsequently administered to the subject. In some examples

of these methods, the resting NK cell is an autologous NK cell obtained from the subject.

In some examples of these methods, the resting NK cell is a haploidentical NK cell

obtained from the subject. In some examples of these methods, the resting NK cell is an

allogeneic resting NK cell. In some examples of these methods, the resting NK cell is an

artificial NK cell. In some examples of any of these methods, the resting NK cell is a

genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor.

In some examples of these methods, the liquid culture medium is a serum-free

liquid culture medium. In some embodiments of any of the methods described herein, the

liquid culture medium is a chemically-defined liquid culture medium. Some examples of

WO wo 2021/247604 PCT/US2021/035285

these methods further include isolating the activated NK cells (and optionally further

administering a therapeutically effective amount of the activated NK cells to a subject,

e.g., any of the subjects described herein). In some embodiments of these methods, the

contacting step is performed for a period of about 2 hours to about 20 days (or any of the

subranges of this range described herein).

In some embodiments of any of the methods described herein, the aging-related

disease or condition is selected from the group of: a cancer, an autoimmune disease, a

metabolic disease, a neurodegenerative disease, a cardiovascular disease, a skin disease, a

progeria disease, and a fragility disease.

Non-limiting examples of cancer include: solid tumor, hematological tumor,

sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,

Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,

B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia

(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute

lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell

lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell

carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast

cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell

head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and

hepatocellular carcinoma.

A non-limiting example of an autoimmune disease is type-1 diabetes.

Non-limiting examples of metabolic disease include: obesity, a lipodystrophy, and

type-2 diabetes mellitus.

Non-limiting examples of neurodegenerative disease include: Alzheimer's

disease, Parkinson's disease, and dementia.

Non-limiting examples of cardiovascular disease include: coronary artery disease,

atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of skin disease include: wound healing, alopecia,

wrinkles, senile lentigo, skin thinning, xeroderma pigmentosum, and dyskeratosis

congenita.

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Non-limiting examples of progeria disease include: progeria and Hutchinson-

Gilford Progeria Syndrome.

Non-limiting examples of fragility disease include: frailty, responsiveness to

vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the aging-related disease or condition described

herein, the aging-related disease or condition is selected from the group of: age-related

macular degeneration, osteoarthritis, adipose atrophy, chronic obstructive pulmonary

disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of

bone mass, sarcopenia, age-associated loss of lung tissue elasticity, osteoporosis, age-

associated renal dysfunction, and chemical-induced renal dysfunction.

In some embodiments of any of the aging-related disease or condition described

herein, the aging-related disease or condition is type-2 diabetes or atherosclerosis.

In some embodiments of any of the methods described herein, the subject has

been diagnosed or identified as having an aging-related disease or condition (e.g., any of

the exemplary aging-related diseases or conditions described herein). Some

embodiments of any of the methods described herein can include a step of selecting a

subject identified or diagnosed as having an aging-related disease or condition (e.g., any

of the exemplary aging-related diseases or conditions described herein).

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 10% decrease to about a 99% decrease,

about a 10% decrease to about a 95% decrease, about a 10% decrease to about a 90%

decrease, about a 10% decrease to about a 85% decrease, about a 10% decrease to about a

80% decrease, about a 10% decrease to about a 75% decrease, about a 10% decrease to

about a 70% decrease, about a 10% decrease to about a 65% decrease, about a 10%

decrease to about a 60% decrease, about a 10% decrease to about a 55% decrease, about a

10% decrease to about a 50% decrease, about a 10% decrease to about a 45% decrease,

about a 10% decrease to about a 40% decrease, about a 10% decrease to about a 35%

decrease, about a 10% decrease to about a 30% decrease, about a 10% decrease to about a

25% decrease, about a 10% decrease to about a 20% decrease, about a 10% decrease to

about a 15% decrease, about a 15% decrease to about a 99% decrease, about a 15%

decrease to about a 95% decrease, about a 15% decrease to about a 90% decrease, about a

15% decrease to about a 85% decrease, about a 15% decrease to about a 80% decrease,

about a 15% decrease to about a 75% decrease, about a 15% decrease to about a 70%

decrease, about a 15% decrease to about a 65% decrease, about a 15% decrease to about a

60% decrease, about a 15% decrease to about a 55% decrease, about a 15% decrease to

about a 50% decrease, about a 15% decrease to about a 45% decrease, about a 15%

decrease to about a 40% decrease, about a 15% decrease to about a 35% decrease, about a

15% decrease to about a 30% decrease, about a 15% decrease to about a 25% decrease,

about a 15% decrease to about a 20% decrease, about a 20% decrease to about a 99%

decrease, about a 20% decrease to about a 95% decrease, about a 20% decrease to about a

90% decrease, about a 20% decrease to about a 85% decrease, about a 20% decrease to

about a 80% decrease, about a 20% decrease to about a 75% decrease, about a 20%

decrease to about a 70% decrease, about a 20% decrease to about a 65% decrease, about a

20% decrease to about a 60% decrease, about a 20% decrease to about a 55% decrease,

about a 20% decrease to about a 50% decrease, about a 20% decrease to about a 45%

decrease, about a 20% decrease to about a 40% decrease, about a 20% decrease to about a

35% decrease, about a 20% decrease to about a 30% decrease, about a 20% decrease to

about a 25% decrease, about a 25% decrease to about a 99% decrease, about a 25%

decrease to about a 95% decrease, about a 25% decrease to about a 90% decrease, about a

25% decrease to about a 85% decrease, about a 25% decrease to about a 80% decrease,

about a 25% decrease to about a 75% decrease, about a 25% decrease to about a 70%

decrease, about a 25% decrease to about a 65% decrease, about a 25% decrease to about a

60% decrease, about a 25% decrease to about a 55% decrease, about a 25% decrease to

about a 50% decrease, about a 25% decrease to about a 45% decrease, about a 25%

decrease to about a 40% decrease, about a 25% decrease to about a 35% decrease, about a wo 2021/247604 WO PCT/US2021/035285

25% decrease to about a 30% decrease, about a 30% decrease to about a 99% decrease,

about a 30% decrease to about a 95% decrease, about a 30% decrease to about a 90%

decrease, about a 30% decrease to about a 85% decrease, about a 30% decrease to about a

80% decrease, about a 30% decrease to about a 75% decrease, about a 30% decrease to

about a 70% decrease, about a 30% decrease to about a 65% decrease, about a 30%

decrease to about a 60% decrease, about a 30% decrease to about a 55% decrease, about a

30% decrease to about a 50% decrease, about a 30% decrease to about a 45% decrease,

about a 30% decrease to about a 40% decrease, about a 30% decrease to about a 35%

decrease, about a 35% decrease to about a 99% decrease, about a 35% decrease to about a

95% decrease, about a 35% decrease to about a 90% decrease, about a 35% decrease to

about a 85% decrease, about a 35% decrease to about a 80% decrease, about a 35%

decrease to about a 75% decrease, about a 35% decrease to about a 70% decrease, about a

35% decrease to about a 65% decrease, about a 35% decrease to about a 60% decrease,

about a 35% decrease to about a 55% decrease, about a 35% decrease to about a 50%

decrease, about a 35% decrease to about a 45% decrease, about a 35% decrease to about a

40% decrease, about a 40% decrease to about a 99% decrease, about a 40% decrease to

about a 95% decrease, about a 40% decrease to about a 90% decrease, about a 40%

decrease to about a 85% decrease, about a 40% decrease to about a 80% decrease, about a

40% decrease to about a 75% decrease, about a 40% decrease to about a 70% decrease,

about a 40% decrease to about a 65% decrease, about a 40% decrease to about a 60%

decrease, about a 40% decrease to about a 55% decrease, about a 40% decrease to about a

50% decrease, about a 40% decrease to about a 45% decrease, about a 45% decrease to

about a 99% decrease, about a 45% decrease to about a 95% decrease, about a 45%

decrease to about a 90% decrease, about a 45% decrease to about a 85% decrease, about a

45% decrease to about a 80% decrease, about a 45% decrease to about a 75% decrease,

about a 45% decrease to about a 70% decrease, about a 45% decrease to about a 65%

decrease, about a 45% decrease to about a 60% decrease, about a 45% decrease to about a

55% decrease, about a 45% decrease to about a 50% decrease, about a 50% decrease to

about a 99% decrease, about a 50% decrease to about a 95% decrease, about a 50%

decrease to about a 90% decrease, about a 50% decrease to about a 85% decrease, about a wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285

50% decrease to about a 80% decrease, about a 50% decrease to about a 75% decrease,

about a 50% decrease to about a 70% decrease, about a 50% decrease to about a 65%

decrease, about a 50% decrease to about a 60% decrease, about a 50% decrease to about a

55% decrease, about a 55% decrease to about a 99% decrease, about a 55% decrease to

about a 95% decrease, about a 55% decrease to about a 90% decrease, about a 55%

decrease to about a 85% decrease, about a 55% decrease to about a 80% decrease, about a

55% decrease to about a 75% decrease, about a 55% decrease to about a 70% decrease,

about a 55% decrease to about a 65% decrease, about a 55% decrease to about a 60%

decrease, about a 60% decrease to about a 99% decrease, about a 60% decrease to about a

95% decrease, about a 60% decrease to about a 90% decrease, about a 60% decrease to

about a 85% decrease, about a 60% decrease to about a 80% decrease, about a 60%

decrease to about a 75% decrease, about a 60% decrease to about a 70% decrease, about a

60% decrease to about a 65% decrease, about a 65% decrease to about a 99% decrease,

about a 65% decrease to about a 95% decrease, about a 65% decrease to about a 90%

decrease, about a 65% decrease to about a 85% decrease, about a 65% decrease to about a

80% decrease, about a 65% decrease to about a 75% decrease, about a 65% decrease to

about a 70% decrease, about a 70% decrease to about a 99% decrease, about a 70%

decrease to about a 95% decrease, about a 70% decrease to about a 90% decrease, about a

70% decrease to about a 85% decrease, about a 70% decrease to about a 80% decrease,

about a 70% decrease to about a 75% decrease, about a 75% decrease to about a 99%

decrease, about a 75% decrease to about a 95% decrease, about a 75% decrease to about a

90% decrease, about a 75% decrease to about a 85% decrease, about a 75% decrease to

about a 80% decrease, about a 80% decrease to about a 99% decrease, about a 80%

decrease to about a 95% decrease, about a 80% decrease to about a 90% decrease, about a

80% decrease to about a 85% decrease, about a 85% decrease to about a 99% decrease,

about a 85% decrease to about a 95% decrease, about a 85% decrease to about a 90%

decrease, about a 90% decrease to about a 99% decrease, about a 90% decrease to about a

95% decrease, or about a 95% decrease to about a 99% decrease) in the number of

senescent cells in a target tissue in the subject, e.g., as compared to the number of

senescent cells in the target tissue in the subject prior to treatment.

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In some embodiments of these methods, the administering results in an increase

(e.g., at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a

20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at

least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55%

increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least

a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase,

at least a 95% increase, or at least a 99% increase, or about a 10% increase to about a

500% increase (or any of the subranges of this range described herein) in the levels of

IFN-y, a cytotoxic granule granzyme, and/or perforin in the subject, as compared to the

levels in a subject prior to treatment or a similar control subject who has not received a

treatment.

In some embodiments, these methods can result in a reduction in the number,

severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as

compared to the number, severity, or frequency of the one or more symptoms of the

cancer in the subject prior to treatment). In some embodiments, these methods can result

in a reduction (e.g., about 1% reduction to about 99% reduction, about 1% reduction to

about 95% reduction, about 1% reduction to about 90% reduction, about 1% reduction to

about 85% reduction, about 1% reduction to about 80% reduction, about 1% reduction to

about 75% reduction, about 1% reduction to about 70% reduction, about 1% reduction to

about 65% reduction, about 1% reduction to about 60% reduction, about 1% reduction to

about 55% reduction, about 1% reduction to about 50% reduction, about 1% reduction to

about 45% reduction, about 1% reduction to about 40% reduction, about 1% reduction to

about 35% reduction, about 1% reduction to about 30% reduction, about 1% reduction to

about 25% reduction, about 1% reduction to about 20% reduction, about 1% reduction to

about 15% reduction, about 1% reduction to about 10% reduction, about 1% reduction to

about 5% reduction, about 5% reduction to about 99% reduction, about 5% reduction to

about 95% reduction, about 5% reduction to about 90% reduction, about 5% reduction to

about 85% reduction, about 5% reduction to about 80% reduction, about 5% reduction to

about 75% reduction, about 5% reduction to about 70% reduction, about 5% reduction to

about 65% reduction, about 5% reduction to about 60% reduction, about 5% reduction to

WO wo 2021/247604 PCT/US2021/035285

about 55% reduction, about 5% reduction to about 50% reduction, about 5% reduction to

about 45% reduction, about 5% reduction to about 40% reduction, about 5% reduction to

about 35% reduction, about 5% reduction to about 30% reduction, about 5% reduction to

about 25% reduction, about 5% reduction to about 20% reduction, about 5% reduction to

about 15% reduction, about 5% reduction to about 10% reduction, about 10% reduction

to about 99% reduction, about 10% reduction to about 95% reduction, about 10%

reduction to about 90% reduction, about 10% reduction to about 85% reduction, about

10% reduction to about 80% reduction, about 10% reduction to about 75% reduction,

about 10% reduction to about 70% reduction, about 10% reduction to about 65%

reduction, about 10% reduction to about 60% reduction, about 10% reduction to about

55% reduction, about 10% reduction to about 50% reduction, about 10% reduction to

about 45% reduction, about 10% reduction to about 40% reduction, about 10% reduction

to about 35% reduction, about 10% reduction to about 30% reduction, about 10%

reduction to about 25% reduction, about 10% reduction to about 20% reduction, about

10% reduction to about 15% reduction, about 15% reduction to about 99% reduction,

about 15% reduction to about 95% reduction, about 15% reduction to about 90%

reduction, about 15% reduction to about 85% reduction, about 15% reduction to about

80% reduction, about 15% reduction to about 75% reduction, about 15% reduction to

about 70% reduction, about 15% reduction to about 65% reduction, about 15% reduction

to about 60% reduction, about 15% reduction to about 55% reduction, about 15%

reduction to about 50% reduction, about 15% reduction to about 45% reduction, about

15% reduction to about 40% reduction, about 15% reduction to about 35% reduction,

about 15% reduction to about 30% reduction, about 15% reduction to about 25%

reduction, about 15% reduction to about 20% reduction, about 20% reduction to about

99% reduction, about 20% reduction to about 95% reduction, about 20% reduction to

about 90% reduction, about 20% reduction to about 85% reduction, about 20% reduction

to about 80% reduction, about 20% reduction to about 75% reduction, about 20%

reduction to about 70% reduction, about 20% reduction to about 65% reduction, about

20% reduction to about 60% reduction, about 20% reduction to about 55% reduction,

about 20% reduction to about 50% reduction, about 20% reduction to about 45%

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reduction, about 20% reduction to about 40% reduction, about 20% reduction to about

35% reduction, about 20% reduction to about 30% reduction, about 20% reduction to

about 25% reduction, about 25% reduction to about 99% reduction, about 25% reduction

to about 95% reduction, about 25% reduction to about 90% reduction, about 25%

reduction to about 85% reduction, about 25% reduction to about 80% reduction, about

25% reduction to about 75% reduction, about 25% reduction to about 70% reduction,

about 25% reduction to about 65% reduction, about 25% reduction to about 60%

reduction, about 25% reduction to about 55% reduction, about 25% reduction to about

50% reduction, about 25% reduction to about 45% reduction, about 25% reduction to

about 40% reduction, about 25% reduction to about 35% reduction, about 25% reduction

to about 30% reduction, about 30% reduction to about 99% reduction, about 30%

reduction to about 95% reduction, about 30% reduction to about 90% reduction, about

30% reduction to about 85% reduction, about 30% reduction to about 80% reduction,

about 30% reduction to about 75% reduction, about 30% reduction to about 70%

reduction, about 30% reduction to about 65% reduction, about 30% reduction to about

60% reduction, about 30% reduction to about 55% reduction, about 30% reduction to

about 50% reduction, about 30% reduction to about 45% reduction, about 30% reduction

to about 40% reduction, about 30% reduction to about 35% reduction, about 35%

reduction to about 99% reduction, about 35% reduction to about 95% reduction, about

35% reduction to about 90% reduction, about 35% reduction to about 85% reduction,

about 35% reduction to about 80% reduction, about 35% reduction to about 75%

reduction, about 35% reduction to about 70% reduction, about 35% reduction to about

65% reduction, about 35% reduction to about 60% reduction, about 35% reduction to

about 55% reduction, about 35% reduction to about 50% reduction, about 35% reduction

to about 45% reduction, about 35% reduction to about 40% reduction, about 40%

reduction to about 99% reduction, about 40% reduction to about 95% reduction, about

40% reduction to about 90% reduction, about 40% reduction to about 85% reduction,

about 40% reduction to about 80% reduction, about 40% reduction to about 75%

reduction, about 40% reduction to about 70% reduction, about 40% reduction to about

65% reduction, about 40% reduction to about 60% reduction, about 40% reduction to

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

about 55% reduction, about 40% reduction to about 50% reduction, about 40% reduction

to about 45% reduction, about 45% reduction to about 99% reduction, about 45%

reduction to about 95% reduction, about 45% reduction to about 90% reduction, about

45% reduction to about 85% reduction, about 45% reduction to about 80% reduction,

about 45% reduction to about 75% reduction, about 45% reduction to about 70%

reduction, about 45% reduction to about 65% reduction, about 45% reduction to about

60% reduction, about 45% reduction to about 55% reduction, about 45% reduction to

about 50% reduction, about 50% reduction to about 99% reduction, about 50% reduction

to about 95% reduction, about 50% reduction to about 90% reduction, about 50%

reduction to about 85% reduction, about 50% reduction to about 80% reduction, about

50% reduction to about 75% reduction, about 50% reduction to about 70% reduction,

about 50% reduction to about 65% reduction, about 50% reduction to about 60%

reduction, about 50% reduction to about 55% reduction, about 55% reduction to about

99% reduction, about 55% reduction to about 95% reduction, about 55% reduction to

about 90% reduction, about 55% reduction to about 85% reduction, about 55% reduction

to about 80% reduction, about 55% reduction to about 75% reduction, about 55%

reduction to about 70% reduction, about 55% reduction to about 65% reduction, about

55% reduction to about 60% reduction, about 60% reduction to about 99% reduction,

about 60% reduction to about 95% reduction, about 60% reduction to about 90%

reduction, about 60% reduction to about 85% reduction, about 60% reduction to about

80% reduction, about 60% reduction to about 75% reduction, about 60% reduction to

about 70% reduction, about 60% reduction to about 65% reduction, about 65% reduction

to about 99% reduction, about 65% reduction to about 95% reduction, about 65%

reduction to about 90% reduction, about 65% reduction to about 85% reduction, about

65% reduction to about 80% reduction, about 65% reduction to about 75% reduction,

about 65% reduction to about 70% reduction, about 70% reduction to about 99%

reduction, about 70% reduction to about 95% reduction, about 70% reduction to about

90% reduction, about 70% reduction to about 85% reduction, about 70% reduction to

about 80% reduction, about 70% reduction to about 75% reduction, about 75% reduction

to about 99% reduction, about 75% reduction to about 95% reduction, about 75%

462 reduction to about 90% reduction, about 75% reduction to about 85% reduction, about

75% reduction to about 80% reduction, about 80% reduction to about 99% reduction,

about 80% reduction to about 95% reduction, about 80% reduction to about 90%

reduction, about 80% reduction to about 85% reduction, about 85% reduction to about

99% reduction, about 85% reduction to about 95% reduction, about 85% reduction to

about 90% reduction, about 90% reduction to about 99% reduction, about 90% reduction

to about 95% reduction, or about 95% reduction to about 99% reduction) in the volume

of one or more solid tumors in the subject (e.g., as compared to the volume of the one or

more solid tumors prior to treatment or at the start of treatment). In some embodiments,

the these methods can reduce (e.g., about 1% reduction to about 99% reduction, or any of

the subranges of this range described herein) the risk of developing a metastasis or

developing one or more additional metastasis in a subject (e.g., as compared to the risk of

developing a metastasis or developing one or more additional metastasis in a subject prior

to treatment or in a similar subject or a population of subjects administered a different

treatment).

In some embodiments, these methods can result in treatment of metabolic disease

in the subject. In some embodiments, the treatment of metabolic disease can result in,

e.g., one or more (e.g., two, three, four, five, or six) improved glucose tolerance,

improved glucose utilization, decreased severity or progression of diabetic

osteoarthropathy, decreased severity or progression of skin lesions, decreased severity or

progression of ketosis, decreased generation of autoantibodies against islet cells,

increased insulin sensitivity, decreased mass, and decreased body mass index. The

response of a subject to treatment can be monitored by determining fasting glucose or

glucose tolerance according to standard techniques. Typically, in accordance with the

method, blood glucose is lowered SO as to achieve a blood glucose level characterized by

a fasting blood glucose of less than 100 mg/dL or a two-hour 75-g oral glucose tolerance

test values of less than 140 mg/dL. In some embodiments, response to treatment may

include determining other factors relevant to pre-diabetes, new-onset diabetes, or active

diabetes including blood pressure, body mass index, PPAR-y function, lipid metabolism,

glycated hemoglobin (H1c), and renal function.

In some embodiments, these methods can eliminate or reduce the risk, lessen the

severity, or delay the outset of the neurodegenerative disease, including biochemical,

histologic and/or behavioral symptoms of the disease, its complications and intermediate

pathological phenotypes presenting during development of the disease.

In some embodiments, effective treatment of a skin disease can be assessed by

any method described herein or known in the art, including inspecting skin conditions

that include skin color, moisture, temperature, texture, mobility and turgor, and skin

lesions, as compared to the skin conditions prior to treatment.

In some embodiments, effective treatment of an autoimmune disease can be

assessed by any method described herein or known in the art, including monitoring full

blood count analysis on freshly isolated PBMCs, total Ig levels, and analysis of serum

autoantibody titers.

In some embodiments, effective treatment of a fragility disease can be assessed by

any method described herein or known in the art, including monitoring bone mineral

density, bone architecture and geometry, biomedical markers of bone turnover, vitamin D

measurement, Karnofsky performance status and ECOG scores, and responsiveness to

vaccination.

Methods of Killing or Reducing the Number of Senescent Cells in a Subject

Provided herein are methods of killing or reducing the number of senescent cells

(e.g. any of the exemplary types of senescent cells described herein or known in the art)

in a subject in need thereof that include administering to the subject a therapeutically

effective amount of one or more NK cell activating agent(s) (e.g. any of the NK cell

activating agent(s) described herein or known in the art).

Also provided herein are methods of killing or reducing the number of senescent

cells (e.g. any of the exemplary types of senescent cells described herein or known in the

art) in a subject in need thereof that include administering to the subject a therapeutically

effective amount of activated NK cells (e.g. any of the activated NK cells described

herein or known in the art).

WO wo 2021/247604 PCT/US2021/035285

Some embodiments of these methods further include: obtaining a resting NK cell;

and contacting the resting NK cell in vitro in a liquid culture medium including one or

more NK cell activating agent(s), where the contacting results in the generation of the

activated NK cells that are subsequently administered to the subject. In some examples

of these methods, the resting NK cell is an autologous NK cell obtained from the subject.

In some examples of these methods, the resting NK cell is a haploidentical NK cell

obtained from the subject. In some examples of these methods, the resting NK cell is an

allogeneic resting NK cell. In some examples of these methods, the resting NK cell is an

artificial NK cell. In some examples of any of these methods, the resting NK cell is a

genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor.

In some examples of these methods, the liquid culture medium is a serum-free

liquid culture medium. In some embodiments of any of the methods described herein, the

liquid culture medium is a chemically-defined liquid culture medium. Some examples of

these methods further include isolating the activated NK cells (and further administering

a therapeutically effective amount of the activated NK cells to a subject, e.g., any of the

subjects described herein). In some embodiments of these methods, the contacting step is

performed for a period of about 2 hours to about 20 days (or any of the subranges of this

range described herein).

In some embodiments of these methods, the senescent cells are senescent cancer

cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent

microglia, senescent neurons, senescent tissue fibroblasts, senescent dermal fibroblasts,

senescent keratinocytes, or other differentiated tissue-specific dividing functional cells.

In some embodiments of these methods, senescent cancer cells are chemotherapy-

induced senescent cells or radiation-induced senescent cells.

In some embodiments of these methods, the subject has been identified or

diagnosed as having an aging-related disease or condition (e.g., any of the aging-related

diseases or conditions described herein or known in the art). In some embodiments of

any of the aging-related disease or condition described herein, the aging-related disease

or condition is selected from the group of: a cancer, an autoimmune disease, a metabolic

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disease, a neurodegenerative disease, a cardiovascular disease, a skin disease, a progeria

disease, and a fragility disease.

Non-limiting examples of cancer include: solid tumor, hematological tumor,

sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,

Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,

B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia

(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute

lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell

lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell

carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast

cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell

head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and

hepatocellular carcinoma.

A non-limiting example of an autoimmune disease is type-1 diabetes.

Non-limiting examples of metabolic disease include: obesity, a lipodystrophy, and

type-2 diabetes mellitus.

Non-limiting examples of neurodegenerative disease include: Alzheimer's

disease, Parkinson's disease, and dementia.

Non-limiting examples of cardiovascular disease include: coronary artery disease,

atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of skin disease include: wound healing, alopecia,

wrinkles, senile lentigo, skin thinning, xeroderma pigmentosum, and dyskeratosis

congenita.

Non-limiting examples of progeria disease include: progeria and Hutchinson-

Gilford Progeria Syndrome.

Non-limiting examples of fragility disease include: frailty, responsiveness to

vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the aging-related disease or condition described

herein, the aging-related disease or condition is selected from the group of: age-related

macular degeneration osteoarthritis, adipose atrophy, chronic obstructive pulmonary

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disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of

bone mass, sarcopenia, age-associated loss of lung tissue elasticity, osteoporosis, age-

associated renal dysfunction, and chemical-induced renal dysfunction.

In some embodiments of any of the aging-related disease or condition described

herein, the aging-related disease or condition is type-2 diabetes or atherosclerosis.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 10% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the number of senescent cells in a

target tissue in the subject, e.g., as compared to the number of senescent cells in the target

tissue in the subject prior to treatment. In some embodiments of these methods, the target

tissue in the subject can be one or more of an adipose tissue, pancreatic tissue, liver

tissue, lung tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye

tissue, skin tissue, muscle tissue, and secondary lympho-organ tissue.

In some embodiments of these methods, the administering results in an increase

(e.g., at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a

20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at

least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55%

increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least

a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase,

at least a 95% increase, or at least a 99% increase, or about a 10% increase to about a

500% increase (or any of the subranges of this range described herein)) in the levels of

IFN-y, a cytotoxic granule granzyme, and/or perforin in the subject, as compared to the

levels in a subject prior to treatment or a similar control subject who has not received a

treatment.

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In some embodiments of these methods, the number of senescent cells in a target

tissue (e.g. any of the target tissues described herein) can be determined by performing

immunostaining on a biopsy sample. In some embodiments of these methods, the number

of senescent cells in a target tissue (e.g. any of the target tissues described herein) can be

observed indirectly through an improvement in one or more symptoms of an aging-

related disease or condition (e.g. any of the symptoms of an aging-related disease or

condition described herein) in a subject.

Senescent Cells

Senescent cells display important and unique properties which include changes in

morphology, chromatin organization, gene expression, and metabolism. There are

several biochemical and functional properties associated with cellular senescence, such as

(i) increased expression of p16 INK4a and p21CIP1, inhibitors of cyclin-dependent kinases,

(ii) presence of senescence-associated B-galactosidase, a marker of lysosomal activity,

(iii) appearance of senescence-associated heterochromatin foci and downregulation of

lamin B1 levels, (iv) resistance to apoptosis caused by an increased expression of anti-

apoptotic BCL-family protein, and (v) upregulation of CD26 (DPP4), CD36 (Scavenger

receptor), forkhead box 4 (FOXO4), and secretory carrier membrane protein 4

(SCAMP4). Senescent cells also express an inflammatory signature, the so-called

senescence-associated secretory phenotype (SASP). Through SASP, the senescent cells

produce a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-B),

chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9) that operate in a

cell-autonomous manner to reinforce senescence (autocrine effects) and communicate

with and modify the microenvironment (paracrine effects). SASP factors can contribute

to tumor suppression by triggering senescence surveillance, an immune-mediated

clearance of senescent cells. However, chronic inflammation is also a known driver of

tumorigenesis, and accumulating evidence indicates that chronic SASP can also boost

cancer metastasis and aging-related diseases.

The secretion profile of senescent cells is context dependent. For instance, the

mitochondrial dysfunction-associated senescence (MiDAS), induced by different

WO wo 2021/247604 PCT/US2021/035285

mitochondrial dysfunction in human fibroblasts, led to the appearance of a SASP that was

deficient in IL-1-dependent inflammatory factors. A decrease in the NAD+/NADH ratio

activated AMPK signaling which induced MiDAS through the activation of p53. As a

result, p53 inhibited NF-kB signaling which is a crucial inducer of pro-inflammatory

SASP. In contrast, the cellular senescence caused by persistent DNA damage in human

cells induced an inflammatory SASP, which was dependent on the activation of ataxia-

telangiectasia mutated (ATM) kinase but not on that of p53. In particular, the expression

and secretion levels of IL-6 and IL-8 were increased. It was also demonstrated that

cellular senescence caused by the ectopic expression p16 INK4a and p21 CIP1 induced the

senescent phenotype in human fibroblasts without an inflammatory SASP indicating that

the growth arrest itself did not stimulate SASP.

One of the most defining characteristics of senescence is stable growth arrest.

This is achieved by two important pathways, the and the p53/p21 CIP1 , both of

which are central in tumor suppression. DNA damage results in: (1) high deposition of

yH2Ax (histone coding gene) and 53BP1 (involved in DNA damage response) in

chromatin: this leads to activation of a kinase cascade eventually resulting in p53

activation, and (2) activation of p16INK4a and ARF (both encoded by CDKN2A) and

P15INK4b (encoded by CDKN2B): p53 induces transcription of cyclin-dependent kinase

inhibitor (p21CIPI) and along with both p16INK4a and p15INK4b block genes for cell

cycle progression (CDK4 and CDK6). This eventually leads to hypophosphorylation of

Retinoblastoma protein (Rb) and cell cycle arrest at the G1 phase.

Selectively killing senescent cells has been shown to significantly improve the

health span of mice in the context of normal aging and ameliorates the consequences of

age-related disease or cancer therapy (Ovadya, J Clin Invest. 128(4):1247-1254, 2018). In

nature, the senescent cells are normally removed by the innate immune cells. Induction

of senescence not only prevents the potential proliferation and transformation of

damaged/altered cells, but also favors tissue repair through the production of SASP

factors that function as chemoattractants mainly for Natural Killer (NK) cells (such as IL-

15 and CCL2) and macrophages (such as CFS-1 and CCL2). These innate immune cells

mediate the immunosurveillance mechanism for eliminating stressed cells. Senescent

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cells usually up-regulate the NK-cell activating receptor NKG2D and DNAMI ligands,

which belong to a family of stress-inducible ligands: an important component of the

frontline immune defense against infectious diseases and malignancies. Upon receptor

activation, NK cells can then specifically induce the death of senescent cells through their

cytolytic machinery. A role for NK cells in the immune surveillance of senescent cells

has been pointed out in liver fibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013),

hepatocellular carcinoma (Iannello, J Exp Med 210(10): 2057-2069, 2013), multiple

myeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cells stressed by

dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer 142(1): 176-190, 2018).

Endometrial cells undergo acute cellular senescence and do not differentiate into decidual

cells. The differentiated decidual cells secrete IL-15 and thereby recruit uterine NK cells

to target and eliminate the undifferentiated senescent cells thus helping to re-model and

rejuvenate the endometrium (Brighton, Elife 6: e31274, 2017). With a similar

mechanism, during liver fibrosis, p53-expressing senescent liver satellite cells skewed the

polarization of resident Kupfer macrophages and freshly infiltrated macrophages toward

the pro-inflammatory M1 phenotype, which display senolytic activity. F4/80+

macrophages have been shown to play a key role in the clearance of mouse uterine

senescent cells to maintain postpartum uterine function.

Senescent cells recruit NK cells by mainly upregulating ligands to NKG2D

(expressed on NK cells), chemokines, and other SASP factors. In vivo models of liver

fibrosis have shown effective clearance of senescent cells by activated NK cells

(Krizhanovsky, Cell 134(4): 657-667, 2008). Studies have described various models to

study senescence including liver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008),

osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci 72(6): 780-785, 2017), and Parkinson's

disease (Chinta, Cell Rep 22(4): 930-940, 2018). Animal models for studying senescent

cells are described in: Krizhanovsky, Cell 134(4): 657-667, 2008; Baker, Nature

479(7372): 232-236, 2011; Farr, Nat Med 23(9): 1072-1079, 2017; Bourgeois, FEBS Lett

592(12): 2083-2097, 2018; Xu, Nat Med 24(8): 1246-1256, 2018).

Senescence is a form of irreversible growth arrest accompanied by phenotypic

changes, resistance to apoptosis and activation of damage-sensing signaling pathways.

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Cellular senescence was first described in cultured human fibroblast cells that lost their

ability to proliferate, reaching permanent arrest after about 50 population doublings

(referred to as the Hayflick limit) (Hayflick et al., Exp. Cell Res. 25:585-621, 1961). He

observed a phenomenon of "replicative senescence" in cultures of non-immortalized

human fibroblasts which is caused by a progressive telomere shortening upon each cell

division and represents a physiological response to prevent genomic instability and

therefore accumulation of DNA damage (He et al., Cell 169(6):1000-1011, 2017).

Senescence is considered a stress response that can be induced by a wide range of

intrinsic and extrinsic insults, including oxidative and genotoxic stress, DNA damage,

telomere attrition, or oncogenic activation, mitochondrial dysfunction, or

chemotherapeutic agents (McHugh et al., J. Cell Biol. 217(1):65-77, 2018). This

accelerated senescence response, independent from the telomere shortening, is known as

premature senescence. Senescence has been linked to various age-related complications

like diabetes, osteoporosis, cardiovascular diseases, dementia, neurodegenerative

disorders, renal failure, and sarcopenia. It is also interesting to note that the aging is the

single biggest risk factor for cancer (McHugh et al., J. Cell Biol. 217(1):65-77, 2018;

Childs et al., Nat. Rev. Drug Discov. 16(10):718-735, 2017).

Senescent cells remain metabolically active and can influence the tissue

hemostasis, disease and aging through their secretory phenotype (He et al., Cell

169(6):1000-1011, 2017). Senescence is considered as a physiologic process and is

important in promoting wound healing, tissue homeostasis (Brighton et al., Elife 6, 2017),

regeneration, embryogenesis, fibrosis regulation, etc. (von Kobbe, Cell Mol. Life Sci.

2018). For instance, transient induction of senescent cells is observed during would

healing and contributes to wound resolution. Perhaps one of the most important roles of

senescence is its role in tumorigenesis suppression (von Kobbe, Cell Mol. Life Sci. 2018).

However, the accumulation of senescent cells also drives aging and aging-related

diseases. The senescent phenotype also can trigger chronic inflammatory responses and

consequently augment chronic inflammatory conditions to promote tumor growth. The

connection between senescence and aging was initially based on observations that

senescent cells accumulate in aged tissue. In the last decade, our understanding of

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senescence's detrimental consequences in aging and age-related pathologies has

expanded significantly. The use of transgenic models enabled the detection of senescent

cells systematically in many age-related pathologies. The development of genetic and

senolytic drugs strategies to selectively eliminate senescent cells has demonstrated that

senescent cells can indeed play a causative role in aging and related pathologies.

Senescent cells display important and unique properties which include changes in

morphology, chromatin organization, gene expression, and metabolism. There are

several biochemical and functional properties associated with cellular senescence, such as

(i) increased expression of p16 INK4a and p21CIP1, inhibitors of cyclin-dependent kinases,

(ii) presence of senescence-associated B-galactosidase, a marker of lysosomal activity,

(iii) appearance of senescence-associated heterochromatin foci and downregulation of

lamin B1 levels, (iv) resistance to apoptosis caused by an increased expression of anti-

apoptotic BCL-family protein, (v) upregulation of CD26 (DPP4) (Kim et al., Genes Dev.

31 1(15):1529-1534, 2017), CD36 (Scavenger receptor) (Chong et al., EMBO Rep. 19(6),

2018), forkhead box 4 (FOXO4) (Bourgeois et al., FEBS Lett. 592(12): 2083-2097,

2018), and secretory carrier membrane protein 4 (SCAMP4) (Kim et al., Genes Dev.

32(13-14): 909-914, 2018), (vi) accumulation of lipofuscin, and (vii) expression of

embryonic chondrocyte-expressed 1 and decoy death receptor 2. Senescent cells also

express an inflammatory signature, the so-called SASP. Through SASP, the senescent

cells produce a wide range of inflammatory cytokines (IL-1a, IL-1B, IL-6, IL-8, TNF-a),

growth factors (TGF-B, PDGF-AA, insulin-like growth factor-binding proteins

(IGFBPs)), chemokines (CCL-2, CCL-20, CCL-7, CXCL-4, CXCL1, and CXCL-12),

and matrix metalloproteinases (MMP-3, MMP-9) that operate in a cell-autonomous

manner to reinforce senescence (autocrine effects) and communicate with and modify the

microenvironment (paracrine effects) (Milanovic et al., Nature 553(7686):96-100, 2018).

IL-1a is considered one of the master regulators of the SASP. The release of IL-1a by

senescent cells transmits senescence to normal cells. IFN can also induce senescence by

triggering DNA damage in the target cells. IGFBs can modulate the insulin-like growth

factor (IGF) pathway, IGF can act as a potent inducer of senescence. TGF-B, secreted as

one of the SASP factors, can induce and maintain a senescent phenotype and age-related

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pathological conditions in an autocrine/paracrine manner. Integrin B3, regulated by the

polycom protein CBX7, was upregulated during senescence, promoted senescence by

activating TGF-B signaling in an autocrine/paracrine manner, and reinforced the SASP in

human fibroblasts. In addition, the TGF-B-mediated accumulation of senescent cells has

been suggested in idiopathic pulmonary fibrosis. A recent report showed that TGF-B

signaling induced the reduction of H4K20me3 abundance, which compromised DNA

damage repair and restored and promoted senescence, by upregulating miR-29a/c and

downregulating its target in Suv4-20h in fibroblasts. This pathway contributed to cardiac

aging in vivo, and the inhibition of TGF-B signaling restored H4K20me3 and improved

cardiac function in older mice.

Matrix metalloproteinases (MMPs) are important elements of SASP, including

MMP-1 and -3, which can act as regulatory elements of senescence. They can cleave IL-

8, IL-1, VEGF, and other CXCL/CCL family chemokines. In addition, senescent cells

secrete serine proteases like urokinase- or tissue-type plasminogen activators.

The SASP is also composed of non-macromolecular elements such as nitric oxide

and reactive oxygen species that can affect the phenotype of neighboring cells.

The secretion profile of senescent cells is context dependent. For instance, the

mitochondrial dysfunction-associated senescence (MiDAS), induced by different

mitochondrial dysfunction in human fibroblasts, led to the appearance of a SASP that was

deficient in IL-1-dependent inflammatory factors (Wiley et al., Cell Metab. 23(2):303-

314,2016). A decrease in the NAD+/NADH ratio activated AMPK signaling which

induced MiDAS through the activation of p53. As a result, p53 inhibited NF-kB

signaling which is a crucial inducer of pro-inflammatory SASP (Salminen et al., Cell

Signal. 24(4):835-845, 2012). In contrast, the cellular senescence caused by persistent

DNA damage in human cells induced an inflammatory SASP, which was dependent on

the activation of ataxia-telangiectasia mutated (ATM) kinase but not on that of p53

(Rodier et al., Nat. Cell Biol. 11(8): 973-979, 2009). In particular, the expression and

secretion levels of IL-6 and IL-8 were increased. It was also demonstrated that cellular

senescence caused by the ectopic expression p16 INK4a and p21CIPI induced the senescent

phenotype in human fibroblasts without an inflammatory SASP indicating that the

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growth arrest itself did not stimulate SASP (Coppe et al., J. Biol. Chem. 286(42): 36396-

36403, 2011). These indicate that the senescent phenotype have a crucial role in the

control of the nature of SASP and its physiological and pathological consequences.

Thus, multiple components of the SASP have the ability to drive senescence in a

paracrine manner in nearby non-senescent cells to increase the overall number of

senescent cells. By means of the SASP, senescent cells can also influence the tissue

microenvironment via paracrine mechanism to influence neighboring proliferating cells

and the recruitment and activation of immune cells in aging tissues and tumors.

SASP factors can contribute to tumor suppression by triggering senescence

surveillance, an immune-mediated clearance of senescent cells. However, chronic

inflammation is also a known driver of tumorigenesis, and accumulating evidence

indicates that chronic SASP can also boost cancer and aging-related diseases. Recently,

it has also been shown that senescent cells affect neighboring cells by direct intercellular

protein transfer (Biran et al., Genes Dev. 29(8):791-802, 2015). Proteins transferred from

senescent cells to recipient neighboring cells triggered activation of signaling pathways in

these cells which led to changes in their cellular behavior. A recent study showed that

chemotherapy-induced senescent cancer cells engulfed neighboring senescent or non-

senescent cancer cells. The engulfment occurred even in the presence of a cell-death

inhibitor p53. The ingested cells are degraded in lysosomes. The senescent cells that ate

their neighbors survived longer in vitro than those that did not. This suggested that the

metabolic building blocks retrieved from the lysosomal digestion of neighboring cells

were being used by senescent cells to promote their survival. The engulfment was

mainly through the phagocytosis rather than the entosis mechanism of action. It was

proposed that cell cannibalism might affect cancer progression by supporting the SASP

response. However, this newly acquired capability of chemotherapy-induced senescent

cancer cells could promote or facilitate cancer-cell metastasis directly by removing

particular cells from the tumor microenvironment. If normal cells are also found to be

removed by senescent cells in aged tissues, this might directly cause tissue degradation.

In summary, all components of SASP contribute to the local inflammatory

environment and may contribute to the inflammaging phenomenon.

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Most of the SASP components are regulated by the nuclear factor kappa light-

chain-enhancer of activated B cells (NF-kB), CCAAT/enhancer-binding protein beta

(CEBP/3) and by mTOR. The transcription factor GATA4, acting upstream of NF-kB, is

also required for senescence establishment and SASP induction. Another regulator of

SASP is the bromodomain and extraterminal domain (BET) family member

bromodomain-containing protein 4 (BRD4) that positively regulates the senescence

secretome and promotes senescence immune clearance. The SASP is also regulated by

signal transducer and activator of transcription 3 (STAT3) in certain tissues. In addition,

the mixed-lineage leukemia 1 (MLL1) has also been reported to enable the SASP, mainly

by inducing genes required for the DNA replication and for the DDR activation. Other

SASP regulators include NOTCH1 and the high mobility group B proteins (HMGB1 and

HMGB2). Recent data also demonstrate that the SASP can be controlled by the

cGAS/STING pathway. cGAS is a DNA sensor that, through the adaptor protein STING,

triggers cellular senescence and the transcription of genes that control the SASP.

One of the most defining characteristics of senescence is stable growth arrest.

This is achieved by the p53/p21c'r1p21cipl and p16TNK4a/Rb pathways (McHugh et al., J.

Cell Biol. 217(1):65-77, 2018). DNA damage and/or DNA damage responses (DDR)

critically control these two pathways.

(1) p53 plays a pivotal role in cellular senescence and its activation

can be DDR-dependent or DDR-independent. In the telomere DDR-dependent case,

telomere attrition, DNA damage, as well as hyperactivation of oncogenes and

inactivation of onco-suppressors (oncogene induced senescence, OIS) resulting from

replicative stress activate the DNA damage repair cascade. DDR activates the stress

sensors' ataxia-telangiectasia mutated kinase (ATM) or ataxia telangiectasia and

Rad3-related (ATR) kinase. ATM/ATR, in turn, activate the p53/p21ClPlp21cipl

axis by phosphorylating both p53 and its ubiquitin ligase Mdm2, leading to the

stabilization of p53 levels. P53 is directly phosphorylated in Ser-15 and indirectly

phosphorylated in Ser-20 via Chk1/2. Many recent studies also demonstrated that

several OIS pathways can actually activate p53p35 bypassing the DDR. These

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demonstrated once again that the crucial role of p53 and p53-triggered senescence

for the suppression of tumorigenesis after the onset of a first mutation.

The stabilization of the p53 protein upregulates p21CIP1 p21clP1p21cip1

p21cipl a member of the mammalian cyclin-dependent kinase (CDK) inhibitor

family, is required for the p53-induced cell cycle arrest at either G1/S or G2/M

checkpoints. p21ClPlp21cip1, encoded by the CDKN1A gene located on chromosome

6 in humans, is a potent cyclin-dependent kinase inhibitor (CKI). It binds to and

inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus

functions as a regulator of cell cycle progression at G1 and S phase. p21clPlp21cipl

also mediates the gene expression modulation of many p53 targets such as CDC25C,

CDC25B, and surviving, mainly through the E4F4 complex recruitment.

p21cPlp21cipl also promotes senescence through the inhibition of apoptosis. It binds

many apoptosis agents, including many caspases. P21clPlP21cip knockout in

senescent cells provokes programmed cell death through the caspase activation

cascade. p21clPlp21cip1 is also capable of inducing senescence independently from

p53 activity. It was shown that Chk2 was able to induce p21cip1 expression in p53-

defective cell lines, contributing to Chk2-mediated senescence.

(2) p16TNK4a/Rb: Three tumor suppressors reside in the INK4/ARF locus: p16TNK4a and

ARF, which are both encoded by the CNDN2A gene, and p151NK4b which is encoded

by CDKN2B gene. p151NK4b and p161NK4a, are CDKIs, like p21CIP1, that affect the cell

cycle by binding and inhibiting CDK4 and CDK6. In contrast, ARF inhibits

MDM2, thereby allowing cross talk with p53/p21clP1 pathways. The INK4/ARF

locus behaves as a senescence sensor. In young, normal cells, the INK4/ARF locus is

epigenetically silenced through deposition of repressive H3K27me3 marks. H3K27

methylation is controlled by polycom repressive complexes, PRC2 and PRC3.

Disrupting PRC1 or PRC2 activity by depleting the expression of some of their

components depresses p16TNK4a and induces senescence. During senescence, the

H3K27 histone demethylase JMJD3 plays a role in removing the repressive marks

around the INK4/ARF locus, facilitating its induction. INK4/ARF induction can be

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observed in tissues during natural aging. In particular, p16 INK4a is considered an

aging biomarker.

In summary, p53 induces transcription of cyclin-dependent kinase inhibitor

p21 CIP1 and along with both p16INK4a and p 15INK4b block genes for cell cycle

progression (CDK4 and CDK6). This eventually leads to hypophosphorylatio of

Retinoblastoma protein (Rb) and cell cycle arrest at the G1 phase (McHugh et al., J. Cell

Biol. 217(1):65-77, 2018).

While the p53/p21clp1 pathway seems to play a key role in the initiation of

senescence, the pathway involving p16 INK4a and the RB family seems to have a central

role in the maintenance of senescence. This was suggested by the observation of a

decrease in p53 levels after induction of senescence, while p16TNK4a levels maintains

steadily high. It has also been shown that the downregulation of p53 in senescent cells

has different effects depending on p16 activity. p53 succeeds in inducing replication and

cell growth in cells with low levels of p16INK4a, while it does not in cells with high

p16 INK4a activity. These findings suggest that the activation of INK4a /Rb pathway is

responsible for drawing a line between two different phases of senescence: the early and

reversible phase is dominated by p53 activity and the irreversible phase is induced by the

p16TNK4a/Rb pathway.

Recently, the cGAS-cGAMP-STING - pathway has emerged as an important link

from DNA damage to inflammation, cellular senescence, and cancer (Tuo et al., J. Exp.

Med. 215(5):1287-1299, 2018). This pathway detects cytoplasmic DNA after DNA

damage and activate type I IFNs and other cytokines. Although both DNase2 and

TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in

pre-senescent cells, the expression of these DNases is downregulated in senescent cells,

resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant

activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through

induction of IFN-B (Takahashi et al., Nature Comm. 9:1249,2018)

The transforming growth factor-ß (TGF-B) is a superfamily of evolutionarily

conserved cytokines that mediate a diverse range of signaling functions to provide tissue-

specific control of cell differentiation and proliferation. They also promote or protect

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against cell death, promote extracellular matrix protein expression, cell motility and

invasion, and control cell metabolism.

The human TGF-B family includes thirty-three genes that encode for

homodimeric or heterodimeric secreted cytokines. The family members include activins,

the bone morphogenetic proteins, the growth differentiation factors, the Mullerian

inhibiting substance, the nodal and the TGF-Bs. The TGF-B family proteins are

synthesized as precursor molecules consisting of a signal peptide, a prodomain (termed

latency-associated peptide (LAP), for TGF-B), and the mature polypeptide. The removal

of the short N-terminal signal peptide allows protein folding, glycosylation, and

processing in subsequent biosynthetic steps during transport from the endoplasmic

reticulum to the Golgi apparatus. Dimerization via disulfide linkage is followed by

proteolytic cleavage of the polypeptide by furin family proteases resulting in the

formation of an N-terminal long dimeric and disulfide-linked LAP, and a C-terminal

short dimeric disulfide-linked mature TGF-B. The LAP and mature TGF-B remain

associated with each other and form the latent form of the ligand called small latency

complex (SLC), structural analysis of this latent form of TGF-B shows that LAP directly

covers the critical amino acids of the C-terminal dimer that are later used for interaction

with the signaling receptors and thus confers inactivation of the mature TGF-B dimer.

Concomitant to the processing of TGF-B polypeptide, crosslinking of the N-terminal LAP

through disulfide bonding to other secreted proteins, latent TGF-B binding proteins

(LTBPs), takes place to form large latent complex (LLC). LTBPs are extracellular

protein, and upon secretion, mediate deposition of LLC to the extracellular matrix (EMC)

via their ability to crosslink with other proteins of the ECM such as fibronectin and

fibrillins. Thus, LTBPs provide the scaffolding units that tether latent TGF-Bs to the

ECM. The latent complexes of the three TGF-Bs require activation mechanisms to

release the mature ligand; however, only the activation of the TGF-B1 complex has been

well characterized. The diverse modes of latent extracellular latent TGF-B activation in

physiologically relevant settings, which include proteolysis, low pH, reactive oxygen

species, bind to other proteins, and mechanical deformation by shear or integrin-mediated

cell pulling, suggest cell type-selective of tissue-selective mechanisms that may be

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depend on the signaling context. In various contexts, one side of the LAP in the SLC is

covalently cross-linked via Cys33 residue to the 8-Cys domain of the LTBP, which in

turn is linked to the ECM. With this resistance, pulling the other end of the LAP via

integrins-notably avß1, avß6, and avß8-enables changes in conformation of latent TGF-B

complex that result in the release of the active TGF-B1 from LLC. Various proteases also

confer activation of latent TGF-B. Many studies strongly suggest that physiological

activation of latent TGF-B1 requires combined activities of integrins and proteases.

Instead of association with an LTBP, the latent TGF-B complex has also been found to be

disulfide-linked to a membrane associated protein named GARP, also known as

LRRC32, or the closely related LRRC33. GARP is primarily expressed in immune cells

such as regulatory T cells (Tregs). The function of GARP has been extensively studied

on Tregs, where it complexes with avß8 integrins to release active TGF-B from the

surface of the cells. GARP was shown to be involved in enhancing Tregs-mediated

peripheral tolerance. In platelets, it has also been shown that thrombin cleaves GARP

resulting in liberation of active TGF-B1 from the GARP-LAP-TGF-B1 complexes.

Once activated from their LAPs, all three TGF-B isoforms act through the same

heteromeric transmembrane TGF-B receptor complex, formed by dimeric TGF-B type I

receptor (RI) alk5 (aka TBR1) and the dimeric TGF-B type II receptor (RII) TGFBRII.

TGF-B associates first with a homodimeric TGFßRII. This interaction causes a

conformational adaption between the ligand and TGFßRII, in a manner that a new high-

affinity binding site is formed for TGFßRI at the interface of ligand and TGFBRII. Upon

recruitment of the two units of TGFßRI, the type II receptor kinase phosphorylates serine

and threonine residues in the juxta-membrane subdomain of TGFßRI that is characterized

by a short glycine-and serine-rich motif (GS), which then induces conformational

changes that release the immunophilin FKBP12 from the GS domain. This dissociation

relieves the inhibitory interaction of the kinase domain with GS domain and activates the

kinase in the type I receptors. Upon ligand-induced receptor activation, the TGFßRI then

activates effector SMADs through phosphorylation of their two C-terminal serine

residues. Specifically, the type I receptor phosphorylates two different SMAD proteins in

the case of TGF-B (and other family members such as activins and nodal), SMAD2 and

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SMAD3, or three different SMAD proteins in the case of BMPs (also some GDFs and

other ligand members), SMAD1, SMAD5, and SMAD8. These "receptor-activated

SMADs" (R-SMADs) then dissociate from the receptor and combine with SMAD4 to

form complexes that translocate into the nucleus, where they cooperate with high-affinity

DNA binding transcription factors and coregulators to activate or repress target genes.

SMAD complexes not only direct gene transcription that then leads to secondary gene

expression changes but also control mRNA splicing, miRNA expression and processing,

and epigenetic changes. With further diversity in SMAD complex formation, the SMAD

signaling pathway is highly versatile, context-dependent, and nuanced pathway that

controls gene expression.

In addition to the canonical SMAD signaling, TGF-B can regulate downstream

cellular responses also via other signal transducers in a context-dependent manner. These

include the ERK MAP kinase pathway, the JNK and p38 MAP kinase pathway (via

TAK1), the PI3-AKT pathway, the JAK-STAT pathway, the Rho-(like) GTPase pathway,

and the TGF-B type I receptor intracellular domain signaling pathway. It is well known

now that the extensive functional versatility and dependence of the SMADS are on these

non-canonical pathways.

Since TGF-Bs control the differentiation of most, if not all, cell lineages and

regulate many aspects of cell and tissue homeostasis, deregulation of TGF-B signaling

leads to developmental anomalies and diseases. Accumulated evidence has indicated that

the impairment of TGF-B signaling in certain cell types and the regulation of TGF-B

ligands contribute to cellular senescence, cell degeneration, tissue fibrosis, inflammation,

decreased regeneration capacity, and metabolic malfunction.

TGF-31, secreted as one of the SASP factors, can induce or accelerate, and

maintain a senescent phenotype in various cell types including fibroblasts, bronchial

epithelial cells, and cancers in an autocrine/paracrine manner. Integrin B3, regulated by

the polycom protein CBX7, was upregulated during senescence, promoted senescence by

activating TGF-B signaling in an autocrine/paracrine manner, and reinforced the SASP in

human fibroblasts. In addition, the TGF-B1-mediated accumulation of senescent cells has

been suggested in idiopathic pulmonary fibrosis. A recent report showed that TGF-31

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signaling induced the reduction of H4K20me3 abundance, which compromised DNA

damage repair and restored and promoted senescence, by upregulating miR-29a/c and

downregulating its target, Suv4-20h in fibroblasts. This pathway contributed to cardiac

aging in vivo, and the inhibition of TGF-B signaling restored H4K20me3 and improved

cardiac function in older mice.

TGF-B1 functions as a senescence driver and induces vascular smooth muscle cell

(VSMC) senescence through reactive oxygen species (ROS)-stimulated activation of NF-

KB signaling pathway and expression of SASP factors, including plasminogen activator

inhibitor type-1 (PAI-1, SERPINE 1). PAI-1 is not only a biomarker of cellular

senescence but also is necessary and sufficient for the replicative senescence downstream

of p53 and is a key inducer of the senescence program. There is evidence suggesting the

existence of a PAI-1/TGF-61-positive feed-forward mechanism, providing for a model

whereby elevated tissue levels of TGF-B1 during the emergence of the senescent

phenotype stimulate expression of PAI-1 that, in turn, reinforces continued TGF-B1

synthesis promoting the maintenance, and perhaps expansion, of the senescent VSMC

population (Seo et al., Am. J. Nephrol. 30:481-490,2009)

It is well known that senescence has tumor suppressive effects that delay clinical

progression following chemotherapy. The last decade has witnessed a big step forward in

the understanding of the biology of senescence, especially from it having a tumor-

suppressing property to a complex, dynamic, and interactive one that may lead to pro-

oncogenic effects on adjacent cancer cells, the stroma and vasculature in the tumor

microenvironment (Hoare et al., Ann. Rev. Cancer Biol. 2:175-194, 2018). A very

elegant study by Milanovic showed in samples from patients with primary B-cell chronic

leukemia that senescent cells also upregulated important stem cell related transcripts

(Milanovic et al., Nature 553(7686):96-100, 2018). Senescent cells have been shown in

acute myeloid leukemia (AML) patients where AML blasts induced a senescent

phenotype in stromal cells and these stromal cells in turn feedback to promote AML blast

survival and proliferation via SASP (Abdul-Aziz et al., Blood 133(5):446-456, 2019).

Tumors are thought to seize pathophysiological programs of growth regulation that are

intended to participate in organ development or tissue repair and 'hijack' this process for

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oncogenic performance instead of creating novel mechanisms for tumor progression

(Milanovic et al., Trends Cell Biol. 28(12):1049-1061, 2018). Epigenetic mechanisms

have been described to be responsible for senescence induction (H3K9 demethylase) and

subsequent stemness (H3K9 demethylase inhibition) acquisition (Yu et al., Cancer Cell

33(2):322-336, 2018).

TGF-B1 triggers epithelial-mesenchymal transitions (EMT) through induction of

the expression of specific transcription factors Snail and Zebl 1/2. EMT provides

migratory and invasive behaviors to the cells due to cell adhesion modifications. This

process involves a loss of epithelial features and the acquisition features leading to

motility and invasive properties. EMT represents an important process leading to the

progression and metastasis of cancer cells.

As an immunosuppressive cytokine, TGF-B1 inhibits the function and

development of innate and adaptive immune systems including macrophages, natural

killer cells, dendritic cells, and T cells. Recent in vivo studies have demonstrated that

exposure to tissue- or tumor-derived TGF-B1 can drive the conversion of circulating NK

cells into an innate lymphoid cell I (ILC-I) -like phenotype, characterized by a reduction

in cytotoxic capacity and the acquisition of several ILC1-associated surface markers.

Interestingly, TGF-B1 also synergizes with IL-15 through MAPK pathways to drive the

conversion of human NK cells to an ILC-1 like phenotype. TGF-B also represses human

NK cell metabolism through its canonical signaling pathway to suppress NK-cell

cytotoxicity. TGF-B1 also stimulates regulatory T cells which suppresses the function of

other lymphocytes. These suppressive functions confer to TGF-B1 one of many cancer

hallmarks with avoiding immune destruction.

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of

the pancreas with an extremely poor prognosis with a five-year survival rate of 7% and a

median survival of less than 11 months. PDAC is highly refractory to all available

antitumor pharmacological options. This is the result of the strong desmoplastic reaction

associated with PDAC progression, displaying a strong activation of pancreatic stellate

cells and formation of dense extracellular matrix that results in insufficient tumor

perfusion and an impenetrable barrier to intravenously infused anticancer drugs or

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chemotherapeutic agents. TGF-B1 contributes to PDAC desmoplasia by enhancing the

conversion of fibroblasts or endothelial cells into myofibroblasts also known as cancer-

associated fibroblasts (CAFs). Aggressiveness is further amplified by infiltrated immune

cells and fibroblasts in the tumor microenvironment, which can produce high levels of

TGF-B. TGF-B induces proangiogenic factors such as vascular endothelial growth factor,

allowing PDAC progression, invasion, and metastasis. The acyl-CoA synthetase long-

chain 3 (ACSL3) was found to be upregulated in PDAC and correlates with increased

fibrosis. The decreased PAI-1 secretion from tumor cells by Acsl3 knockout markedly

reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells, increases

cytotoxic T cell infiltration in mice. This study also found that PAI-1 expression in

PDAC positively correlates with markers of fibrosis and immunosuppression and predicts

poor patient survival. Since PAI-1 is a key component of SASP and a mediator of

cellular senescence and is regulated by TGF-B1, it is conceivable that TGF-B1 plays a

role in this ACSL3-PAI-1 signaling axis mediating tumor-stroma cross-talk that promotes

pancreatic cancer progression.

In fibrotic disease, excessive deposition of extracellular matrix (EMC) proteins

compromises tissue integrity and interferes with normal organ function. Fibrosis can

arise in any tissue that suffered chronic insults but most frequently observed in the

kidneys, livers, lung, and heart. Fibrosis is primarily driven by inflammatory cytokines

including the interleukins and members of the TGF-B superfamily. Many of these

ligands are expressed by infiltrating inflammatory cells which are attracted to the

damaged tissue. Overexpression of TGF-B1 induces fibrosis via activation of both

canonical (SMAD-based) and non-canonical (non SMAD-based) signaling pathways,

which result in activation of myofibroblasts, excessive production of ECM and inhibition

of ECM degradation. Activation of SMAD 2/3 regulates the expression of several

profibrotic genes including collagens (COLIAL, COL3A1, COL5A2, COL6A1, COL6A3,

and COL7A1), PAI-1, various proteoglycans, integrins, connective tissue growth factor,

and matrix metalloproteases. This results in excessive deposits of ECM that

compromises the local tissue architecture.

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Although signaling through the SMAD pathway is believed to play a central role

in TGF-B's fibrogenesis, emerging evidence indicates that reactive oxygen species (ROS)

are also involved in modulating TGF-B's signaling through different pathways including

the SMAD pathway. TGF-B1 increases mitochondrial ROS production in different type

of cells, which mediate TGF-B-induced cell apoptosis, senescence, EMT, fibrotic gene

expression and myofibroblast differentiation. TGF-B has been shown to induce the

expression of several NADPH oxidases (Noxs) enzymes (including Nox1, Nox2, and

Nox 4 in different types of cells), which are a group of heme-containing transmembrane

proteins important in ROS production for both phagocytic and non-phagocytic cell.

Nox4-derived ROS mediate TGF-B's fibrogenic effects, including fibroblast

activation/myofibroblast differentiation, epithelial and endothelial cell apoptosis, EMT,

and the expression of fibroblastic/profibrotic genes. An increase in Nox4 expression has

also been detected in fibrotic diseases including IPF, which correlates with increased

expression of myofibroblast marker, a-SMA, further supporting the role of Nox4 in

fibrotic diseases. Several pathways have been shown to be involved in the induction of

Nox4 by TGF-B. These include the SMAD pathway, PI3K pathway, MAPK pathways,

and RHOA/ROCK pathway. Emerging evidence suggests that there is crosstalk between mitochondria and

NADPH oxidases. Mitochondria-derived ROS contribute to the increase in NOX

expression in response to TGF-B whereas NOX-generated ROS cause mitochondria

dysfunction and increase mitochondrial ROS production. The cross-talk between

mitochondria and Nox enzyme has also been shown to mediate TGF-B's profibrogenic

effect. A feed-forward interaction between mitochondria and Nox4 in TGF-B-induced

ROS production is implicated (Jain et al., Journal of Biological Chemistry. 288:770-777,

2013).

By 2030, more than 20% of the population will be age 65 or older (see,

census.gov/content/dam/Census/library/publications/2014/demo/p23-212.pdf): and

approximately 40% will be obese (Finkelstein et al., Am. J. Prev. Med. 42(6):563-570,

2012). Metabolic diseases impact the capacity of the cell to conduct vital processes that

involve transport or processing of proteins, carbohydrates and lipids. Aging and obesity

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are key risk factors for chronic conditions that predispose to conditions including

diabetes, cardiovascular disease and hepatic steatosis, all of which are leading causes of

death and therefore pose a significant public health concern (Must et al., "The Disease

Burden Associated with Overweight and Obesity," In: Feingold KR, Anawalt B., Boyce

A., et al., eds., Endotext, South Dartmouth (MA), 2000; Martin et al., Nat. Rev. Cardiol.

14(3):132,2017).

Excessive calorie intake promoted oxidative stress in adipose tissue in mice and

resulted in features of Type-2 diabetes concomitantly with the expression of senescence

markers such as p53, beta galactosidase in mice (Minamino et al., Nat. Med. 15(9):1082-

1087,2009) Senescence also promoted biological decline in adipose tissue by

preventing adipogenic differentiation (Mitterberger et al., Gerontol. A Biol. Sci. 69(1):13-

24, ,2014). Another recent study has shown that obesity-induced senescence can lead to

anxiety and impaired neurogenesis by increasing fat deposits in the brain and clearance of

these senescent cells led to improvement in obesity-induced anxiety-like behavior in mice

(Ogrodnik et al., Cell Metab. 29(5):1061-1077, 2019). Other studies have shown that

obesity also impairs functions of immune cells. NK cell effector function was shown to

be impaired due to lipid accumulation in these cells and reversal of this process restored

function (Michelet et al., Nat. Immunol. 19(12):1330-1340, 2018). Additional studies

have shown that impairment of NK cells in obesity is independent of age as similar

defects were observed in young and older obese individuals (Tobin et al., JCI Insight

2(24):e94939, 2017; Michelet et al., Nat. Immunol. 19(12):1330-1340, 2018).

In mice, increased calorie intake leads to fat deposition in blood vessels which in

turn recruit monocytes that engulf these lipids and turn into foamy macrophages that

eventually accumulate in the subendothelial spaces leading to atherosclerotic plaques

(Bennett et al., Nat. Rev. Cardiol. 14(3):132, 2017; Katsuumi et al., Front. Cardiovasc.

Med. 5:18, 2018). Mice fed on Western high fat diet (diet consisting of 42% calories

from fat) also showed that the burden of senescent cells were directly proportional to the

formation of plaques (lipid laden macrophages). Successful elimination of these

senescent cells in transgenic mice led to significant reduction in plaque formation (Childs

et al., Science 354(6311):472-477, 2016).

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Age, obesity and other factors linked to alterations in glucose levels, growth

hormone (IGF) can lead to diabetes (Palmer et al., Diabetes 64(7):2289-2298, 2015).

Upregulation of senescent markers like p53 in mice fed with high fat diet correlated with

insulin resistance whereas inhibition of p53 activity in adipose tissue led to decrease in

senescence markers and correlated with improved insulin resistance in mice models

(Minamino et al., Nat. Med. 15(9):1082-1087, 2009). Concomitantly, pancreatic B-cell

senescence has been shown to be a contributor to type 2 diabetes in obese mice (Sone et

al., Diabetologia 48(1):58-67, 2005).

The hypothalamic production of TGF-B is excessive under high-fat diet

conditions. This leads to hypothalamic inflammation, hyperglycemia, and glucose

intolerance. The data suggest that the excessive amount of TGF-B induces a

hypothalamic RNA stress response, leading to the accelerated mRNA decay of IkBa.

IkBa is an inhibitor of NF-kB (Yan et al., Nature Medicine 20:1001-1008, 2014). Thus,

TGF-B signaling exacerbates obesity and diabetes through actions on the peripheral and

central nervous systems.

Aging is a major risk factor for developing many neurodegenerative diseases.

Accumulation of senescent cells in the nervous system has been shown with aging and

neurodegenerative disease and may predispose a person to the appearance of a

neurodegenerative condition or may aggravate its course (Kritsilis et al., Int. J. Mol. Sci.

19(10:2937, 2018). Cellular senescence can impede cellular function by: 1. Promotion of

chronic inflammation (Huell et al., Acta Neuropathol. 89(6):544-551, 1995; Nelson et al.,

Aging Cell 11(2):345-349, 2012), 2. Exhaustion of neuron regeneration (Cipriani et al.,

Cereb. Cortex 28(7):2458-2478, 2018), 3. Loss of function (De Stefano et al., J. Neurol.

Neurosurg. Psychiatry 87(1):93-99, 2016) and 4. Blood brain barrier dysfunction

(Yamazaki et al., Stroke 47(4):1068-1077, 2016). Studies have shown the accumulation

of AB peptide containing amyloid plaques and misfolded tau protein in Alzheimer's

disease (AD), the most prevalent neurodegenerative disease in humans (Musi et al.,

Aging Cell 17(6):e12840, 2018). These changes eventually affect neurons leading to

cognitive impairment and neurodegeneration. Astrocytes cultured from AD patients

showed high expression of well known senescent markers CDKi p16INK4A and MMP-1

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and IL-6 (Bhat et al., PLoS One 7(9):e45069, 2012; Myung et al., Age 30(4):209-215,

2008). Clinical trials targeting amyloid proteins have been disappointing (Mehta et al.,

Expert Opin. Invest. Drugs 26(6):735-739, 2017). Recent studies have shown the

presence of senescent cells to be responsible for neuronal disorders in animal models

(Crews et al., Hum. Mol. Genet. 19(R1):R12-R20, 2010; Chinta et al., Cell Rep.

22(4):930-940, 2018). Studies in animal models reflecting human AD has shown

encouraging results. Clearance of senescent cells in transgenic mice prevented

neurofibrillary tangles and abnormal accumulations of a tau protein inside neurons thus

preserving cognitive function (Bussian et al., Nature 562(7728):578-582, 2018). Patients

with Parkinson's disease (PD), the second most common neurodegenerative disease

demonstrate loss of motor control due to loss of dopamine-producing neurons in the

substantia nigra. Astrocytes, the most abundant cell type within the CNS is important for

providing structural, metabolic support to neurons and also plays a role in control of the

blood brain barrier and blood flow. A recent ground-breaking study showed a senescent

phenotype in astrocytes in postmortem brain samples from patients with PD (Chinta et

al., Cell Rep. 22(4):930-940, 2018). This study also developed an animal model of PD

induced by an environmental neurotoxin (Parquat, which induces senescence through

oxidative stress) which showed neuropathology linked to PD. The authors showed that

elimination of senescent cells in the transgenic mice lead to abrogation of paraquat-

induced neuropathology.

Aging of the human skin can be either: 1. intrinsic (chronological), which is a

consequence of physiologic and genetic changes over time or 2. extrinsic; caused by

exposure to external factors such as ultraviolet (UV) radiation, environmental toxins and

other agents that can induce DNA damage (Cavinato et al., Exp. Gerontol. 94:78-82,

2017). Among the changes that affect cutaneous tissue with age, the loss of elastic

properties caused by changes in elastin production, increased degradation and/or

processing produces a substantial impact on tissue esthetics and health (Wang et al.,

Front. Genet. 9:247, 2018). Acute UV exposure leads to sunburns, aberrant pigmentation,

visible appearance of blood vessels under the skin (telangiectasia) and immune

suppression while long term exposure may lead to premature skin aging and even risk of

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developing malignancies (Rittie et al., Cold Spring Harb. Perspective 5(1):a015370,

2015). There is a direct correlation between the evolution of medicine and population

growth, which is characterized by an increase in the number of middle-aged and elderly

individuals and therefore a significant demand for anti-aging treatments (Weihermann et

al., Int. J. Cosmet. Sci. 39(3):241-247, 2017). UVB from sunlight is mutagenic and

directly induces DNA damage during DNA replication. The hallmark of photodamaged

skin is accumulation of amorphous elastic fibers along with disorganized dermal

collagen. Studies have shown that this could result from either impaired elastic and

fibrillin production or elevated breakdown of Matrix metalloproteinases (MMP) secreted

by senescent cells that have undergone DNA damage (Pittayapruek et al., Int. J. Mol. Sci.

17(6):868, 2016). Reactive oxygen species (ROS) production following UVB radiation

leads to activation of factors central to senescence such as nuclear factor-kappa (NF-kB)

and mitogen-activated protein kinase (MAPK) (Pittayapruek et al., Int. J. Mol. Sci.

17(6):868,2016). UVB irradiation can alter TGF-B signaling pathway in human dermal

fibroblasts mainly by decreasing the synthesis of transforming growth factor-ß receptor II

(TBRII) (Purohit et al., J. Dermatol. 83(1):80-83, 2016). Several studies have shown the

presence of senescent cells in aged as well as skin exposed to UV both in vitro and in

vivo. Keratinocytes and skin fibroblasts have been extensively studied as models of

photoaging which express markers of senescence such as p16 INK4asd beta galactosidase,

Lamin B1 and Senescence associated secretory phenotype (SASP) (Waaijer et al., Aging

10(2):278-289, 2018; Dimri et al., Proc. Natl. Acad. Sci. U.S.A. 92(20):9363-9367, 1995;

Wang et al., Sci. Rep. 7(1): :15678, 2017; Ghosh et al., J. Invest. Dermatol. 136(11):2133-

2139, 2016). As senescent cells are known to express NK ligands, induction of NK cells

along with activation of other immune cells (T regulatory cells) would represent an

attractive strategy to clear senescent cells and maintain healthy skin (Carr et al., Clin.

Immunol. 105(2):126-140, 2002; Ali et al., Immunology 152(3):372-381, 2017).

The confirmation that selectively killing senescent cells significantly improves the

health span of mice in the context of normal aging and ameliorates the consequences of

age-related disease or cancer therapy has ignited interest in the identification of

compounds that can clear senescent cells. In nature, the senescent cells are normally

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removed by the innate immune cells. Induction of senescence not only prevents the

potential proliferation and transformation of damaged/altered cells, but also favors tissue

repair through the production of SASP factors (Munoz-Espin et al., Nat. Rev. Mol. Cell

Biol. 15(7):482-496, 2014) that function as chemoattractants mainly for natural killer

(NK) cells (such as IL-15 and CCL2) and macrophages (such as CFS-1 and CCL2).

These innate immune cells mediate the immunosurveillance mechanism for eliminating

stressed cells. Senescent cells usually up-regulate the NK-cell activating receptor

NKG2D and DNAMI ligands, which belong to a family of stress-inducible ligands, an

important component of the frontline immune defense against infectious diseases and

malignancies. Upon receptor activation, NK cells can then specifically induce the death

of senescent cells through their cytolytic machinery. A role for NK cells in the immune

surveillance of senescent cells has been pointed out in liver fibrosis (Sagiv et al.,

Oncogene 32(15):1971-1977, 2013), hepatocellular carcinoma (Iannello et al., J. Exp.

Med. 210(10):2057-2069, 2013), multiple myeloma (Soriani et al., Blood 113(15):3503-

3511, 2009), and glioma cells stressed by dysfunction of the mevalonate pathway

(Ciaglia et al., Int. J. Cancer 142(1):176-190, 2018). In cancer, combination

chemotherapy was shown to upregulate markers of senescence and NK ligands on

KRAS- mutant lung tumors suggesting that NK cells are required for targeting these cells

(Ruscetti et al., Science 362(6421):1416-1422, 2018). Endometrial cells undergo acute

cellular senescence and do not differentiate into decidual cells. The differentiated

decidual cells secrete IL-15 and thereby recruit uterine NK cells to target and eliminate

the undifferentiated senescent cells thus helping to re-model and rejuvenate the

endometrium (Brighton et al., Elife 6, 2017). With a similar mechanism, during liver

fibrosis, p53-expressing senescent liver satellite cells skewed the polarization of resident

Kupfer macrophages and freshly infiltrated macrophages toward the pro-inflammatory

M1 phenotype, which display senolytic activity. F4/80+ macrophages have been shown

to play a key role in the clearance of mouse uterine senescent cells to maintain

postpartum uterine function (Lujambio et al., Cell 153(2):449-460, 2013).

The strategies of senescent cell clearance mainly fall into three categories:

senolytics, immunotherapy and SASP inhibition (He et al., Cell 169(6):1000-1011,

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2017). There is a growing body evidence suggesting the efficacy of senolytics to clear

senescent cells. Senolytics in general, act by targeting the senescent cell anti-apoptotic

pathways (SCAP) like the BCL-2 protein family, the p53/p21clP1p21 axis, PI3K/AKT,

receptor tyrosine kinases, and the HSP90 proteins. In mice, senolytics alleviate a range of

conditions that have been associated with effects of senescent cells. So far, these include

effects on cardiac, vascular, metabolic, neurological, radiation-induced, chemotherapy-

induced, renal, and pulmonary functions as well as mobility and frailty in several animal

models (Kirkland et al., EBioMedicine 21:21-28, 2017). A number of additional

senolytic drugs are currently being developed. Recently, a FOXO4-related peptide that

inhibits the PI3K/AKT/p53/p21 pathway was described and showed encouraging results

both in vitro human fibroblast and mouse models. Other senolytics include ABT-737 and

ABT-263 which act on BCL-2 protein (Tse et al., Cancer Res. 68(9):3421-3428, 2008)

and A1331852 and A1155463 which target the BCL-XL pathway (Zhu et al., Aging

(Albany NY) 9(3):955-963, 2017), dasatinib and quercitin which target tyrosine kinase

have demonstrated senescent cell clearance (Farr et al., Nat. Med. 23(9):1072-1079,

2017). BCL-2 family inhibitors may potentially cause side effects like neutropenia and

thrombocytopenia. As many of the senolytics are only in their pre-clinical phase, studies

are warranted on possible side-effects before they move into clinical phase trials.

Blocking SASP factors is an alternative strategy to prevent the detrimental role of

senescent cells. These factors include inflammatory chemokines and cytokines, growth

factors, and matrix-remodeling proteases. The central pathways involved in these effects

are the NF-kB and the C/EBPB pathways. mTOR inhibitors, such as rapamycin and its

analogs, can abolish SASP by reducing the expression of membrane-bound IL-1a. Two

other notable drugs used to inhibit the NF-kB and the C/EBPB pathways in vivo mouse

models are Metformin and Ruxolitinib respectively (Moiseeva et al., Aging Cell

12(3):489-498, 2013; Xu et al., Proc. Natl. Acad. Sci. U.S.A. 112(46):E6301-6310, 2015).

Other drugs like siltuximab or tocilizumab block cytokines like IL-6, another SASP

factors. Again, as with the use of some senolytics, treatment with anti-inflammatory

drugs can give rise to potential side effects (Karkera et al., Prostate 71 (13): 1455-1465,

2011). A recent Phase I clinical trial using senolytics (dasatinib plus quercetin) in patients

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with pulmonary fibrosis did not lead to any conclusive results (Justice et al.,

EBioMedicine 40:554-563, 2019).

The third strategy, which is potentially superior than those described above is

immune-mediated interventions. As mentioned above, cells recruited to clear senescent

cells include NK cells, macrophages and neutrophils. Senescent cells recruit NK cells by

mainly upregulating ligands to NKG2D (expressed on NK cells), chemokines and other

SASP factors. In vivo models of liver fibrosis have shown effective clearance of

senescent cells by activated NK cells (Krizhanovsky et al., Cell 134(4):657-667, 2008).

Senescent cells resist NK cell mediated clearance by upregulating decoy receptor DCR2

which inhibits apoptosis and restricting their clearance mainly by granzyme and perforin

mediated pathways (Sagiv et al., Oncogene 32(15):1971-1977, 2013). Recent data has

shown that lipid accumulation in NK cells seen in obese individuals leads to reduction in

both their frequencies and effector cytotoxic function and this was independent of age

(Michelet et al., Nat. Immunol. 19(12):1330-1340, 2018; Tobin et al., JCI Insight

2(24):e94939, 2017). NK cell-mediated antibody-dependent cell cytotoxicity (ADCC)

has been demonstrated in vitro human senescent cells against dipeptidyl peptidase 4

(DPP4/CD26), a recently described senescence marker (Kim et al., Genes Dev.

1(15):1529-1534, 2017). Other strategies include using CAR-T cells to redirect immune

responses against senescent cells (Grupp et al., N. Engl. J. Med. 368(16):1509-1518,

2013; Yousefzadeh et al., Nature, published online on May 12, 2021).

Studies have described various models to study senescence including liver fibrosis

(Krizhanovsky et al., Cell 134(4):657-667, 2008), osteoarthritis (Xu et al., J. Gerontol. A

Biol. Sci. Med. Sci. 72(6):780-785, 2017), Parkinson's (Chinta et al., Cell Rep.

22(4):930-940, 2018), obesity induced anxiety (Ogrodnik et al., Cell Metab. 29(5):1061-

1077,2019), atherosclerosis (Childs et al., Science 354(6311):472-477, 2016), and

diabetes (Sone et al., Diabetologia 48(1):58-67,2005) One recent study showed that

transplanting in-vitro senescence-induced cells into young mice led to physical

dysfunction (Xu et al., Nat. Med. 24(8):1246-1256, 2018). The question that lingers is

which type of therapy is effective in clearing senescent cells in different tissues. Majority

of the available data are based on in vitro experiments and few mouse studies

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(Krizhanovsky et al., Cell 134(4):657-667, 2008; Xu et al., Nat. Med. 24(8):1246-1256,

2018; Baker et al., Nature 479(7372):232-236, 2011; Farr et al., Nat. Med. 23(9):1072-

1079, 2017; Xu et al., J. Gerontol. A Biol. Sci. Med. Sci. 72(6):780-785, 2017; Bourgeois

et al., FEBS Lett. 592(12):2083-2097, 2018). NK cells provide an attractive strategy to

counter senescent cell accumulation. However, very few studies in senescence models

have explored this strategy (Krizhanovsky et al., Cell 134(4):657-667, 2008). Various

clinical trials have shown the success of utilizing adoptive transfer of NK cells to treat

various forms of cancer (Sakamoto et al., J. Transl. Med. 13:277, 2015; Miller et al.,

Blood 105(8):3051-3057, 2005; Cifaldi et al., Trends Mol. Med. 23(12): 1156-1175, 2017;

Li et al., Cytotherapy 20(1):134-148, 2018). Of importance is the recent clinical trial of

utilizing autologous ex-vivo expanded NK cells in patients with colon cancer (Li et al.,

Cytotherapy 20(1):134-148, 2018). The authors showed that NK cell therapy in

combination with chemotherapy prevented recurrence and prolonged survival with

acceptable adverse effects (Li et al., Cytotherapy 20(1):134-148, 2018). Transfer of

cytokine activated-NK cells by cytokines such as IL-15, IL-12, IL-18 and IL-21 can be

used as a potential immunotherapeutic strategy to clear senescent cells with minimal side-

effects (Romee et al., Blood 120(24): 4751-4760, 2012; Song et al., Eur. J. Immunol.

48(4):670-682, 2018). Moreover, the safety of using NK cells has been shown in acute

myeloid leukemia (Romee et al., Blood 120(24): 4751-4760, 2012; Fehniger et al., Biol.

Blood Marrow Transplant. 2018). Other approaches would be to block circulating SASP

factors like TGF-B, IL-8 and IL-6 (Ganesh et al., Immunity 48(4):626-628, 2018;

Georgilis et al., Cancer Cell 34(1):85-102, 2018). The models of senescence mentioned

above would be ideal to test these approaches. Therefore, more consideration should be

given to such strategies that avoid unwanted side-effects from using foreign compounds

and drugs as a solution to age-related pathologies.

Cellular senescence is a series of progressive and phenotypically diverse cellular

states that are acquired after initial growth arrest (Van Deursen, Nature 509(7501): 439-

446, 2014). Thus, senescent cells are heterogeneous populations of cells with few shared

core properties (Dou et al., Nature 550(7676):402-406, 2017). Identifying common

senolytic drug targets, therefore, is difficult. This further precludes the achievement of a

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goal of developing senolytics that selectively, safety, and effectively eliminate senescent

cells upon systemic administration. As described above, immune cells are the effector

cells to remove senescent cells naturally after the fulfillment of senescent-cell

physiological roles (Brighton et al., Elife 6, 2017). The weakening of the immune system

during the aging process allows the accumulation of senescent cells (Karin et al., Nat

Commun 10(1):5495, 2019) (Chambers et al., Allergy Clin Immunol 145(5): 1323-1331,

2020). In addition, TGF-B, a component of the SASP of senescent cells, plays a caustic

role in cellular senescence and aging-related pathologies when it is produced in an

excessive amount in tissues (Tominaga et al., Int. J. Mol. Sci. 20(20), 2019). Provided

herein are methods of using complexes of common gamma-chain cytokines and their

cognate receptors to promote and to activate immune cells, and TGF-BRII to reduce the

amount of the active form of TGF-B in the aging tissues and tumor microenvironment

through subcutaneous administration to regain their capabilities of reducing senescent

cells and to lower the chronic inflammation in vivo effectively, selectively, and safely.

In some embodiments of any of the methods described herein, the methods result

in rejuvenation of aged immune cells in the subject (e.g., one or more of: increased

metabolic activity (e.g., increased oxidative phosphorylation, increased glycolysis, and

increased oxygen consumption) of aged immune cells in the subject; decreased level(s) of

one or more of p16, p21, and a SASP factor in aged immune cells in the subject; and

increased cytolytic activity of the aged immune cells in the subject, e.g., as compared to

the level(s) in the subject prior to treatment). As used herein, the term "aged immune

cell" means an immune cell that has one or more of: reduced metabolic activity (e.g.,

reduced oxidative phosphorylation, reduced glycolysis, and reduced oxygen

consumption); increased level(s) of one or more of p16, p21, and a SASP factor; and

decreased cytolytic activity, e.g., as compared to a control immune cell obtained from a

healthy subject (non-immune compromised subject) whose age is less than half the

average life span of the subject's population. Non-limiting examples of aged immune

cells include aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes,

aged macrophages, aged neutrophils, aged basophils, aged eosinophils, Kupffer cells, and

aged microgial cells.

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In some embodiments of any of the methods described herein, the methods result

in an increase in the naive T cell to memory T cell ratio in the subject. In some

embodiments of any of the methods described herein, the methods result in a decrease in

the ratio of CD4+ T cells to CD8+ T cells in the subject.

In some embodiments, the rejuvenation of the aged immune cells results in a

reduction of number of diseased cells or infectious agents in the subject. In some

embodiments, the aged immune cells include one or more of aged NK cells, aged NKT

cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils,

aged basophils, aged eosinophils, aged Kupffer cells, and aged microgial cells. In some

embodiments, the diseased cells include cancer cells, virally-infected cells, and

intracellularly-bacterially-infected cells. In some embodiments, the infectious agents

include virus, bacterium, fungus, and parasite.

Provided herein are also methods of using complexes of common gamma-chain

cytokines and their cognate receptors and/or agents that result in a decrease in the

activation of a TGF-B receptor to rejuvenate the immune system.

Methods of Improving the Texture and/or Appearance of Skin and/or Hair

Also provided herein are methods of improving the texture and/or appearance of

skin and/or hair in a subject in need thereof over a period of time (e.g. any of the periods

of time described herein) that include administering to the subject a therapeutically

effective amount of one or more natural killer (NK) cell activating agent(s) (e.g. any of

the NK cell activating agent(s) described herein or known in the art).

Also provided herein are methods of improving the texture and/or appearance of

skin and/or hair in a subject in need thereof over a period of time (e.g. any of the periods

of time described herein) that include administering to the subject a therapeutically

effective number of activated NK cells (e.g. any of the activated NK cells described

herein or known in the art).

Some embodiments of these methods further include: obtaining a resting NK cell;

and contacting the resting NK cell in vitro in a liquid culture medium including one or

more NK cell activating agent(s), where the contacting results in the generation of the

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activated NK cells that are subsequently administered to the subject. In some examples

of these methods, the resting NK cell is an autologous NK cell obtained from the subject.

In some examples of these methods, the resting NK cell is a haploidentical NK cell

obtained from the subject. In some examples of these methods, the resting NK cell is an

allogeneic resting NK cell. In some examples of these methods, the resting NK cell is an

artificial NK cell. In some examples of any of these methods, the resting NK cell is a

genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor.

In some examples of these methods, the liquid culture medium is a serum-free

liquid culture medium. In some embodiments of any of the methods described herein, the

liquid culture medium is a chemically-defined liquid culture medium. Some examples of

these methods further include isolating the activated NK cells (and further administering

a therapeutically effective amount of the activated NK cells to a subject, e.g., any of the

subjects described herein). In some embodiments of these methods, the contacting step is

performed for a period of about 2 hours to about 20 days (or any of the subranges of this

range described herein).

In some embodiments of these methods, the method provides for an improvement

in the texture and/or appearance of skin of the subject over the period of time (e.g. any of

periods of time described herein).

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the rate of formation of wrinkles

in the skin of the subject over the period of time (e.g., any of the periods of time

described herein), e.g., as compared to the rate of formulation of wrinkles in the subject

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prior to treatment or the rate of formulation of wrinkles in a similar subject not receiving

a treatment.

In some embodiments of these methods, the method results in an improvement in

the coloration of skin of the subject over the period of time (e.g. any of the periods of

time described herein).

In some embodiments of these methods, the method results in an improvement in

the texture of skin of the subject over the period of time (e.g. any of the periods of time

described herein).

In some embodiments of these methods, the method provides for an improvement

in the texture and/or appearance of hair of the subject over the period of time (e.g. any of

the periods of time described herein).

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the rate of formation of gray hair

in the subject over the period of time (e.g. any of the range of time period described

herein), e.g., as compared to the rate of formulation of gray hair in the subject prior to

treatment or the rate of formulation of gray hair in a similar subject not receiving a

treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

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any of the subranges of this range described herein)) in the number of gray hairs of the

subject over the period of time (e.g. any of the periods of time described herein), e.g., as

compared to the number of gray hairs in the subject prior to treatment or the rate of

formation of gray hairs in a similar subject not receiving a treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the rate of hair loss in the subject

over the period of time (e.g., any of the periods of time described herein), e.g., as

compared to the rate of hair loss in the subject prior to treatment or the rate of hair loss in

a similar subject not receiving a treatment.

In some embodiments of these methods, the method results in an improvement in

the texture of hair of the subject over the period of time (e.g. any of the periods of time

described herein).

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the number of senescent dermal

fibroblasts in the skin of the subject over the period of time (e.g., any of the periods of

time described herein), e.g., as compared to the number of senescent dermal cells in the

subject prior to treatment or the number of senescent dermal cells in a similar subject not

receiving a treatment.

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In some embodiments of these methods, improvement in the texture and/or

appearance of skin of the subject over the period of time (e.g. any of the periods of time

described herein) can be assessed by any method described herein or known in the art,

including inspecting the presence, size and shape of skin lesions, skin color and

pigmentation, skin moisture, temperature, elasticity, and vascularity.

In some embodiments of these methods, improvement in the texture and/or

appearance of hair of the subject over the period of time (e.g., any of periods of time

described herein) can be assessed by any method described herein or known in the art,

In some embodiments of these methods, the period of time is, e.g., one month to

ten years, one month to nine years, one month to eight years, one month to seven years,

one month to six years, one month to five years, one month to four years, one month to

three years, one month to two years, one month to eighteen months, one month to twelve

months, one month to ten months, one month to eight months, one month to six months,

one month to four months, one month to two months, one month to six weeks, six weeks

to ten years, six weeks to nine years, six weeks to eight years, six weeks to seven years,

six weeks to six years, six weeks to five years, six weeks to four years, six weeks to three

years, six weeks to two years, six weeks to eighteen months, six weeks to twelve months,

six weeks to ten months, six weeks to eight months, six weeks to six months, six weeks to

four months, six weeks to two months, two months to ten years, two months to nine

years, two months to eight years, two months to seven years, two months to six years,

two months to five years, two months to four years, two months to three years, two

months to two years, two months to eighteen months, two months to twelve months, two

months to ten months, two months to eight months, two months to six months, two

months to four months, four months to ten years, four months to nine years, four months

to eight years, four months to seven years, four months to six years, four months to five

years, four months to four years, four months to three years, four months to two years,

four months to eighteen months, four months to twelve months, four months to ten

months, four months to eight months, four months to six months, six months to ten years,

six months to nine years, six months to eight years, six months to seven years, six months

to six years, six months to five years, six months to four years, six months to three years,

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six months to two years, six months to eighteen months, six months to twelve months, six

months to ten months, six months to eight months, eight months to ten years, eight

months to nine years, eight months to eight years, eight months to seven years, eight

months to six years, eight months to five years, eight months to four years, eight months

to three years, eight months to two years, months to eighteen months, eight months to

twelve months, eight months to ten months, ten months to ten years, ten months to nine

years, ten months to eight years, ten months to seven years, ten months to six years, ten

months to five years, ten months to four years, ten months to three years, ten months to

two years, ten months to eighteen months, ten months to twelve months, twelve months

to ten years, twelve months to nine years, twelve months to eight years, twelve months to

seven years, twelve months to six years, twelve months to five years, twelve months to

four years, twelve months to three years, twelve months to two years, twelve months to

eighteen months, eighteen months to ten years, eighteen months to nine years, eighteen

months to eight years, eighteen months to seven years, eighteen months to six years,

eighteen months to five years, eighteen months to four years, eighteen months to three

years, eighteen months to two years, two years to ten years, two years to nine years, two

years to eight years, two years to seven years, two years to six years, two years to five

years, two years to four years, two years to three years, three years to ten years, three

years to nine years, three years to eight years, three years to seven years, three years to

six years, three years to five years, three years to four years, four years to ten years, four

years to nine years, four years to eight years, four years to seven years, four years to six

years, four years to five years, five years to ten years, five years to nine years, five years

to eight years, five years to seven years, five years to six years, six years to ten years, six

years to nine years, six years to eight years, six years to seven years, seven years to ten

years, seven years to nine years, seven years to eight years, eight years to ten years, eight

years to nine years, or nine years to ten years.

In some embodiments of these methods, the age of the subject is between about

30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50

to about 55, about 55 to about 60, about 60 to about 65, about 65 to about 70, about 70 to

about 75, about 75 to about 80, about 80 to about 85, about 85 to about 90, about 90 to about 95, about 95 to about 100, about 100 to about 105, about 105 to about 110, about

110 to about 115, or about 115 to about 120.

Methods of Assisting in the Treatment of Obesity in a Subject

Provided herein are methods of assisting in the treatment of obesity in a subject in

need thereof over a period of time (e.g. any of the range of time period described herein),

that include administering to the subject a therapeutically effective amount of one or

more natural killer (NK) cell activating agent(s) (e.g. any of the NK cell activating

agent(s) described herein or known in the art).

Also provided herein are methods of assisting in the treatment of obesity in a

subject in need thereof over a period of time (e.g. any of the range of time period

described herein) that include administering to the subject a therapeutically effective

number of activated NK cells (e.g. any of the activated NK cells described herein or

known in the art).

Some embodiments of these methods further include: obtaining a resting NK cell;

and contacting the resting NK cell in vitro in a liquid culture medium including one or

more NK cell activating agent(s), where the contacting results in the generation of the

activated NK cells that are subsequently administered to the subject. In some examples

of these methods, the resting NK cell is an autologous NK cell obtained from the subject.

In some examples of these methods, the resting NK cell is a haploidentical NK cell

obtained from the subject. In some examples of these methods, the resting NK cell is an

allogeneic resting NK cell. In some examples of these methods, the resting NK cell is an

artificial NK cell. In some examples of any of these methods, the resting NK cell is a

genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor.

In some examples of these methods, the liquid culture medium is a serum-free

liquid culture medium. In some embodiments of any of the methods described herein, the

liquid culture medium is a chemically-defined liquid culture medium. Some examples of

these methods further include isolating the activated NK cells (and further administering

a therapeutically effective amount of the activated NK cells to a subject, e.g., any of the subjects described herein). In some embodiments of these methods, the contacting step is performed for a period of about 2 hours to about 20 days (or any of the subranges of this range described herein).

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the mass of the subject over the

period of time (e.g. any of the periods of time described herein), e.g., as compared to the

mass of the subject prior to treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the body mass index (BMI) of the

subject over the period of time (e.g. any of periods of time described herein), e.g., as

compared to the BMI of the subject prior to treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

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any of the subranges of this range described herein)) in the rate of progression from pre-

diabetes to type 2 diabetes in the subject, e.g., as compared to the rate of progression

from pre-diabetes to type 2 diabetes in the subject prior to treatment or the rate of

progression from pre-diabetes to type 2 diabetes in a similar subject not receiving a

treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in fasting serum glucose level in the

subject, e.g., as compared to the fasting serum glucose level in the subject prior to

treatment.

In some embodiments of these methods, the method results in an increase (e.g., at

least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20%

increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least

a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase,

at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75%

increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least

a 95% increase, or at least a 99% increase, or about a 10% increase to about a 500%

increase (or any of the subranges of this range described herein) in insulin sensitivity in

the subject, e.g., as compared to the insulin sensitivity in the subject prior to treatment.

In some embodiments of these methods, the method results in a decrease (e.g., at

least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20%

decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at

least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

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decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the severity of atherosclerosis in

the subject, e.g., as compared to the severity of atherosclerosis in the subject prior to

treatment.

In some embodiments of these methods, treatment of obesity in the subject over

the period of time (e.g. any of the periods of time described herein) can be assessed by

any method described herein or known in the art, including, e.g., measurement of body

weight and/or body dimensions, total body fat, total or regional adiposity, and body mass

index (BMI).

In some embodiments of these methods, the response of a subject to the treatment

can be monitored by determining fasting serum glucose level or glucose tolerance

according to standard techniques. In some embodiments of these methods, insulin

sensitivity can be measured using any method described herein or known in the art,

including hyperinsulinemic euglycemic clamp and intravenous glucose tolerance test,

homeostasis model assessment (HOMA), and quantitative insulin sensitivity check index

(QUICKI).

In some embodiments of these methods, the severity of atherosclerosis in the

subject can be measured using any method described herein or known in the art,

including cardiac catheterization, Doppler sonography, blood pressure comparison,

MUGA/radionuclide angiography, Thallium/myocardial perfusion scan, and

computerized tomography.

In some embodiments of these methods, the period of time is one month to ten

years (or any of the subranges of this range described herein).

In some embodiments of these methods, the age range for the subject is between

about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about

20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40

to about 45, about 45 to about 50, about 50 to about 55, about 55 to about 60, about 60 to

about 65, about 65 to about 70, about 70 to about 75, about 75 to about 80, about 80 to

about 85, about 85 to about 90, about 90 to about 95, about 95 to about 100, about 100 to

about 105, about 105 to about 110, about 110 to about 115, or about 115 to about 120.

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Additional Therapeutic Agents

Some embodiments of any of the methods described herein can further include

administering to a subject (e.g., any of the subjects described herein) a therapeutically

effective amount of one or more additional therapeutic agents. The one or more

additional therapeutic agents can be administered to the subject at substantially the same

time as the NK cell activating agent(s) or activated NK cells (e.g., administered as a

single formulation or two or more formulations to the subject). In some embodiments,

one or more additional therapeutic agents can be administered to the subject prior to

administration of the NK cell activating agent(s) or activated NK cells. In some

embodiments, one or more additional therapeutic agents can be administered to the

subject after administration of the NK cell activating agent(s) or activated NK cells to the

subject.

Non-limiting examples of additional therapeutic agents include: anti-cancer drugs,

activating receptor agonists, immune checkpoint inhibitors, agents for blocking HLA-

specific inhibitory receptors, Glucogen Synthase Kinase (GSK) 3 inhibitors, and

antibodies.

Non-limiting examples of anticancer drugs include antimetabolic drugs (e.g., 5-

fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine,

fludarabine, gemcitabine, hydroxycarbamide, methotrexate, 6-thioguanine, cladribine,

nelarabine, pentostatin, or pemetrexed), plant alkaloids (e.g., vinblastine, vincristine,

vindesine, camptothecin, 9-methoxycamptothecin, coronaridine, taxol, naucleaorals,

diprenylated indole alkaloid, montamine, schischkiniin, protoberine, berberine,

sanguinarine, chelerythrine, chelidonine, liriodenine, clivorine, 3-carboline, antofine,

tylophorine, cryptolepine, neocryptolepine, corynoline, sampangine, carbazole,

crinamine, montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide, irinotecan,

topotecan, or acridone alkaloids), proteasome inhibitors (e.g., lactacystin, disulfiram,

epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912),

delanzomib (CEP-18770), epoxomicin, MG132, beta-hydroxy beta-methylbutyrate,

bortezomib, carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin,

daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin, plicamycin, mitomycin,

WO wo 2021/247604 PCT/US2021/035285

or bleomycin), histone deacetylase inhibitors (e.g., vorinostat, panobinostat, belinostat,

givinostat, abexinostat, depsipeptide, entinostat, phenyl butyrate, valproic acid,

trichostatin A, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol, tenovin 1,

tenovin 6, sirtinol, ricolinostat, tefinostat, kevetrin, quisinostat, resminostat, tacedinaline,

chidamide, or selisistat), tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafinib,

erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), and chemotherapeutic agents

(e.g., all-trans retinoic acid, azacitidine, azathioprine, doxifluridine, epothilone,

hydroxyurea, imatinib, teniposide, tioguanine, valrubicin, vemurafenib, and

lenalidomide). Additional examples of chemotherapeutic agents include alkylating

agents, e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide,

thiotepa, hexamethylmelamine, busulfan, altretamine, procarbazine, dacarbazine,

temozolomide, carmustine, lumustine, streptozocin, carboplatin, cisplatin, and

oxaliplatin.

Non-limiting examples of activating receptor agonists include any agonists for

activating receptors which activate and enhance the cytotoxicity of NK cells, including

anti-CD16 antibodies (e.g., anti-CD16/CD30 bispecific monoclonal antibody (BiMAb))

and Fc-based fusion proteins. Non-limiting examples of checkpoint inhibitors include

anti-PD-1 antibodies (e.g., MEDI0680), anti-PD-L1 antibodies (e.g., BCD-135, BGB-

A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-

1316, anti-PD-L1/CTLA-4 bispecific antibody KN046, anti-PD-L1/TGF3RII fusion

protein M7824, anti-PD-L1/TIM-3 bispecific antibody LY3415244, atezolizumab, or

avelumab), anti-TIM3 antibodies (e.g., TSR-022, Sym023, or MBG453) and anti-CTLA-

4 antibodies (e.g., AGEN1884, MK-1308, or an anti-CTLA-4/OX40 bispecific antibody

ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory

receptors include monalizumab (e.g., an anti-HLA-E NKG2A inhibitory receptor

monoclonal antibody). Non-limiting examples of GSK3 inhibitor include tideglusib or

CHIR99021. Non-limiting examples of antibodies that can be used as additional

therapeutic agents include anti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and

any other antibody or antibody construct that can bind to and activate an Fc receptor (e.g.,

CD16) on a NK cell. In some embodiments, an additional therapeutic agent can be

insulin or metformin.

Exemplary Methods that Include Administration of One or More Common

Gamma-Chain Family Cytokine Receptor Activating Agent(s)

Provided herein are methods of killing or reducing the number of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effectively amount of one or more common gamma-chain

family cytokine receptor activating agent(s).

Also provided herein are methods of decreasing the accumulation of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effectively amount of one or more common gamma-chain

family cytokine receptor activating agent(s).

Also provided herein are methods of decreasing a level of a marker of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more common gamma-chain

family cytokine receptor activating agent(s). In some embodiments, a marker of

naturally-occurring and/or treatment-induced senescent cells is p21clPlp21 and CD26.

Additional markers of naturally-occurring and/or treatment-induced senescent cells are

described herein. Additional markers of naturally-occurring and/or treatment-induced

senescent cells are known in the art.

Also provided herein are methods of reducing the activity of naturally-occurring

and/or treatment-induced senescent cells in a subject that include administering to the

subject a therapeutically effective amount of one or more common gamma-chain family

cytokine receptor activating agent(s).

Also provided herein are methods of decreasing levels and/or activity of one or

more senescence-associated secretory phenotype (SASP) factor(s) derived from

naturally-occurring and/or treatment-induced senescent cells in a subject that include

administering to the subject a therapeutically effective amount of one or more common

gamma-chain family cytokine receptor activating agent(s). In some embodiments,

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senescent cells express an inflammatory signature, where the inflammatory signature is

aSASP factor. In some embodiments, the senescence-associated secretory phenotype

(SASP) factor includes, but is not limited to, inflammatory cytokines (e.g., IL-1a, IL-1B,

IL-6, IL-8, and TNF-a), growth factors (e.g., TGF-B, PDGF-AA, and insulin-like growth

factor-binding proteins (IGFBPs)), chemokines (e.g., CCL-2, CCL-20, CCL-7, CXCL-4,

CXCL1, and CXCL-12), and matrix metalloproteinases (e.g., MMP-3, MMP-9) that

operate in a cell-autonomous manner to reinforce senescence (autocrine effects) and

communicate with and modify the microenvironment (paracrine effects). In some

embodiments, the method decreases expression levels and/or activity of one or more

(e.g., two, three, four, or five) of the senescence-associated secretory phenotype (SASP)

factor(s). In some embodiments, the expression level or activity of a SASP factor is

determined using enzyme-linked immunosorbent assay (ELISA). In some embodiments,

the expression level or activity of a SASP factor is determined using immunoblotting.

In some embodiments of any of the methods described herein, the subject has

been previously diagnosed or identified as having an aging-related disease (e.g. any of

the exemplary types of aging-related disease or condition described herein or known in

the art) or an inflammatory disease (e.g. any of the exemplary types of aging-related

disease or condition described herein or known in the art).

In some embodiments, the aging-related disease is inflamm-aging related.

In some embodiments, the aging-related disease is a cancer (e.g. any of the

exemplary types of cancer described herein or known in the art).

In some embodiments of any of the methods described herein, the inflammatory

disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel

disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury,

Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Crohn's disease,

multiple sclerosis, Guillain-Barre syndrome, psoriasis, Grave's disease, ulcerative colitis,

nonalcoholic steatohepatitis, mood disorders and cancer treatment-related cognitive

impairment.

In some examples of these methods, the treatment-induced senescent cells are

chemotherapy-induced senescent cells.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the number of naturally-occurring

and/or treatment-induced senescent cells in a target tissue (e.g., any of the exemplary

types of target tissues described herein or known in the art) in the subject, e.g., as

compared to the number of naturally-occurring and/or treatment-induced senescent cells

in the target tissue in the subject prior to treatment.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the accumulation of naturally-

occurring and/or treatment-induced senescent cells in the subject (e.g., any of the periods

of time described herein), e.g., as compared to the accumulation of naturally-occurring

and/or treatment-induced senescent cells in the subject prior to treatment or the

accumulation of naturally-occurring and/or treatment-induced senescent cells in a similar

subject not receiving a treatment.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

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least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in a level of one or more (e.g., two,

three, or four) marker(s) of naturally-occurring and/or treatment-induced senescent cells

in the subject, e.g., as compared to the level of the one or more marker(s) of naturally-

occurring and/or treatment-induced senescent cells in the subject prior to treatment.

"Naturally-occurring senescent cells" as described herein are senescent cells that

are generated as a result of normal aging or inflammatory processes. Naturally-occurring

senescent cells may accumulate in various tissues and organs of an individual over time.

Naturally-occurring senescent cells can be any of the exemplary types of senescent cells

described herein that are not induced by a therapeutic treatment (e.g., chemotherapy or

radiation).

"Treatment-induced senescent cells" as described herein are senescent cells that

are generated as a result of therapeutic treatment (e.g., chemotherapy or radiation).

Common Gamma-Chain Family Cytokine Receptor Activating Agents

Provided herein are methods that include the use or administration of one or more

common gamma-chain family cytokine receptor activating agent(s). In some

embodiments, the common gamma-chain family cytokine receptor activating agent is a

single-chain chimeric polypeptide (e.g. any of the exemplary single-chain chimeric

polypeptides described herein), a multi-chain chimeric polypeptide (e.g. any of the

exemplary multi-chain chimeric polypeptides described herein), a soluble IL-15 or IL-15

agonist (e.g., any of the soluble IL-15 or IL-15 agonists described herein), a soluble IL-2

or IL-2 agonist (e.g., any of the soluble IL-2 or IL-2 agonists described herein), a

complex of a common gamma-chain family cytokine (or a functional fragment thereof)

and an antibody (or antibody fragment) that binds specifically to the common gamma-

chain family cytokine or the functional fragment thereof, an antibody or an antigen-

binding antibody fragment that binds specifically to a common gamma-chain family

cytokine.

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Exemplary Single-Chain Chimeric Polypeptide

Non-limiting examples of common gamma-chain family cytokine receptor

activating agents are single-chain chimeric polypeptides that include: (i) a first target-

binding domain, (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein or known in the art), and (iii) as second target-

binding domain, wherein one or both of the first target-binding domain and the second

target-binding domain is a soluble common gamma-chain family cytokine, an agonistic

antigen-binding domain that binds specifically to a common gamma-chain family

cytokine receptor, a soluble common gamma-chain family cytokine receptor, or an

antigen-binding domain that binds specifically to a common gamma-chain family

cytokine.

Some embodiments of any of the single-chain chimeric polypeptides described

herein can further include one or more (e.g., two, three, four, five, six, seven, eight, nine,

or ten) additional target-binding domains (e.g., any of the exemplary target-binding

domains described herein or known in the art) at its N- and/or C-terminus.

In some embodiments of any of the single-chain chimeric polypeptide described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is a soluble common gamma-chain

family cytokine. Non-limiting examples of soluble common gamma-chain family

cytokines include soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15,

and soluble IL-21.

In some embodiments, one or both of the first target-binding domain and the

second target-binding domain includes a soluble common gamma-chain family cytokine

receptor (e.g., a soluble receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21).

In some embodiments of any of the single-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

WO wo 2021/247604 PCT/US2021/035285

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is an agonistic antigen-binding

domain that binds specifically to a common gamma-chain family cytokine receptor.

Non-limiting examples of common gamma-chain family cytokine receptors include a

receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

Multi-Chain Chimeric Polypeptide

Non-limiting examples of common gamma-chain family cytokine receptor

activating agents are multi-chain chimeric polypeptides that include: (a) a first chimeric

polypeptide including: (i) a first target-binding domain; (ii) a soluble tissue factor

domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric

polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second

target-binding domain, where one or both of the first target-binding domain and the

second target-binding domain is a soluble common gamma-chain family cytokine, an

agonistic antigen-binding domain that binds specifically to a common gamma-chain

family cytokine receptor, a soluble common gamma-chain family cytokine receptor, or an

antigen-binding domain that binds specifically to a common gamma-chain family

cytokine.

In some embodiments of any of the multi-chain chimeric polypeptides, the first

chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven,

eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary target-

binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain (e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) is a soluble common gamma-chain family cytokine.

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Non-limiting examples of soluble common gamma-chain family cytokines include

soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more of the first target-binding domain (e.g., any of the exemplary target-

binding domains described herein or known in the art), the second target-binding domain

(e.g., any of the exemplary target-binding domains described herein or known in the art),

and the one or more additional target-binding domains (e.g., any of the exemplary target-

binding domains described herein or known in the art) is an agonistic antigen-binding

domain that binds specifically to a common gamma-chain family cytokine receptor.

Non-limiting examples of common gamma-chain family cytokine receptors include a

receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

In some embodiments of any of the multi-chain chimeric polypeptides described

herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first

target-binding domain (e.g., any of the exemplary target-binding domains described

herein or known in the art), the second target-binding domain (e.g., any of the exemplary

target-binding domains described herein or known in the art), and the one or more

additional target-binding domains (e.g., any of the exemplary target-binding domains

described herein or known in the art) is a soluble common gamma-chain family cytokine

receptor.

In some embodiments of the multi-chain chimeric polypeptides described herein,

the first domain or the second domain of a pair of affinity domains is a soluble common

gamma-chain family cytokine or an antigen-binding domain that binds specifically to a

common gamma-chain family cytokine receptor.

Soluble Common Gamma-Chain Family Cytokines

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain can be a soluble common gamma-chain family cytokine.

In some embodiments, a common gamma-chain family cytokine receptor activating agent

can be a soluble common gamma-chain family cytokine. Non-limiting examples of soluble common gamma-chain family cytokines include soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21. Non-limiting examples of sequences for soluble IL-2, soluble IL-7, soluble IL-15, and soluble IL-21 are described herein. Non-limiting examples of soluble IL-4 and IL-9 sequences are shown below.

Human soluble IL-4 (SEQ ID NO: 335)

LTEQKTLCTE LTVTDIFAAS HKCDITLQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC KNTTEKETFC RAATVLRQFY RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS

Human soluble IL-9 (SEQ ID NO: 336)

QGCPTLAGILDI NFLINKMQED PASKCHCSAN VTSCLCLGIP SDNCTRPCFS ERLSQMTNTT MQTRYPLIFS RVKKSVEVLK NNKCPYFSCE QPCNQTTAGN ALTFLKSLLE IFQKEKMRGM RGKI

Antigen-Binding Domains

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain. In some embodiments of

any of the single-chain or multi-chain chimeric polypeptides described herein, the first

target-binding domain and the second target-binding domain are each antigen-binding

domains. In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, the antigen-binding domain includes or is a scFv or a

single domain antibody (e.g., a VaHH or a VNAR domain).

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is an agonistic antigen-binding domain that binds

specifically to a common gamma-chain family cytokine receptor. In some examples, an

agonistic antigen-binding domain (e.g., any of the antigen-binding domains described

herein) can bind specifically to a receptor for IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21.

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The antigen-binding domains present in any of the single-chain or multi-chain

chimeric polypeptides described herein are each independently selected from the group

consisting of: a VHH domain, a VNAR domain, and a scFv. In some embodiments, any

of the antigen-binding domains described herein is a BiTe, a (scFv)2, a nanobody, a

nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-

scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. Additional examples of antigen-

binding domains that can be used in any of the single-chain or multi-chain chimeric

polypeptide are known in the art.

In some embodiments, each of the antigen-binding domains in the single-chain or

multi-chain chimeric polypeptides described herein are both VHH domains, or at least

one antigen-binding domain is a VHH domain. In some embodiments, each of the

antigen-binding domains in the single-chain or multi-chain chimeric polypeptides

described herein are both VNAR domains, or at least one antigen-binding domain is a

VNAR domain. In some embodiments, each of the antigen-binding domains in the

single-chain or multi-chain chimeric polypeptides described herein are both scFv

domains, or at least one antigen-binding domain is a scFv domain.

In some embodiments, two or more of polypeptides present in the single-chain or

multi-chain chimeric polypeptide can assemble (e.g., non-covalently assemble) to form

any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of

an antibody (e.g., any of the antigen-binding fragments of an antibody described herein),

a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-

in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light

chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a

LUZ-Y, a Fcab, a kh-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-

(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-

IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-

CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-

scFv, a F(ab')2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a

Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a 1mmTAC, an IgG-IgG

conjugate, a Cov-X-Body, and a scFvl-PEG-scFv2. See, e.g., Spiess et al., Mol. Immunol,

WO wo 2021/247604 PCT/US2021/035285

67:95-106, 2015, incorporated in its entirety herewith, for a description of these elements.

Non-limiting examples of an antigen-binding fragment of an antibody include an Fv

fragment, a Fab fragment, a F(ab')2 fragment, and a Fab' fragment. Additional examples

of an antigen-binding fragment of an antibody is an antigen-binding fragment of an IgG

(e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-

binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2,

IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment

of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a

human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an

antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of

an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-

binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized

IgM).

Soluble IL-15 and IL-15 Agonists

Non-limiting examples of common gamma-chain family cytokine receptor

activating agents are soluble IL-15 or IL-15 agonists. IL-15 functions through the

trimeric IL-15 receptor complex, which consists of a high affinity unique binding IL-

15Ra chain that confers receptor specificity for IL-15 and the common IL-15RB and y-

chains (also known as IL-2RB/y) shared with IL-2.

In some embodiments, the soluble IL-15 is at least 90% (e.g., at least 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 82. In some

embodiments, the soluble IL-15 is a recombinant soluble human IL-15. In some

embodiments, the soluble IL-15 is a mutant IL-15 having one or more amino acid

substitutions as compared to a wild type IL-15 (e.g., SEQ ID NO: 82). The mutant IL-15

can, for example, include a D8N or a D8A amino acid substitution as compared to a wild

type IL-15. In some embodiments, soluble IL-15 can be conjugated to a polymer (See,

e.g. Miyazaki et al., Proceed. Annual Meeting AACR, 2019, Abstract 3265).

Some examples of the IL-15 agonists described herein can include a complex of

IL-15 and all or a portion of a soluble IL-15 receptor (IL-15R). The complex of IL-15

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and all or a portion of a soluble IL-15R may have prolonged half-life and/or higher

potency as compared to free IL-15. In some embodiments, the IL-15 agonists described

herein further include an Fc domain (e.g., any of the exemplary Fc domains described

herein).

In some embodiments, the portion of a soluble IL-15R is IL-15Ra. For example,

IL-15 can be associated with an IL-15Ra-Fc fusion to form an IL-15:IL-15Ra-Fc

complex (See, e.g., those described in Stoklasek et al., J. Immunology 177:6072-80,

2006; Dubios et al., J. Immunol. 180:2099-106, 2008; Epardaud et al., Cancer Res.

68:2972-83, 2008; Rubinstein et al., Proc. Natl. Acad. Sci. U.S.A. 103:9166-71, 2006).

In some embodiments, the soluble IL-15 and IL-15Ra forms a heterodimer (see, e.g.

Colon et al., Cancer Res. 79(13 Supplement):CT082 July 1, 2019).

In some embodiments, the portion of a soluble IL-15R is a portion of IL-15Ra

(e.g., a sushi domain of IL-15Ra).

The IL-15 in the complex can be a wild type IL-15 or a mutant IL-15. For

example, mutant IL-15 containing the N72D mutation can be used to complex with all or

a portion of a soluble IL-15R (e.g., a sushi domain of IL-15Ra). In some embodiments,

the complex is ALT-803, which includes a human IL-15 mutant IL-15N72D complexed

with IL-15Ra sushi-Fc fusion (see, e.g. Zhu et al., J. Immunol. 183(6):3598-607, 2009).

Non-limiting examples of IL-15 agonists include ALT-803/N-803 (Altor

Bioscience/ImmunityBio), BNZ-1 (Bioniz Therapeutics), NIZ985 (Novartis), RTX-212

(Rubius Therapeutics), AM0015 (rhIL-15) (Lilly), IGM-7354 (IGM), XmAb24306

(Roche/Xencor), KD033 (srKD033) (Kadmon), OXS-C3550 (GT Biopharma), and

NKTR-255 (Nektar Therapeutics).

Soluble IL-2 and IL-2 Agonists

Non-limiting examples of common gamma-chain family cytokine receptor

activating agents are soluble IL-2 or IL-2 agonists. IL-2 is a cytokine centrally involved

in immune tolerance and immune activation by its effects on CD4+ T regulatory cells and

cytotoxic effector lymphocytes such as CD8+ T cells and NK cells. IL-2 acts on cells

expressing either dimeric IL-2 receptors (IL-2R) consisting of IL-2RB and Y chains, or

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trimeric aBy receptor (IL-2RaBy), with the trimeric receptor displaying 10-100 fold

higher affinity for IL-2 compared to dimeric IL-2Rs. CD4+ T regulatory cells are

characterized by strong constitutive expression of IL-2Ra, which enables the cells to

express IL-2RaBy and thereby use low levels of IL-2. Dimeric IL-2Rs are most

prominent on antigen-experienced (memory) CD8+ T cells and NK cells. High levels of

IL-2 therefore strongly stimulate CD8+ T cells and NK cells, in addition to activating

Treg cells.

In some embodiments, the soluble IL-2 is at least 90% (e.g., at least 95%

identical, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to

SEQ ID NO: 78. In some embodiments, the soluble IL-2 is a recombinant human IL-2.

The soluble IL-2 can be an IL-2 variant. For example, an IL-2 variant can bind more

effectively (e.g., at least 50, 100, 150 or 200 times more effectively) to IL-2RB than to

IL-2Ra. An exemplary IL-2 variant is MDNA109 (see, e.g., Rafei et al., J. Clin. Oncol.

37(15 Suppl.), 2019). In some embodiments, the IL-2 variant has abolished CD25

binding. For example, residues F42, Y45, and L72 which are involved in CD25 binding

can be mutated (see, e.g., Klein et al., Oncoimmunology 6(3):e1277306, 2017).

In some embodiments, the IL-2 agonist is a PEGylated IL-2 that has limited

binding to the IL2Ra subunit and preferentially binds the dimeric IL2RBY (see, e.g.,

Bentebibel et al., Cancer Discov. 9(6):711-721, 2019).

Some examples of IL-2 agonists described herein are fusion proteins that include

an IL-2. In some embodiments, the fusion proteins include IL-2 or a variant thereof

linked to all or a portion of a soluble IL-2R. In some embodiments, the portion of a

soluble IL-2R is IL-2Ra (See, e.g., Vaishampayan et al., J. Clin. Oncol. 35 (15 Suppl.),

2017). The fusion proteins can, for example, selectively activate the dimeric IL-2RBY.

Further examples of IL-2 fusion proteins include those fused to a toxin (e.g., a diphtheria

toxin).

In some embodiments, the fusion proteins include an IL-2 or a variant thereof

(e.g., any of the IL-2 variant described herein) linked to an antibody (e.g., a monoclonal

antibody or an scFv). Non-limiting examples of antibodies that can be linked to an IL-2

or a variant thereof include a human monoclonal antibody against fibroblast activation

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protein-alpha (FAP) (see, e.g., Soerensen et al., J. Clin. Oncol. 36, No. 15 Suppl.), an

anti-CD20 monoclonal antibody (see, e.g., Lansigan et al., Blood 128(22):620, 2016), an

scFv against the A1 domain of tenascin-C (see, e.g. Catania et al., Cell Adh. Migr. 9(1-

2):14-21, , 2015); and an anti-CEA antibody (See, e.g., Klein et al., Oncoimmunol.

6(3):e1277306, 2017).

Additional examples of IL-2 agonists include Proleukin (Clinigen), pulmoleukin

(Immunservice), NKTR-214 (Nektar Therapeutics), DI-Leu16-IL2 (Alopexx/Provenance

Biopharmaceuticals), RG7461 (Roche), Teleukin (Philogen), ALT-801803 (Altor

Bioscience), ALT-801 (Altor Bioscience), ALKS 4230 (Alkermes), cergutuzumab

amunaleukin (RG7813) (Roche), Camidanlumab tesirine (ADC Therapeutics/Genbmab),

NHS-IL2-LT/EMD 521873 (Merck KGaA), NIZ985 (Novartis), MDNA109 (Medicenna

Therapeutics), Angeloxin (Angelica Therapeutics), PB101 (Pivotal Biosciences), Anti-

IL-2 Program (Xoma), NKTR-255 (Nektar Therapeutics), NKTR-358/LY3471851

(Nektar Therapeutics/Lilly), CYP 0150 (Cytunepharma), NL-201 (Neoleukin), THOR-

809 (Sanofi/Synthorx), BNT151/153 (BioNTech), TransCon IL-2 B/y (Ascendis Pharma),

ILT-101 (Servier/ILT-101) and AM0015 (Lilly). Additional examples of IL-2 agonists

are known in the art.

Complexes of Common Gamma-Chain Family Cytokine and an Antibody or Antibody

Fragment Non-limiting examples of common gamma-chain family cytokine receptor

activating agents are complexes including a common gamma-chain family cytokine (e.g.,

any of the common gamma-chain family cytokines described herein) and an antibody or

antigen-binding antibody fragment that binds specifically to the common gamma-chain

family cytokine.

In some embodiments, the complex of a common gamma-chain family cytokine

and antibody or antigen-binding antibody fragment binding specifically to the common

gamma-chain family cytokine can enhance the activity of the common gamma-chain

family cytokines, and lead to expansion of CD8+ T cells and/or NK cells. In some

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embodiments, the complex has longer half-life in circulation than the free common

gamma-chain family cytokine.

In some embodiments, the complex can comprise soluble IL-2 (e.g., recombinant

soluble human IL-2) or a functional fragment thereof, and an anti-IL-2 antibody or an

antigen-binding antibody fragment thereof. Non-limiting examples of complexes of

soluble IL-2 and anti-IL-2 antibodies include soluble IL-2 complexed with anti-IL-2

antibodies S4B6, JES6-5, or MAB602, respectively (see, e.g., Tomala et al., J. Immunol.

183:4904-4912, 2009; and Boyman et al., Science 311, 2006).

In some embodiments, the complex can comprise soluble IL-4 (e.g., recombinant

soluble human IL-4) and an anti-IL-4 antibody or an antigen-binding antibody fragment

thereof. Non-limiting examples of anti-IL-4 antibodies include those described in e.g.,

Sato et al., J. Immunol. 150:2717-2723, 1993, and Finkelman et al., J. Immunol.

151:1235-1244, 1993.

In some embodiments, the complex can comprise soluble IL-7 (e.g., recombinant

soluble human IL-7) and an anti-IL-7 antibody or an antigen-binding antibody fragment

thereof. Non-limiting examples of anti-IL-7 antibodies include those described in e.g.,

Finkelman et al., J. Immunol. 151:1235-1244, 1993, and Boyman et al., J. Immunol.

180:7265-75, 2008.

In some examples of the complexes, the common gamma-chain family cytokine

(or a functional fragment thereof) and the antibody (or an antigen-binding antibody

fragment thereof) can be administered separately, and the complex between the common

gamma-chain family cytokine and the antibody or the antigen-binding antibody fragment

can be formed in vivo.

Additional example of common gamma-chain family cytokines and

corresponding antibodies or antigen-binding antibody fragments that binds to the same

are known in the art.

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Exemplary Methods that Include Administration of One or More Agent(s) that

Result in a Decrease in the Activation of a TGF-B Receptor

Provided herein are methods of killing or reducing the number of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing the accumulation of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing a level of a marker of naturally-

occurring and/or treatment-induced senescent cells in a subject that include administering

to the subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor. In some embodiments, a marker of

naturally-occurring and/or treatment-induced senescent cells is p21clPlp21 and CD26.

Additional markers of naturally-occurring and/or treatment-induced senescent cells are

described herein. Additional markers of naturally-occurring and/or treatment-induced

senescent cells are known in the art.

Also provided herein are methods of reducing the activity of naturally-occurring

and/or treatment-induced senescent cells in a subject that include administering to the

subject a therapeutically effective amount of one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor.

Also provided herein are methods of decreasing levels and/or activity of one or

more SASP factor(s) derived from naturally-occurring and/or treatment-induced

senescent cells in a subject that include administering to the subject a therapeutically

effective amount of one or more agent(s) that result(s) in a decrease in the activation of a

TGF-B receptor. In some embodiments, senescent cells express an inflammatory

signature, where the inflammatory signature is a SASP factor. In some embodiments, the

SASP factor includes, but is not limited to, inflammatory cytokines (e.g., IL-1a, IL-1B,

IL-6, IL-8, and TNF-a), growth factors (e.g., TGF-B, PDGF-AA, and insulin-like growth

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factor-binding proteins (IGFBPs)), chemokines (e.g., CCL-2, CCL-20, CCL-7, CXCL-4,

CXCL1, and CXCL-12), and matrix metalloproteinases (e.g., MMP-3 and MMP-9) that

operate in a cell-autonomous manner to reinforce senescence (autocrine effects) and

communicate with and modify the microenvironment (paracrine effects). In some

embodiments, the method decreases expression levels or activity of one or more of the

SASP factor(s). In some embodiments, the expression level or activity of a SASP factor

is determined using enzyme-linked immunosorbent assay (ELISA). In some

embodiments, the expression level or activity of a SASP factor is determined using

immunoblotting.

In some embodiments of any of the methods described herein, the subject has

been previously diagnosed or identified as having an aging-related disease (e.g. any of

the exemplary types of aging-related disease or condition described herein or known in

the art) or an inflammatory disease (e.g. any of the exemplary types of aging-related

disease or condition described herein or known in the art).

In some embodiments, the aging-related disease is inflamm-aging related.

In some embodiments, the aging-related disease is a cancer (e.g. any of the

exemplary types of cancer described herein or known in the art).

In some embodiments of any of the methods described herein, the inflammatory

disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel

disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury,

Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Crohn's disease,

multiple sclerosis, Guillain-Barre syndrome, psoriasis, Grave's disease, ulcerative colitis,

nonalcoholic steatohepatitis, mood disorders and cancer treatment-related cognitive

impairment.

In some examples of these methods, the treatment-induced senescent cells are

chemotherapy-induced senescent cells.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

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decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the number of naturally-occurring

and/or treatment-induced senescent cells in a target tissue (e.g., any of the exemplary

types of target tissues described herein or known in the art) in the subject, e.g., as

compared to the number of naturally-occurring and/or treatment-induced senescent cells

in the target tissue in the subject prior to treatment.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in the accumulation of naturally-

occurring and/or treatment-induced senescent cells in the subject (e.g., any of the periods

of time described herein), e.g., as compared to the accumulation of naturally-occurring

and/or treatment-induced senescent cells in the subject prior to treatment or the

accumulation of naturally-occurring and/or treatment-induced senescent cells in a similar

subject not receiving a treatment.

In some embodiments of these methods, the administering results in a decrease

(e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a

20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease,

at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55%

decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at

least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90%

decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or

any of the subranges of this range described herein)) in a level of one or more (e.g., two,

three, or four) marker(s) of naturally-occurring and/or treatment-induced senescent cells in the subject, e.g., as compared to the level of the one or more marker(s) of naturally- occurring and/or treatment-induced senescent cells in the subject prior to treatment.

In some embodiments, the TGF-B receptor is a TGF-B receptor II (TGF-BRII).

In some embodiments, the TGF-B receptor is a TGF-BRIII.

In some embodiments, at least one of the one or more agent(s) that result(s) in a

decrease in the activation of a TGF-B receptor is a soluble TGF-B receptor, an

extracellular domain of TGF-B receptor, an antibody that binds specifically to TGF-B, an

antagonistic antibody that binds to a TGF-B receptor, an agent that binds to a LAP, or an

agent that binds to a TGF-B/LAP complex. In some embodiments, the one or more

agent(s) that result(s) in a decrease in the activation of a TGF-B receptor decrease(s) the

activation of a TGF-B receptor through binding to a LAP, or to a TGF-B/LAP complex.

Non-limiting examples of agents that result in a decrease in the activation of a TGF-B

receptor are described below.

Agent(s) that Result in a Decrease in the Activation of a TGF-B Receptor

Provided herein are methods that include the use or administration of one or more

agent(s) that result(s) in a decrease in the activation of a TGF-B receptor. In some

embodiments, the agent that results in a decrease in the activation of a TGF-B receptor is

a single-chain chimeric polypeptide (e.g. any of the exemplary single-chain chimeric

polypeptides described herein), a multi-chain chimeric polypeptide (e.g. any of the

exemplary multi-chain chimeric polypeptides described herein), a soluble TGF-B

receptor, an extracellular domain of TGF-B receptor, an antibody (or antibody fragment)

that binds specifically to TGF-B, an antagonistic antibody that binds to a TGF-B receptor,

an agent that binds to a LAP, or an agent that binds to a TGF-B/LAP complex.

Exemplary Single-Chain Chimeric Polypeptide

Non-limiting examples of agents that result in a decrease in the activation of a

TGF-B receptor are single-chain chimeric polypeptides that include: (i) a first target-

binding domain, (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble

tissue factor domains described herein or known in the art), and (iii) a second target-

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binding domain, where one or both of the first target-binding domain and the second

target-binding domain binds specifically to a ligand of a TGF-B receptor; or one or both

of the first target-binding domain and the second target-binding domain is an antagonistic

antigen-binding domain that binds specifically to a TGF-B receptor. In some

embodiments, the TGF-B receptor is TGF-BRII. In some embodiments, the TGF-B

receptor is TGF-BRIII.

Some embodiments of any of the single-chain chimeric polypeptides described

herein can further include one or more (e.g., two, three, four, five, six, seven, eight, nine,

or ten) additional target-binding domains (e.g., any of the exemplary target-binding

domains described herein or known in the art) at its N- and/or C-terminus.

In some embodiments of any of the single-chain chimeric polypeptide described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and/or the second target-binding domain (e.g., any

of the exemplary target-binding domains described herein or known in the art) is a

soluble TGF-B receptor. Non-limiting examples of soluble TGF-B receptors include

soluble TGFBRI, soluble TGFßRII, soluble TGFßRIII, and soluble endoglin. Non-

limiting sequences for an exemplary soluble TGFßRII are described herein.

Exemplary Multi-Chain Chimeric Polypeptide

Non-limiting examples of agents that result in a decrease in the activation of a

TGF-B receptor are multi-chain chimeric polypeptides that include: (a) a first chimeric

polypeptide including: (i) a first target-binding domain; (ii) a soluble tissue factor

domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric

polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second

target-binding domain, where one or both of the first target-binding domain and the

second target-binding domain binds specifically to a ligand of a TGF-B receptor; or one

or both of the first target-binding domain and the second target-binding domain is an

antagonistic antigen-binding domain that binds specifically to a TGF-B receptor.

In some embodiments of any of the multi-chain chimeric polypeptides, the first

chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven,

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eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary target-

binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides, the second

chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven,

eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary target-

binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptide described

herein, the first target-binding domain (e.g., any of the exemplary target-binding domains

described herein or known in the art) and/or the second target-binding domain (e.g., any

of the exemplary target-binding domains described herein or known in the art) is a

soluble TGF-B receptor. Non-limiting examples of soluble TGF-B receptors include

soluble TGFßRI, soluble TGFßRII, soluble TGFßRIII, and soluble endoglin.

In some embodiments of any of the multi-chain chimeric polypeptide described

herein, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15

receptor (IL15Ra) and a soluble IL-15. In some embodiments of any of the multi-chain

chimeric polypeptide described herein, the soluble IL-15 has a D8N or D8A amino acid

substitution. In some embodiments, the soluble IL-15 comprises a mutation to reduce or

eliminate IL-15 activity.

In some embodiments of any of the multi-chain chimeric polypeptide described

herein, the pair of affinity domains is selected from the group consisting of: barnase and

barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I

fragments, and SNARE modules based on interactions of the proteins syntaxin,

synaptotagmin, synaptobrevin, and SNAP25. In some embodiments of any of the multi-

chain chimeric polypeptide described herein, the first domain or the second domain of a

pair of affinity domains is a soluble common gamma-chain family cytokine or an

antigen-binding domain that binds specifically to a common gamma-chain family

cytokine receptor.

Non-limiting examples of multi-chain chimeric polypeptides that are agents that

result in a decrease in the activation of a TGF-B receptor are those described in

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subsections herein titled "Exemplary Multi-Chain Chimeric Polypeptides-Type B, G, I,

K, L, M, N, O, and P."

Soluble TGF-B receptors

In some embodiments, one or more agent(s) that result(s) in a decrease in the

activation of a TGF-B receptor is a soluble TGF-B receptor. In some embodiments, one

or more agent(s) that result(s) in a decrease in the activation of a TGF-B receptor is a

soluble TGF-B receptor. Non-limiting examples of soluble TGF-B receptors include

soluble TGFßRI, soluble TGFBRII, soluble TGFßRIII, and soluble endoglin.

In some embodiments, the TGF-ß receptor is a TGF-B receptor II (TGFBRII). In

some embodiments, the TGFB receptor is a TGFBRIII.

TGFßRI, the type I receptor is a membrane-bound serine/threonine kinase that

requires the presence of TGFßRII to bind TGF-B. TGFBRII, the type II receptor is a

membrane-bound serine/threonine kinase that binds TGF-B 1 and TGF-B 3 with high

affinity and TGF-B2 with a much lower affinity. In some embodiments, signal

transduction requires the cytoplasmic domains of both TGFßRI and TGFBRII. TGF-

BRIII, the type III receptor is a proteoglycan that exists in membrane-bound and soluble

forms, and binds TGF-B1, TGF-B2, and TGF-B3, but does not appear to be involved in

signal transduction. Non-limiting examples of sequences for soluble TGFßRII are

described herein.

Antigen-Binding Domains

In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain. In some embodiments of

any of the single-chain or multi-chain chimeric polypeptides described herein, the first

target-binding domain and the second target-binding domain are each antigen-binding

domains. In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, the antigen-binding domain includes or is a scFv or a

single domain antibody (e.g., a VHH or a VNAR domain).

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In some embodiments of any of the single-chain or multi-chain chimeric

polypeptides described herein, one or both of the first target-binding domain and the

second target-binding domain is an antagonistic antigen-binding domain that binds

specifically to a TGF-B receptor. In some examples, an antagonistic antigen-binding

domain (e.g., any of the antigen-binding domains described herein) can bind specifically

to a soluble TGFßRI, soluble TGFßRII, soluble TGF3RIII, or soluble endoglin.

In some embodiments, any of the antigen-binding domains described herein is a

BiTe, a (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a

scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HSA, or a tandem-scFv.

Additional examples of antigen-binding domains that can be used in any of the single-

chain or multi-chain chimeric polypeptide are known in the art.

In some embodiments, two or more of polypeptides present in the single-chain or

multi-chain chimeric polypeptide can assemble (e.g., non-covalently assemble) to form

any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of

an antibody (e.g., any of the antigen-binding fragments of an antibody described herein),

a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-

in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light

chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a

LUZ-Y, a Fcab, a ka-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-

(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-

IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-

CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-

scFv, a F(ab')2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a

Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG

conjugate, a Cov-X-Body, and a scFvl-PEG-scFv2. See, e.g., Spiess et al., Mol. Immunol.

67:95-106, 2015, incorporated in its entirety herewith, for a description of these elements.

Non-limiting examples of an antigen-binding fragment of an antibody include an

Fv fragment, a Fab fragment, a F(ab')2 fragment, and a Fab' fragment. Additional

examples of an antigen-binding fragment of an antibody is an antigen-binding fragment

of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an

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antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1,

IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding

fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized

IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD

(e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding

fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or

an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or

humanized IgM).

Agents that Bind to a Latency-Associated Peptide (LAP)

Non-limiting examples of agents that bind to a latency-associated peptide (LAP)

are TGF-B1, thrombospondin-1 (TSP-1), integrin avß6, or KRFK peptide. In some

embodiments, LAP binds TGF-B1, forming a latent complex, wherein LAP is presumed

to function as a sequestering agent for active TGF-B1. In some embodiments, LAP of the

latent TGF-B complex also interacts with thrombospondin-1 (TSP-1) as part of a

biologically active complex. TSP-1/LAP complex formation involves the activation

sequence of TPS-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that

is conserved in TGF- 31-5. The interactions of LAP with TSP-1 through the LSKL and

KRFK sequences are important for thrombospondin-mediated activation of latent TGF-B

since LSKL peptides can competitively inhibit latent TGF- activation by TSP-1 or

KRFK-containing peptides. In some embodiments, integrin avß6 has been shown to

have high affinity for the TGF-B1 LAP and to participate in the activation of the TGF-31

latent complex.

Agents that Bind to a TGF-B/LAP Complex

Non-limiting examples of agents that bind to a TFG-B/LAP complex are latent

TGF-B binding proteins (LTBP). In some embodiments, the latent TGF-B binding protein

(LTBP) binds a TFG-B/LAP complex, forming a larger complex called large latent

complex (LLC). In some embodiments, LTBPs include LTBP-1, LTBP-2, LTBP-3 and

LTBP-4. In some embodiments, LTBP-1 forms a disulfide linked complex with the

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TGFß propeptide (e.g., LAP) in the endoplasmic reticulum. In some embodiments,

LTBP-4 binds only to TGF-B1, thus, mutation in LTBP-4 can lead to TGF-B associated

complications which are specific to tissues that predominantly involve TGF-B1.

Methods of Administration

Some embodiments of the methods described herein include administering one or

two or more (e.g., three or more, four or more, five or more, six or more, seven or more,

eight or more, nine or more, or ten or more) doses of the one or more agent(s) that

result(s) in a decrease in the activation of a TGF-B receptor to the subject. In some

embodiments of these methods, any two consecutive doses of the two or more doses are

administered about 1 week to about one year apart (e.g., about 1 week to about 11

months, about 1 week to about 10 months, about 1 week to about 9 months, about 1 week

to about 8 months, about 1 week to about 7 months, about 1 week to about 6 months,

about 1 week to about 5 months, about 1 week to about 4 months, about 1 week to about

3 months, about 1 week to about 2 months, about 1 week to about 1 months, about 1

week to about 3 weeks, about 1 week to about 2 weeks, about 2 weeks to about 12

months, about 2 weeks to about 11 months, about 2 weeks to about 10 months, about 2

weeks to about 9 months, about 2 weeks to about 8 months, about 2 weeks to about 7

months, about 2 weeks to about 6 months, about 2 weeks to about 5 months, about 2

weeks to about 4 months, about 2 weeks to about 3 months, about 2 weeks to about 2

months, about 2 weeks to about 1 months, about 2 weeks to about 3 weeks, about 3

weeks to about 12 months, about 3 weeks to about 11 months, about 3 weeks to about 10

months, about 3 weeks to about 9 months, about 3 weeks to about 8 months, about 3

weeks to about 7 months, about 3 weeks to about 6 months, about 3 weeks to about 5

months, about 3 weeks to about 4 months, about 3 weeks to about 3 months, about 3

weeks to about 2 months, about 3 weeks to about 1 month, about 1 month to about 12

months, about 1 month to about 11 months, about 1 month to about 10 months, about 1

month to about 9 months, about 1 month to about 8 months, about 1 month to about 7

months, about 1 month to about 6 months, about 1 month to about 5 months, about 1

month to about 4 months, about 1 month to about 3 months, about 1 month to about 2

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months, about 2 months to about 12 months, about 2 months to about 11 months, about 2

months to about 10 months, about 2 months to about 9 months, about 2 months to about 8

months, about 2 months to about 7 months, about 2 months to about 6 months, about 2

months to about 5 months, about 2 months to about 4 months, about 2 month to about 3

months, about 3 months to about 12 months, about 3 months to about 11 months, about 3

months to about 10 months, about 3 months to about 9 months, about 3 months to about 8

months, about 3 months to about 7 months, about 3 months to about 6 months, about 3

months to about 5 months, about 3 months to about 4 months, about 4 months to about 12

months, about 4 months to about 11 months, about 4 months to about 10 months, about 4 months to about 9 months, about 4 months to about 8 months, about 4 months to about 7

months, about 4 months to about 6 months, about 4 months to about 5 months, about 4

months to about 4 months, about 5 months to about 12 months, about 5 months to about

11 months, about 5 months to about 10 months, about 5 months to about 9 months, about

5 months to about 8 months, about 5 months to about 7 months, about 5 months to about

6 months, about 6 months to about 12 months, about 6 months to about 11 months, about

6 months to about 10 months, about 6 months to about 9 months, about 6 months to about

8 months, about 6 months to about 7 months, about 7 months to about 12 months, about 7

months to about 11 months, about 7 months to about 10 months, about 7 months to about

9 months, about 7 months to about 8 months, about 8 months to about 12 months, about 8

months to about 11 months, about 8 months to about 10 months, about 8 months to about

9 months, about 9 months to about 12 months, about 9 months to about 11 months, about

9 months to about 10 months, about 10 months to about 12 months, about 10 months to

about 11 months, or about 11 months to about 12 months apart).

In some embodiments of any of the methods described herein, the one or two or

more doses are administered by subcutaneous administration. In some embodiments of

any of the methods described herein, the one or two or more doses are administered by

intramuscular administration.

In some embodiments of any of the methods described herein, the two or more

doses are administered over a period of time of about 1 year to about 60 years (e.g., about

1 year to about 55 years, about 1 year to about 50 years, about 1 year to about 45 years,

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about 1 year to about 40 years, about 1 year to about 35 years, about 1 year to about 30

years, about 1 year to about 25 years, about 1 year to about 20 years, about 1 year to

about 15 years, about 1 year to about 10 years, about 1 year to about 5 years, about 5

years to about 60 years, about 5 years to about 55 years, about 5 years to about 50 years,

about 5 years to about 45 years, about 5 years to about 40 years, about 5 years to about 35

years, about 5 years to about 30 years, about 5 years to about 25 years, about 5 years to

about 20 years, about 5 years to about 15 years, about 5 years to about 10 years, about 10

years to about 60 years, about 10 years to about 55 years, about 10 years to about 50

years, about 10 years to about 45 years, about 10 years to about 40 years, about 10 years

to about 35 years, about 10 years to about 30 years, about 10 years to about 25 years,

about 10 years to about 20 years, about 10 years to about 15 years, about 15 years to

about 60 years, about 15 years to about 55 years, about 15 years to about 50 years, about

15 years to about 45 years, about 15 years to about 40 years, about 15 years to about 35

years, about 15 years to about 30 years, about 15 years to about 25 years, about 15 years

to about 20 years, about 20 years to about 60 years, about 20 years to about 55 years,

about 20 years to about 50 years, about 20 years to about 45 years, about 20 years to

about 40 years, about 20 years to about 35 years, about 20 years to about 30 years, about

20 years to about 25 years, about 25 years to about 60 years, about 25 years to about 55

years, about 25 years to about 50 years, about 25 years to about 45 years, about 25 years

to about 40 years, about 25 years to about 35 years, about 25 years to about 30 years,

about 30 years to about 60 years, about 30 years to about 55 years, about 30 years to

about 50 years, about 30 years to about 45 years, about 30 years to about 40 years, about

30 years to about 35 years, about 35 years to about 60 years, about 35 years to about 55

years, about 35 years to about 50 years, about 35 years to about 45 years, about 35 years

to about 40 years, about 40 years to about 60 years, about 40 years to about 55 years,

about 40 years to about 50 years, about 40 years to about 45 years, about 45 years to

about 60 years, about 45 years to about 55 years, about 45 years to about 50 years, about

50 years to about 60 years, about 50 years to about 55 years, or about 55 years to about

60 years).

In some embodiments of these methods, each of the one or two or more doses are

administered at a dosage of about 0.01 mg of each agent that results in a decrease in the

activation of a TGF-B receptor/kg to about 10 mg of each agent that results in a decrease

in the activation of a TGF-B receptor/kg (e.g., about 0.01 mg/kg to about 9 mg/kg, about

0.01 mg/kg to about 8 mg/kg, about 0.01 mg/kg to about 7 mg/kg, about 0.01 mg/kg to

about 6 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4 mg/kg,

about 0.01 mg/kg to about 3 mg/kg, about 0.01 mg/kg to about 2 mg/kg, about 0.01

mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about

0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 10 mg/kg,

about 0.05 mg/kg to about 9 mg/kg, about 0.05 mg/kg to about 8 mg/kg, about 0.05

mg/kg to about 7 mg/kg, about 0.05 mg/kg to about 6 mg/kg, about 0.05 mg/kg to about 5

mg/kg, about 0.05 mg/kg to about 4 mg/kg, about 0.05 mg/kg to about 3 mg/kg, about

0.05 mg/kg to about 2 mg/kg, about 0.05 mg/kg to about 1 mg/kg, about 0.05 mg/kg to

about 0.5 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 10

mg/kg, about 0.1 mg/kg to about 9 mg/kg, about 0.1 mg/kg to about 8 mg/kg, about 0.1

mg/kg to about 7 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about 5

mg/kg, about 0.1 mg/kg to about 4 mg/kg, about 0.1 mg/kg to about 3 mg/kg, about 0.1

mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.5

mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 9 mg/kg, about 0.5

mg/kg to about 8 mg/kg, about 0.5 mg/kg to about 7 mg/kg, about 0.5 mg/kg to about 6

mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 4 mg/kg, about 0.5

mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 1

mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about 1 mg/kg

to about 8 mg/kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6 mg/kg,

about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about

3 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 10 mg/kg, about 2

mg/kg to about 9 mg/kg, about 2 mg/kg to about 8 mg/kg, about 2 mg/kg to about 7

mg/kg, about 2 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg

to about 4 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 10 mg/kg,

about 3 mg/kg to about 9 mg/kg, about 3 mg/kg to about 8 mg/kg, about 3 mg/kg to about

7 mg/kg, about 3 mg/kg to about 6 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3

mg/kg to about 4 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4 mg/kg to about 9

mg/kg, about 4 mg/kg to about 8 mg/kg, about 4 mg/kg to about 7 mg/kg, about 4 mg/kg

to about 6 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg,

about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg, about 5 mg/kg to about

7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6

mg/kg to about 9 mg/kg, about 6 mg/kg to about 8 mg/kg, about 6 mg/kg to about 7

mg/kg, about 7 mg/kg to about 10 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7 mg/kg

to about 8 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9 mg/kg, or

about 8 mg/kg to about 10 mg/kg of each agent that results in a decrease in the activation

of a TGF-B receptor).

In some embodiments of these methods, a single or first dose of the one or more

agent(s) that result(s) in a decrease in the activation of a TGF-B receptor begins when the

subject reaches an age of at least 30 years (e.g., at least 32, 34, 36, 38, 40, 42, 44, 46, 48,

50, 52, 54, 56, 58, 60, 62, 65, 70, 75, or 80 years).

In some embodiments of any of the methods described herein, the subject is not

diagnosed or identified as having an aging-related disease (e.g., any of the aging-related

disease or condition described herein or known in the art) or an inflammatory disease

(e.g., any of the inflammatory diseases described herein or known in the art). In some

embodiments of any of the methods described herein, the subject has not been previously

treated with a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described

herein or known in the art). In some embodiments of any of the methods described

herein, the subject has not been previously treated with a therapeutic agent that induces

cellular senescence (e.g. any of the additional therapeutic agents that induce cellular

senescence described herein).

Some embodiments of the methods described herein include administering one or

two or more (e.g., three or more, four or more, five or more, six or more, seven or more,

eight or more, nine or more, or ten or more) doses of the one or more common gamma-

chain family cytokine receptor activating agent(s) to the subject. In some embodiments of

these methods, any two consecutive doses of the two or more doses are administered

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about 1 week to about one year apart (e.g., about 1 week to about 11 months, about 1

week to about 10 months, about 1 week to about 9 months, about 1 week to about 8

months, about 1 week to about 7 months, about 1 week to about 6 months, about 1 week

to about 5 months, about 1 week to about 4 months, about 1 week to about 3 months,

about 1 week to about 2 months, about 1 week to about 1 months, about 1 week to about

3 weeks, about 1 week to about 2 weeks, about 2 weeks to about 12 months, about 2

weeks to about 11 months, about 2 weeks to about 10 months, about 2 weeks to about 9

months, about 2 weeks to about 8 months, about 2 weeks to about 7 months, about 2

weeks to about 6 months, about 2 weeks to about 5 months, about 2 weeks to about 4

months, about 2 weeks to about 3 months, about 2 weeks to about 2 months, about 2

weeks to about 1 months, about 2 weeks to about 3 weeks, about 3 weeks to about 12

months, about 3 weeks to about 11 months, about 3 weeks to about 10 months, about 3

weeks to about 9 months, about 3 weeks to about 8 months, about 3 weeks to about 7

months, about 3 weeks to about 6 months, about 3 weeks to about 5 months, about 3

weeks to about 4 months, about 3 weeks to about 3 months, about 3 weeks to about 2

months, about 3 weeks to about 1 month, about 1 month to about 12 months, about 1

month to about 11 months, about 1 month to about 10 months, about 1 month to about 9

months, about 1 month to about 8 months, about 1 month to about 7 months, about 1

month to about 6 months, about 1 month to about 5 months, about 1 month to about 4

months, about 1 month to about 3 months, about 1 month to about 2 months, about 2

months to about 12 months, about 2 months to about 11 months, about 2 months to about

10 months, about 2 months to about 9 months, about 2 months to about 8 months, about 2

months to about 7 months, about 2 months to about 6 months, about 2 months to about 5

months, about 2 months to about 4 months, about 2 month to about 3 months, about 3

months to about 12 months, about 3 months to about 11 months, about 3 months to about

10 months, about 3 months to about 9 months, about 3 months to about 8 months, about 3

months to about 7 months, about 3 months to about 6 months, about 3 months to about 5

months, about 3 months to about 4 months, about 4 months to about 12 months, about 4

months to about 11 months, about 4 months to about 10 months, about 4 months to about

9 months, about 4 months to about 8 months, about 4 months to about 7 months, about 4

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months to about 6 months, about 4 months to about 5 months, about 4 months to about 4

months, about 5 months to about 12 months, about 5 months to about 11 months, about 5

months to about 10 months, about 5 months to about 9 months, about 5 months to about 8

months, about 5 months to about 7 months, about 5 months to about 6 months, about 6

months to about 12 months, about 6 months to about 11 months, about 6 months to about

10 months, about 6 months to about 9 months, about 6 months to about 8 months, about 6

months to about 7 months, about 7 months to about 12 months, about 7 months to about

11 months, about 7 months to about 10 months, about 7 months to about 9 months, about

7 months to about 8 months, about 8 months to about 12 months, about 8 months to about

11 months, about 8 months to about 10 months, about 8 months to about 9 months, about

9 months to about 12 months, about 9 months to about 11 months, about 9 months to

about 10 months, about 10 months to about 12 months, about 10 months to about 11

months, or about 11 months to about 12 months apart).

In some embodiments of any of the methods described herein, the one or two or

more doses are administered by subcutaneous administration. In some embodiments of

any of the methods described herein, the one or two or more doses are administered by

intramuscular administration.

In some embodiments of any of the methods described herein, the two or more

doses are administered over a period of time of about 1 year to about 60 years (e.g., about

1 year to about 55 years, about 1 year to about 50 years, about 1 year to about 45 years,

about 1 year to about 40 years, about 1 year to about 35 years, about 1 year to about 30

years, about 1 year to about 25 years, about 1 year to about 20 years, about 1 year to

about 15 years, about 1 year to about 10 years, about 1 year to about 5 years, about 5

years to about 60 years, about 5 years to about 55 years, about 5 years to about 50 years,

about 5 years to about 45 years, about 5 years to about 40 years, about 5 years to about 35

years, about 5 years to about 30 years, about 5 years to about 25 years, about 5 years to

about 20 years, about 5 years to about 15 years, about 5 years to about 10 years, about 10

years to about 60 years, about 10 years to about 55 years, about 10 years to about 50

years, about 10 years to about 45 years, about 10 years to about 40 years, about 10 years

to about 35 years, about 10 years to about 30 years, about 10 years to about 25 years,

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about 10 years to about 20 years, about 10 years to about 15 years, about 15 years to

about 60 years, about 15 years to about 55 years, about 15 years to about 50 years, about

15 years to about 45 years, about 15 years to about 40 years, about 15 years to about 35

years, about 15 years to about 30 years, about 15 years to about 25 years, about 15 years

to about 20 years, about 20 years to about 60 years, about 20 years to about 55 years,

about 20 years to about 50 years, about 20 years to about 45 years, about 20 years to

about 40 years, about 20 years to about 35 years, about 20 years to about 30 years, about

20 years to about 25 years, about 25 years to about 60 years, about 25 years to about 55

years, about 25 years to about 50 years, about 25 years to about 45 years, about 25 years

to about 40 years, about 25 years to about 35 years, about 25 years to about 30 years,

about 30 years to about 60 years, about 30 years to about 55 years, about 30 years to

about 50 years, about 30 years to about 45 years, about 30 years to about 40 years, about

30 years to about 35 years, about 35 years to about 60 years, about 35 years to about 55

years, about 35 years to about 50 years, about 35 years to about 45 years, about 35 years

to about 40 years, about 40 years to about 60 years, about 40 years to about 55 years,

about 40 years to about 50 years, about 40 years to about 45 years, about 45 years to

about 60 years, about 45 years to about 55 years, about 45 years to about 50 years, about

50 years to about 60 years, about 50 years to about 55 years, or about 55 years to about

60 years).

In some embodiments of these methods, each of the one or two or more doses are

administered at a dosage of about 0.01 mg of each common gamma-chain family

cytokine receptor activating agent/kg to about 10 mg of each common gamma-chain

family cytokine receptor activating agent/kg (e.g., about 0.01 mg/kg to about 9 mg/kg,

about 0.01 mg/kg to about 8 mg/kg, about 0.01 mg/kg to about 7 mg/kg, about 0.01

mg/kg to about 6 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4

mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.01 mg/kg to about 2 mg/kg, about

0.01 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to

about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 10

mg/kg, about 0.05 mg/kg to about 9 mg/kg, about 0.05 mg/kg to about 8 mg/kg, about

0.05 mg/kg to about 7 mg/kg, about 0.05 mg/kg to about 6 mg/kg, about 0.05 mg/kg to

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about 5 mg/kg, about 0.05 mg/kg to about 4 mg/kg, about 0.05 mg/kg to about 3 mg/kg,

about 0.05 mg/kg to about 2 mg/kg, about 0.05 mg/kg to about 1 mg/kg, about 0.05

mg/kg to about 0.5 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to

about 10 mg/kg, about 0.1 mg/kg to about 9 mg/kg, about 0.1 mg/kg to about 8 mg/kg,

about 0.1 mg/kg to about 7 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to

about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, about 0.1 mg/kg to about 3 mg/kg,

about 0.1 mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to

about 0.5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 9 mg/kg,

about 0.5 mg/kg to about 8 mg/kg, about 0.5 mg/kg to about 7 mg/kg, about 0.5 mg/kg to

about 6 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 4 mg/kg,

about 0.5 mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to

about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about

1 mg/kg to about 8 mg/kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6

mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg

to about 3 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 10 mg/kg,

about 2 mg/kg to about 9 mg/kg, about 2 mg/kg to about 8 mg/kg, about 2 mg/kg to about

7 mg/kg, about 2 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2

mg/kg to about 4 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 10

mg/kg, about 3 mg/kg to about 9 mg/kg, about 3 mg/kg to about 8 mg/kg, about 3 mg/kg

to about 7 mg/kg, about 3 mg/kg to about 6 mg/kg, about 3 mg/kg to about 5 mg/kg,

about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4 mg/kg to

about 9 mg/kg, about 4 mg/kg to about 8 mg/kg, about 4 mg/kg to about 7 mg/kg, about 4

mg/kg to about 6 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10

mg/kg, about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg, about 5 mg/kg

to about 7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 10 mg/kg,

about 6 mg/kg to about 9 mg/kg, about 6 mg/kg to about 8 mg/kg, about 6 mg/kg to about

7 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7

mg/kg to about 8 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9

mg/kg, or about 8 mg/kg to about 10 mg/kg of each common gamma-chain family

cytokine receptor activating agent).

In some embodiments of these methods, a single or first dose of the one or more

common gamma-chain family cytokine receptor activating agent(s) begins when the

subject reaches an age of at least 30 years (e.g., at least 32, 34, 36, 38, 40, 42, 44, 46, 48,

50, 52, 54, 56, 58, 60, 62, 65, 70, 75, or 80 years).

In some embodiments of any of the methods described herein, the subject is not

diagnosed or identified as having an aging-related disease (e.g., any of the aging-related

disease or condition described herein or known in the art) or an inflammatory disease

(e.g., any of the inflammatory diseases described herein or known in the art). In some

embodiments of any of the methods described herein, the subject has not been previously

treated with a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described

herein or known in the art). In some embodiments of any of the methods described

herein, the subject has not been previously treated with a therapeutic agent that induces

cellular senescence (e.g. any of the additional therapeutic agents that induce cellular

senescence described herein).

EXAMPLES The invention is further described in the following examples, which do not limit

the scope of the invention described in the claims.

Example 1: Immunostimulation in C57BL/6 mice using a multi-chain polypeptide

Materials and Methods

An exemplary multi-chain polypeptide (a type A multi-chain polypeptide

described herein) was generated that includes a first polypeptide and a second

polypeptide, where the first polypeptide is a soluble fusion of two TGFßRII domains, a

human tissue factor 219 fragment, and a human IL-15, and the second polypeptide is a

soluble fusion of two TGFßRII domains and the sushi domain of human IL-15Ra chain.

Results

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Immunostimulation in C57BL/6 mice

Wild type C57BL/6 mice were treated subcutaneously with either a control PBS

solution or with the multi-chain polypeptide at a dosage of 0.3 mg/kg, 1 mg/kg, 3 mg/kg,

or 10 mg/kg, respectively. Four days after treatment, spleen weight and the percentages

of various immune cell types present in the spleen were evaluated. Specifically, single

splenocyte suspensions were generated and stained with fluorochrome-conjugated

antibodies including anti-CD4, anti-CD8, anti-NK1.1, and anti-CD19. The percentages of

CD4+ T cells, CD8+ T cells, Natural Killer (NK) cells, and CD19 E cells present in the

spleen of mice treated with either the control solution or the multi-chain polypeptide were

evaluated using flow cytometry. As shown in Figure 1A, the spleen weight in mice

treated with the multi-chain polypeptide increased with increasing dosage of the multi-

chain polypeptide. Moreover, the spleen weight in mice treated with 1 mg/kg, 3 mg/kg,

and 10 mg/kg of the multi-chain polypeptide were significantly higher as compared to

mice treated with the control solution, respectively. As shown in Figure 1B, in the

spleens of mice treated with the multi-chain polypeptide, the percentages of CD8+ T cells

and NK cells both increased with increasing dosage of the multi-chain polypeptide.

Specifically, the percentages of CD8+ T cells were higher in mice treated with 0.3 mg/kg,

3 mg/kg, and 10 mg/kg of the multi-chain polypeptide compared to control-treated mice,

and the percentages of NK cells were higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3

mg/kg, and 10 mg/kg of the multi-chain polypeptide compared to control-treated mice.

These results demonstrate that the exemplary multi-chain polypeptide is able to stimulate

immune cells in the spleen, in particular CD8+ T cells and NK cells.

Pharmacokinetics

The pharmacokinetics of the exemplary multi-chain polypeptide were evaluated

in wild type C57BL/6 mice. Mice were treated subcutaneously with the multi-chain

polypeptide at a dosage of 3 mg/kg. Blood was collected at various time points via tail

vein, and serum was prepared. The concentration of the multi-chain polypeptide in the

serum was determined with ELISA. Briefly, the multi-chain polypeptide was captured

using an anti-human tissue factor antibody, and detected using a biotinylated anti-human

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TGFß receptor, a peroxidase conjugated streptavidin, and ABTS substrate. The results

showed that the half-life of the exemplary multi-chain polypeptide was 12.66 hours.

Immunostimulation over time in C57BL/6 mice

To evaluate the effect of immunostimulation by the multi-chain polypeptide over

time, mice were treated with a single dose of the multi-chain polypeptide at 3 mg/kg and

the spleen weight and percentages of immune cell types present in the spleen were

evaluated immediately upon treatment and at 16, 24, 48, 72, and 92 hours after treatment,

using techniques described above. As shown in Figure 2A, the spleen weight of mice

treated with the multi-chain polypeptide increased at 48 hours after treatment, and

continued to increase over the next 44 hours. Moreover, as shown in Figure 2B, in the

spleens of mice treated with the multi-chain polypeptide, the percentages of CD8+ T cells

and NK cells both increased at 48 hours after treatment and continued to increase over

the next 44 hours. These results further demonstrate that the exemplary multi-chain

polypeptide is able to stimulate immune cells in the spleen, in particular CD8+ T cells and

NK cells, over time.

Increased proliferation and Granzyme B expression by CD8+ T cells and NK cells

To evaluate the proliferation and cytotoxic potential of the immune cells induced

by the multi-chain polypeptide, mice were treated with a single dose of the multi-chain

polypeptide at 3 mg/kg, and the spleens of these mice were evaluated immediately after,

and at 16, 24, 48, 72, and 92 hours after treatment. Briefly, single splenocyte suspensions

were generated and stained with fluorochrome-conjugated antibodies for the various cell

types including anti-CD4, anti-CD8, anti-NK1.1, and anti-CD19, and with an anti-Ki67

antibody (i.e. a cell proliferation marker) and an anti-Granzyme B antibody (i.e. a

cytotoxic marker). The mean fluorescent intensity (MFI) of Ki67 and Granzyme B for

each immune cell type was analyzed by flow cytometry. As shown in Figures 3A and 3B,

the expression of Ki67 and Granzyme B by NK cells showed an increase at 24 hours as

well as each time point evaluated thereafter as compared to immediately after treatment

(0 hours). Moreover, the expression of Ki67 and Granzyme B by CD8+ T cells showed an

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increase at 48 hours as well as each time point evaluated thereafter as compared to

immediately after treatment (0 hours). As such, a single dose of the multi-chain

polypeptide resulted in proliferation of CD8+ T cells and NK cells for up to at least 4

days post-treatment.

These results demonstrate that the multi-chain polypeptide not only increased the

number of CD8+ T cells and NK cells in the spleen, but also enhanced the proliferation

and cytotoxicity of these cells.

Cytotoxicity against tumor cells

Next, the cytotoxicity of the splenocytes activated by the multi-chain polypeptide

against tumor cells were evaluated in C57BL/6 mice. Mouse Moloney leukemia cells

(Yac-1) were labeled with CellTrace Violet and used as tumor target cells. C57BL/6 mice

were treated with a single dose of the multi-chain polypeptide at 3 mg/kg, and

splenocytes were prepared at various time points thereafter and used as effector cells. The

target tumor cells were mixed with the effector cells at an effector:target (E:T) ratio of

10:1, and incubated at 37°C for 20 hours. Target cell viability was assessed by analyzing

Propidium Iodide (PI)-positive, violet-labeled Yac-1 cells using flow cytometry. The

percentage of Yac-1 tumor inhibition was calculated using the formula:

Percentage of Yac-1 tumor inhibition = (1-viable Yac-1 cell number in

experimental sample/viable Yac-1 cell number in the sample without splenocytes) X 100

As shown in Figure 4, splenocytes from mice after 24-hour or more treatment

with the multi-chain polypeptide showed increased cytotoxicity against Yac-1 cells as

compared to the splenocytes from untreated mice.

Example 2: Immunostimulation in C57BL/6 mice using a high fat diet-based Type-2

diabetes mouse model

Materials and Methods

TGFRt15-TGFRs is a multi-chain chimeric polypeptide (a type A multi-chain

chimeric polypeptide described herein) that includes two TGF3-binding domains which a

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soluble human TGFßRII dimer (aa24-159). 21t15-TGFRs is a multi-chain chimeric

polypeptide (a type A multi-chain chimeric polypeptide described herein) that includes

IL-21 and a TGFB-binding domain. 2t2 is a chimeric polypeptide (a type B chimeric

polypeptide described herein) that include two IL-2 polypeptides.

Results

To evaluate the effect of TGFRt15-TGFRs, 2t2, and 21t15-TGFRs in treating

Type-2 diabetes, a high fat diet-based Type-2 diabetes mouse model (B6.129P2-

ApoEtmIUnc/J from The Jackson Laboratory) was used. Mice were fed either a control

diet or a high fat diet for 11 weeks. A subset of mice fed with the high fat diet were also

treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. Mice fed the control diet, high fat

diet, and mice fed with the high fat diet and treated with TGFRt15-TGFRs, 2t2, or 21t15-

TGFRs were evaluated 4 days post-treatment. Briefly, single splenocyte suspensions

were generated and stained with fluorochrome-conjugated antibodies including anti-CD4,

anti-CD8, anti-NK1.1, and anti-CD19. The percentages of CD4+ T cells, CD8+ T cells,

Natural Killer (NK) cells, and CD19+B cells present in the spleen of mice in each group

were evaluated using flow cytometry.

As shown in Figure 5A, in mice fed a high fat diet, the percentage of NK cells in

PBMCs was significantly increased after treatment with TGFRt15-TGFRs or 2t2

compared to untreated mice, but not after treatment with 21t15-TGFRs. Furthermore, the

percentage of CD8+ T cells in PBMCs was significantly increased after treatment with

TGFRt15-TGFRs, 2t2, or 21t15-TGFRs compared to untreated mice. Moreover, the

proliferation of CD4+ T cells, CD8+ T cells, Natural Killer (NK) cells, and CD19+ B cells

in PBMCs were also evaluated using an anti-Ki67 antibody. As shown in Figure 5B, the

number of proliferating NK cells, CD4+ T cells, and CD8+ T cells were significantly

increased after treatment with TGFRt15-TGFRs, but not after treatment with 2t2 or

21t15-TGFRs.

To examine the effect of TGFRt15-TGFRs, 2t2 and 21t15-TGFRs on the

appearance and texture of skin and hair in animals, mice were fed either a control or a

high fat diet for 7 weeks, and a subset of the mice fed a high fat diet were also treated

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with TGFRt15-TGFRs, 2t2 or 21t15-TGFRs. One week post-treatment, the appearance of

the mice was evaluated. Mice fed a high fat diet and untreated, or a high diet and treated

with 21t15-TGFRs appeared ungroomed and ruffled, and had increased gray hair/hair

loss as compared to mice fed a control diet (Figure 6A, 6B and 6E). Surprisingly, mice

fed a high fat diet that received TGFRt15-TGFRs or 2t2 treatment appeared groomed and

healthier (less gray hair/hair loss) (Figure 6C and 6D) as compared to mice fed a high fat

diet that did not receive TGFRt15-TGFRs or 2t2 treatment (Figure 6B). Specifically,

TGFRt15-TGFRs or 2t2-treated mice showed superior skin and hair appearance and

texture as compared to control mice. These results demonstrate that treatment with

TGFRt15-TGFRs or 2t2 improves the appearance and texture of skin and hair in

mammals. Next, mice were fed either a control or high fat diet for 9 weeks, and a subset of

the mice fed a high fat diet were treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs.

Four days post-treatment, the fasting body weight of mice in each group were measured.

The fasting body weight of mice fed with the high fat diet and untreated, as well as mice

fed with the high fat diet and treated with 21t15-TGFRs were significantly increased

compared to mice fed a control diet. However, the fasting body weight of mice fed a high

fat diet and treated with TGFRt15-TGFRs or 2t2 were decreased compared to the other

two high fat diet groups mentioned above. The fasting body weight of the mice at the

end of the study (9 weeks) is shown in Figure 7.

To evaluate the fasting glucose levels in the mice of each group, mice were fed

either a control or a high fat diet and were either untreated or treated with TGFRt15-

TGFRs, 2t2, or 21t15-TGFRs on days 44, 59 and 73. The fasting blood glucose in the

mice of each group were measured 4 days post-treatment. As shown in Figure 8, after the

second and third doses (on Days 59 and 73, respectively), the fasting blood glucose level

was significantly reduced for mice fed a high fat diet and treated with 2t2 (red line) as

compared to mice fed a high fat diet but untreated (yellow line). The fasting blood

glucose level remained constant for mice fed a high fat diet and treated with TGFRt15-

TGFRs (green line), whereas the fasting blood glucose level increased for mice fed a high

fat diet and treated with 21t15-TGFRs (blue line).

Example 3: Chemotherapy-induced Senescent B16F10 Melanoma Cells express NK

ligands

Material and Methods

Cellular senescence in B16F10 melanoma cells was induced by treating the cells

with docetaxel (7.5 M M, Sigma) for 3 days followed by recovery in complete media for 4

days. Cellular senescence was accessed by staining the cells with senescence-associated

B-galactosidase SA B-gal). Briefly, B16F10 control and senescence cells (B16F10-SNC)

were washed once with PBS, fixed with 0.5% glutaraldehyde (PBS (pH 7.2)), for 30

minutes. Cells were stained in X-gal solution (1 mg/mL X-gal, 0.12 mM K3Fe [CN]6,

0.12 mM K4Fe[CN]6, and 1 mM MgCl2 in PBS at pH 6.0) overnight at 37 °C, and were

imaged using a Nikon optical light microscope.

Results

Cellular senescence in B16F10 melanoma cells was induced using chemotherapy

as described above. As shown in Figure 9A, chemotherapy-induced senescent B16F10

cells (B16F10-SNC) were positive for SA B-gal staining, while the control B16F10 cells

were not stained. Next, expression of senescence genes was analyzed using RT-qPCR

with RNA isolated on day 0 or following senescence induction on days 4, 8, 12 and 16,

respectively. The expression levels were normalized to control B16F10 cells. As shown

in Figures 9B-9D, the expression of p21, IL6 and DPP4 were upregulated in RNA

isolated from the senescent cells over the duration of the experiment. Moreover, as shown

in Figures 9E and 9F, the expression of RATE1E and ULBP1 (NK activating receptor

NKG2D ligands) were also induced in senescent cells, with the highest expression level

being on day 16. These results demonstrate that the chemotherapy-induced senescent

B16F10 cells are subjected to stronger cytotoxicity of activated NK cells than control

B16F10 cells.

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Acquisition of Stem-cell Properties in Chemotherapy-induced Senescent B16F10

Melanoma Cells

To examine whether chemotherapy-induced senescent B16F10 melanoma cells

acquired stem cell properties, a colony formation assay was performed. Briefly, 1000

cells/well were seeded on a six well plate, and the media was changed every third day. As

shown in Figure 10A (images taken at 100x magnification), after 5 weeks in culture the

senescent cells were able to form colonies. To evaluate stem cell marker expression by

the colonies, RNA was isolated from the colonies and the expression of Oct4 and Notch4

mRNA were determined by RT-qPCR. As compared to control B16F10 cells,

chemotherapy-induced senescent B16F10 melanoma cells showed upregulation of Oct4

and Notch 4, which are cancer stem cell markers (Figures 10B and 10C). Moreover, cell

surface expression of stem cell markers CD44, CD24 and CD133 were evaluated by

staining with antibodies against CD44, CD24, and CD133 followed by flow cytometry.

As shown in Figures 10D-10F, double positive populations (CD44*CD24*,

CD44*CD133T, and CD24*CD133*) were increased in the chemotherapy induced

senescence stem cells (B16F10-SNC-CSC) compared to control B16F10.

Chemotherapy-induced senescent (CIS) melanoma cells with stem cell properties are

more "Migratory" and "Invasive" than control B16F10 cells

The migratory properties of chemotherapy-induced senescent (CIS) melanoma

cells with stem cell properties (B16F10-SNC-CSC) were analyzed using a migration

assay. Briefly, control B16F10 cells and B16F10-SNC-CSC cells were plated on six well

plates and wounded with a p20 pipette tip. Movement of cells were imaged at 0, 12, and

24 hours after. As shown in Figure 11A, chemotherapy-induced senescent (CIS)

melanoma cells with stem cell properties (B16F10-SNC-CSC) were more migratory in

the in vitro migration assay, as compared to control B16F10 cells.

Next, the invasive properties of chemotherapy-induced senescent cells with stem

cell properties (B16F10-SNC-CSC) were analyzed using an invasion assay. The invasion

6 assay was carried out on 24-well transwell inserts coated with Matrigel. Briefly, 0.5x10

control B16F10 cells and B16F10-SNC-CSC cells were seeded in serum-free media onto

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the upper chamber, and the lower chamber was filled with media supplemented with 10%

FBS. After 16 hours of incubation, the cells on the upper surface of the filter were

removed, and cells underneath the filter were fixed and stained with a 0.02% crystal

violet solution. The number of cells were counted in three fields at 100x magnification.

As shown in Figures 11B and 11C, chemotherapy-induced senescent cells with stem cell

properties were more aggressive in invading the Matrigel coated membrane as compared

to control B16F10 cells. These results demonstrate that chemotherapy-induced senescent

B16F10 tumor cells are able to regain their proliferation capability, obtain stem-cell

features, and have increased migratory abilities and invasiveness for metastasis.

Cytotoxic Activity of Mouse NK Cells on Chemotherapy-induced Senescent Cells with

Stem Cell Properties

To expand NK cells in vivo, C57BL/6 mice were injected subcutaneously with

TGFRt15-TGFRs (10 mg/kg) for 4 days. The spleens from these mice were obtained and

NK cells were purified using MACS Miltenyi column. The purified NK cells were then

expanded in vitro with 2t2 (Figure 12A).

To evaluate the cytotoxicity of the expanded NK cells, chemotherapy-induced

senescent stem cells (B16F10-SNC-CSC) or control B16F10 cells were labelled with

CellTrace violet and incubated with in vitro activated 2t2 mouse NK cells (isolated from

spleen of C57BL/6 mice injected with 10 mg/kg TGFRt15-TGFRs for 4 days) at various

E:T ratios for 16 hrs. The B16F10-SNC-CSC and control B16F10 cells were trypsinized,

washed and re-suspended in complete media containing a Propidium Iodide (PI) solution,

and cytotoxicity was accessed by flow cytometry. As shown in Figure 12B, NK cells

were more effective at killing chemotherapy-induced senescent cells with stem cell

properties (B16F10-SNC-CSC), as compared to control B16F10 cells.

Combination Treatment in Melanoma Mouse Model

The effect of TGFRt15-TGFRs in treating melanoma was evaluated in a mouse

melanoma model. Briefly, 5x105 B16F10 cells were injected subcutaneously into

C57BL/6 mice. When the tumor volume reached ~100 mm mice were treated with

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docetaxel (chemotherapy) (5 mg/kg) or TA99 (200 ug) either as a single agent or in

combination every third day, and TGFRt15-TGFRs (3 mg/kg) was given once a week

(Figure 13A). Mice that received saline, docetaxel (chemotherapy)/TA99 alone, or

TGFRt15-TGFRs alone were used as controls. Five mice were tested in each

experimental and control group. Tumor volume was measured every third day. As shown

in Figures 13B and 13C, combinations of TGFRt15-TGFRs with either chemotherapy or

TA99 slowed down tumor progression as compared to mice treated with saline or mice

treated with chemotherapy or TA99 alone in the syngeneic melanoma mouse model.

Example 4: Chemotherapeutic Induction of Senescence in Human Pancreatic Cell

Line SW1990 Materials and Methods

B-galactosidase staining: Confirmation of chemotherapy induced senescence was

carried out by standard B-galactosidase staining at pH 6.0 using commercially available kit

(Cell Signaling Technology) according to manufacturer's instructions. The following day,

the staining solution was removed, and cells were washed with phosphate buffered saline,

and 70% glycerol was added to the wells. The B-galactosidase positive cells will be stained

blue, while control untreated cells will not stain.

Flow cytometry: One million control and senescent cells were obtained and stained

using commercially available antibodies to surface markers of stem cells such as anti-CD44

and anti-CD24 antibodies (Biolegend) according to manufacturers' instructions. The cells

were then washed and analyzed using the BD Celesta flow cytometer. Cells showing stem

cell-like properties will be doubly positive for both CD44 and CD24.

Gene expression assay: One million control and senescent cells were obtained and

lysed using Trizol (Thermofisher), followed by RNA purification using an RNA isolation

kit (Qiagen). The RNA was quantified and converted to cDNA using a Qiagen cDNA

Quantitect kit. The cDNA was then used as a template for standard Taqman gene

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expression assays (Thermofisher) to quantify the relative abundance of senescent, stem cell

markers as well as NK ligands.

NK cell cytotoxicity assay: NK cells were isolated from healthy human donors

(n=2) using a commercially available NK isolation kit (Stem Cell), and were activated

overnight using the cytokine fusion molecule 18t15-12s (100nM). On the following day,

NK cells were washed to remove cytokine molecules and mixed with either CellTrace

Violet labelled control untreated tumor cells or chemotherapy-induced senescent tumor

cells at an E:T ratio of 4:1 for 20 hours. On the following day, cells were trypsinized, and

complete contents of each well were analyzed using flow cytometry and percent inhibition

of cells was analyzed.

Results

Senescence in the human pancreatic tumor cell line SW1990 was induced through

treatment with chemotherapeutic drugs Abraxane (Celgene) and Gemcitabine (Sigma

Aldrich) for 3 days at 2.5uM and 6.25uM, respectively. SW1990 cells that were untreated

were used as controls. Media was changed after 3 days and cells were allowed to rest in

the culture media for 4 days. As shown in Figure 14, senescent cells treated with the

chemotherapeutic drugs were positive for B-galactosidase staining (blue), while control

cells were not stained. Senescent cells and control cells were evaluated for their expression

of senescence and stem cell markers at 4 days, 11 days, and 22 days post-treatment. As

shown in Figure 14, senescent cells showed increased double positive staining for CD44

and CD24 over time as compared to the control cells. Moreover, the chemotherapy-induced

senescent SW1990 cells were also analyzed for their expression of senescent markers

including DPP4, IL6, and p21, stem cell markers including Oct3/4, CD24, and CD44, and

NK ligands including Nectin and MICA, on day 0, and days 2, 4, and 24 post-treatment

using the gene expression assay described above. As shown in Figure 15, the expression

of all of the markers mentioned showed an increase over time.

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Cytotoxicity of in vitro activated Human NK Cells

To evaluate the cytotoxicity of in vitro activated human NK Cells (treated with

18t15-12s), senescence in the human pancreatic tumor cell line SW1990 was induced

through treatment with chemotherapeutic drugs Abraxane (Celgene) and Gemcitabine

(Sigma Aldrich) for 3 days at 2.5 M M and 6.25 MM, respectively. SW1990 cells that were

untreated were used as controls. Media was changed after 3 days and cells were allowed to

rest in the culture media for 30 days. The culture media was changed every 4 days.

Activated NK cells were obtained and their cytotoxicity for chemotherapy-induced

senescent tumor cells and untreated control tumor cells were evaluated using the NK cell

cytotoxicity assay described above. As shown in Figure 16, activated NK cells showed

increased cytotoxicity against both control SW1990 cells (SW1990) and senescent

SW1990 cells (SW1990s).

Example 5: Creation of an IL-12/IL-15RaSu DNA construct

In a non-limiting example, an IL-12/IL-15RaSu DNA construct was created

(Figure 17). The human IL-12 subunit sequences, human IL-15RaSu sequence, human

IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were

obtained from the UniProt website and DNA for these sequences was synthesized by

Genewiz. A DNA construct was made linking the IL-12 subunit beta (p40) to IL-12

subunit alpha (p35) with a GS (3) linker to generate a single chain version of IL-12 and

then directly linking the IL-12 sequence to the IL-15RaSu sequence. The final IL-12/IL-

15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence of the IL12/IL-15RaSu construct (including signal

peptide sequence) is as follows (SEQ ID NO: 181):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT ACTCC (Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG

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ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATG CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT GGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT AGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG GGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATO CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTC. CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTO AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCO AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTA CCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTA ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT TCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTO CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC PATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT wo WO 2021/247604 PCT/US2021/035285

GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCAT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG AGCTATTTAAACGCCAGC (Human IL-15Rasushidomain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

Example 6: Creation of an IL-18/TF/IL-15 DNA construct

In a non-limiting example, an IL-18/TF/IL-15 construct was made (Figure 18)

linking the IL-18 sequence to the N-terminus coding region of tissue factor 219, and

further linking the IL-18/TF construct with the N-terminus coding region of IL-15. The

nucleic acid sequence of the IL-18/TF/IL-15 construct (including leader sequence),

synthesized by Genewiz, is as follows (SEQ ID NO: 177):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC GACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATO CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATO GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC

GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTO AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA GGAT (Human Tissue Factor 219)

551

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA, ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGO AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTO CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 7: Secretion of IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins

The IL-12/IL-15RaSu and IL-18/TF/IL-15 DNA constructs were cloned into a

pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene

Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of a soluble IL-18/TF/IL-15:IL-12/IL-15RaSu

protein complex (referred to as 18t15-12s; Figure 19 and Figure 20). The 18t15-12s

protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non- aggregated) protein complexes consisting of IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins.

The amino acid sequence of the IL12/IL-15RaSu fusion protein (including signal

peptide sequence) is as follows (SEQ ID NO: 180):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERV GDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKI RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVP PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

(Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DTKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE

DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA: LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including signal

peptide sequence) is as follows (SEQ ID NO: 176):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-18)

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YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader (signal sequence) peptide is cleaved from the intact

polypeptide to generate the mature form that may be soluble or secreted.

Example 8: Purification of 18t15-12s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE HealthcareTM AKTA

Avant protein purification system. The flow rate was 4 mL/min for all steps except the

elution step, which was 2 mL/min.

Cell culture harvest of 18t15-12s was adjusted to pH 7.4 with 1M Tris base and

loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of

PBS. After loading the sample, the column was washed with 5 column volumes PBS,

followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280

nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding 1M Tris

base. The neutralized sample was then buffer exchanged into PBS using Amicon

centrifugal filters with a 30 KDa molecular weight cutoff. Figure 21 shows that the

18t15-12s complex binds the anti-TF antibody affinity column, wherein TF is an 18t15-

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12s binding partner. The buffer-exchanged protein sample is stored at 2-8°C for further

biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6

column volumes 0. 1M glycine, pH 2.5. The column was then neutralized using 10

column volumes PBS, 0.05% sodium azide and stored at 2-8°C.

Example 9: Size exclusion chromatography of 18t15-12s

A GE Healthcare Superdex 200 Increase 10/300 GL gel filtration column was

connected to a GE Healthcare AKTATM Avant protein purification system. The column

was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A

capillary loop was used to inject 200uL of 1 mg/mL of 18t15-12s complex onto the

column. The injection was chased with 1.25 column volumes of PBS. The SEC

chromatograph is shown in Figure 22. There is a main 18t15-12s protein peak with a

minor high molecular weight peak, likely due to differing degrees of glycosylation of

18t15-12s dimers or aggregates.

Example 10: SDS-PAGE of 18t15-12s

To determine the purity and protein molecular weight, the purified 18t15-12s

protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The

gel was stained with InstantBlueTM for about 30 min, followed by destaining overnight in

purified water. Figure 23 shows an example SDS gel of anti-TF antibody affinity

purified 18t15-12s, with bands at the expected molecular weights (66 kDa and 56 kDa).

Example 11: Glycosylation of 18t15-12s in CHO-K1 cells

Glycosylation of 18t15-12s in CHO-K1 cells was confirmed using the Protein

Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer's

instructions. Figure 24 shows an example SDS PAGE of deglycosylated and non-

deglycosylated 18t15-12s. Deglycosylation reduces the molecular weight of 18t15-12s as

seen in Figure 24, lane 4.

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Example 12: Recombinant protein quantitation of 18t15-12s complexes

The 18t15-12s complex was detected and quantified using standard sandwich

ELISA methods (Figures 25-28). Anti-human tissue factor antibody served as the

capture antibody and biotinylated anti-human IL-12, IL-15, or IL-18 antibody (BAF 219,

BAM 247, D045-6, all R&D Systems) served as the detection antibody. Tissue factor in

purified 18t15-12s protein complexes was also detected using an anti-human tissue factor

capture antibody (143), and anti-human tissue factor antibody detection. The I43/anti-TF

antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 13: Immunostimulatory capacity of the 18t15-12s complex

To assess the IL-15 immunostimulatory activity of the 18t15-12s complex,

increasing concentrations of 18t15-12s was added to 32DB cells (104 cell/well) in 200 uL

IMDM: 10% FBS media. The 32DB cells were incubated for 3 days at 37°C. On the

fourth day, WST-1 proliferation reagent (10 uL/well) was added and after 4 hours,

absorbance was measured at 450 nm to determine cell proliferation based on cleavage of

WST-1 to a soluble formazan dye. Bioactivity of human recombinant IL-15 was assessed

as a positive control. As shown in Figure 29, 18t15-12s demonstrated IL-15-dependent

cell proliferation of 32DB cells. The 18t15-12s complex demonstrated reduced activity

compared to human recombinant IL-15, possibly due to the linkage of IL-18 and tissue

factor to the IL-15 domain.

In order to assess the individual activities of IL-12 and IL-18 in the 18t15-12s

complex, 18t15-12s was added to HEK-Blue IL-12 and HEK-Blue IL-18 reporter cells

(5x104 cell/well; hkb-il12 and hkb-hmil18, InvivoGen) in 200 uL IMDM: 10% heat-

inactivated FBS media. Cells were incubated for overnight at 37°C. 20 ul of induced

HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell supernatant was added to 180 ul of

QUANTI-Blue (InvivoGen), and incubated for 1-3 hours at 37°C. IL-12 or IL-18 activity

was assessed by measuring absorbance at 620 nm. Human recombinant IL-12 or IL-18

was assessed as a positive or negative control. As shown in Figure 30 and Figure 31,

each of the cytokine domains of the 18t15-12s complex retain specific biological activity.

The activity of 18t15-12s was reduced compared to that of human recombinant IL-18 or

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IL-12, possibly due to linkage of IL-15 and tissue factor to the IL-18 domain and linkage

of IL-12 to the IL-15Ra sushi domain.

Example 14: Induction of cytokine-induced memory-like NK cells by the 18t15-12s

complex Cytokine-induced memory-like NK cells can be induced ex vivo following

overnight stimulation of purified NK cells with saturating amounts of IL-12 (10 ng/mL),

IL-15 (50 ng/mL), and IL-18 (50 ng/mL). These memory-like properties have been

measured through expression of IL-2 receptor a (IL-2Ra, CD25), CD69 (and other

activation markers), and increased IFN-y production. To evaluate the ability of 18t15-12s

complexes to promote generation of cytokine-induced memory-like NK cells, purified

human NK cells (>95% CD56+) were stimulated for 14-18 hours with 0.01nM to

10000nM of the 18t15-12s complex or a combination of individual cytokines

(recombinant IL-12 (10 ng/ml), IL-18 (50 ng/ml), and IL-15 (50 ng/ml)). Cell-surface

CD25 and CD 69 expression and intracellular IFN-y levels were assessed by antibody-

staining and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells

were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The

purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were

counted and resuspended in 0.2 X 106/mL in a 96 well flat bottom plate in 0.2 mL of

complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a mixture of

cytokines hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D Systems) and hIL-

15 (50 ng/mL) (NCI) or with 0,01 nM to 10000nM of the 18t15-12s at 37°C, 5% CO2 for

14-18 hrs. The cells were then harvested and surface stained for CD56-BV421, CD16-

BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend) for 30 minutes.

After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in

FACS buffer (1X PBS (Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium

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azide (Sigma)). After two washes, cells were analyzed using a BD FACSCelestaTM flow

cytometer (Plotted Data-Mean Fluorescence Intensity; Figure. 32A and Figure 32B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells

were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The

purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were

counted and resuspended in 0.2 X 106/mL in a 96 well flat bottom plate in 0.2 mL of

complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a cytokine

mix of hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D), and hIL-15 (50

ng/mL) (NCI), or 0.01 nM to 10000 nM of the 18t15-12s complex at 37°, 5% CO2 for

14-18 hrs. The cells were then treated with 10 ug/mL of Brefeldin A (Sigma) and 1X of

Monensin (eBioscience) for 4 hrs before harvesting and staining for CD56-BV421,

CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After

staining, cells were washed (1500 RPM for 5 minutes in room temperature) in FACS

buffer (1X PBS (Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium azide

(Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed

(1500 RPM for 5 minutes in room temperature) in 1x permeabilized buffer (eBioscience)

and stained with IFN-y- PE (Biolegend) for 30 minutes at room temperature. Cells were

washed once again with 1x permeabilized buffer and then washed with FACS buffer. Cell

pellets were resuspended in 300 uls of FACS buffer and analyzed using a BD

FACSCelestaTM flow cytometer (Plotted % of IFN-y Positive Cells; Figure 33).

Example 15: In vitro cytotoxicity of NK cells against human tumor cells

Human myelogenous leukemia cells, K562 (CellTrace violet labelled), were

incubated with purified human NK cells in the presence of increasing concentrations of

the 18t15-12s complex or a mixture of cytokines as a control. After 20 hours, the

cultures were harvested, stained with propidium iodide (PI), and assessed by flow

cytometry. As shown in Figure 34, the 18t15-12s complex induced human NK cytotoxicity against K562, at levels similar or greater than the cytokine mixture, wherein both the 18t15-12s complex and the cytokine mixture induced greater cytotoxicity than the medium control.

Example 16: Creation of IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15DN

constructs

In a non-limiting example, IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15

DNA constructs were created (Figure 35 and Figure 36). The human IL-12 subunit

sequences, human IL-15RaSu sequence, human IL-15 sequence, human tissue factor 219

sequence, and human IL-18 sequence were synthesized by Genewiz. A DNA construct

was made linking the IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3)

linker to generate a single chain version of IL-12, directly linking the IL-12 sequence to

the IL-15RaSu sequence, and directly linking the IL-12/IL-15RaSu construct to the N-

terminus coding region of aCD16scFv.

The nucleic acid sequence of the IL-12/IL-15RaSu/aCD16scFv construct is as

follows (SEQ ID NO: 226):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Human IL-12 - subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAG CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCC GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA

GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGO wo 2021/247604 WO PCT/US2021/035285

TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA

GCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTC. AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA CTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTC TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTA CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTA ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTO ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCT CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCT AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT

CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCAT AGCTATTTAAACGCCAGC (Human IL-15Rasushidomain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTO

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGC' CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (anti-Human CD16 light chain variable domain)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC

GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT wo WO 2021/247604 PCT/US2021/035285

ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTO CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAA0 CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG

CTGACCGTGGGCCAT (Linker)

GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC (anti-Human CD16 heavy chain variable domain)

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGG GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGG CTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCA TGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCAT TGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCAT CAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTC ACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCC ACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCC TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCT TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCT GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

Constructs were also made linking the IL-18 sequence to the N-terminus coding

region of tissue factor 219, and linking the IL-18/TF construct with the N-terminus

coding region of IL-15 (Figure 36). The nucleic acid sequence of the IL-18/TF/IL-15

construct (including leader sequence) is as follows (SEQ ID NO: 177):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAA0

GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATG7 ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTO AAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC

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GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTO GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA GGAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT CACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA CAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA TCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGO CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACA AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA ACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTO AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human I IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCO

Example 17: Secretion of IL-12/IL-15RaSu/aCD16scFy and IL-18/TF/IL-15fusion

proteins

The IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 constructs were cloned

into a pMSGV-1 modified retrovirus expression vector (Hughes, Hum Gene Ther

16:457-72, 2005, herein incorporated by reference), and the expression vector was

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

resulted in secretion of a soluble IL-18/TF/IL-15:IL-12/IL-15RaSu/aCD16scFv protein

complex (referred to as 18t15-12s/a.CD16; Figure 37 and Figure 38). Co-expression of

the two constructs in CHO-K1 cells resulted in secretion of the soluble IL-18/TF/IL-

15:IL-12/IL-15RaSu/aCD16scFv protein complex (referred to as 18t15-12s/aCD16;

Figure 37 and Figure 38), which can be purified by anti-TF Ab affinity and other

chromatography methods. In some cases, the signal peptide is cleaved from the intact

polypeptide to generate the mature form.

The amino acid sequence of the IL-12/IL-15RaSu/aCD16scFv fusion protein

(including signal peptide sequence) is as follows (SEQ ID NO: 225):

(Signal peptide)

MKWVTFISLLFLFSSAYS MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS

GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPI RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS, TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE wo WO 2021/247604 PCT/US2021/035285

DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT

NVAHWTTPSLKCIR (anti-Human CD16 light chain variable domain)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTK TVGH (Linker)

GGGGSGGGGSGGGGS (anti-Human CD16 heavy chain variable domain)

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEW SGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRS LLFDYWGQGTLVTVSR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including leader

sequence) is as follows (SEQ ID NO: 221):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFISM YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE

(Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 18: Creation of IL-18/IL-15RoSu and IL-12/TF/IL-15 DNA constructs

In a non-limiting example, IL-18/IL-15RaSu and IL-12/TF/IL-15 DNA constructs

were created. The human IL-18 subunit sequences, human IL-15RaSu sequence, human

IL-12 sequence, human tissue factor 219 sequence, and human IL-15 sequence were

synthesized by Genewiz. A DNA construct was made linking IL-18 directly to IL-

15RaSu. An additional construct was also made linking IL-12 sequence to the N-

terminus coding region of human tissue factor 219 form, and further linking the IL-12/TF

construct to the N-terminus coding region of IL-15. As described above, a single-chain

version of IL-12 (p40-linker-p35) was used.

The nucleic acid sequence of the IL-18/IL-15RaSu construct (including signal

peptide sequence) is as follows (SEQ ID NO: 320):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC GACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA PAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAAT AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGO AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTC AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA

GGAT wo WO 2021/247604 PCT/US2021/035285

(Human IL-15Rasushidomain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence of the IL-12/TF/IL-15 construct (including leader

sequence) is as follows (SEQ ID NO: 321):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCC'

ACTCC (Human IL-12 subunit beta(p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAA ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGA

GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTG TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCC GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG GGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGC. AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTO CTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTC wo 2021/247604 WO PCT/US2021/035285

(Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35))

GTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAG CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC CCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATO TCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGO CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATO

AGCTATTTAAACGCCAGC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGA0 CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAG TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAG ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAG

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACAC, AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTG0 AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAC (Human IL-15) wo 2021/247604 WO PCT/US2021/035285

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCO TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 19: Secretion of IL-18/IL-15RaSu and IL-12/TF/IL-15 fusion proteins

The IL-18/IL-15RaSu and IL-12/TF/IL-15 constructs were cloned into a pMSGV-

1 modified retrovirus expression vector (Hughes, Hum Gene Ther 16:457-72, 2005

herein incorporated by reference), and the expression vector was transfected into CHO-

K1 cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of a

soluble IL-12/TF/IL-15:IL-18/IL-15RaSu protein complex (referred to as 12t15/s18),

which can be purified by anti-TF Ab affinity and other chromatography methods.

The amino acid sequence of the IL-18/IL-15RaSu fusion protein (including signal

peptide sequence) is as follows (SEQ ID NO: 322):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFLSM YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIFFQRSVPG YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human IL-15Rasushidomain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-12/TF/IL-15 fusion protein (including leader

sequence) is as follows (SEQ ID NO: 323):

(Signal peptide) wo WO 2021/247604 PCT/US2021/035285

MKWVTFISLLFLFSSAYS MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS TLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN ITFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERY RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVE PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE OLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS

LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKG YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTE ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYV ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW

KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

Example 20: Recombinant protein quantitation of the 18t15-12s16 complex

The 18t15-12s16 complex (comprising IL-12/IL-15RaSu/aCD16scFv;IL-

18/TF/IL-15) was detected and quantified using standard sandwich ELISA methods

(Figure 39). Anti-human tissue factor antibody/IL-2 or anti-TF Ab/IL-18 served as the

capture antibody and biotinylated anti-human IL-12 or IL-18 antibody (BAF 219, D045-

6, both R&D Systems) served as the detection antibody. Tissue factor was also detected

using an anti-human tissue factor antibody (143), and anti-human tissue factor antibody

detection.

Example 21: Creation of TGFBRII/IL-15RaSu and IL-21/TF/IL-15 DNA constructs

In a non-limiting example, a TGF3RII/IL-15RaSu DNA construct was created

(Figure 40). The human TGFßRII dimer and human IL-21 sequences were obtained from

the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA

construct was made linking the TGFßRII to another TGFßRII with a linker to generate a

single chain version of TGFßRII and then directly linking the TGFßRII single chain

dimer sequence to the N-terminal coding region of IL-15RaSu.

The nucleic acid sequences of the TGF3RII/IL-15RaSu construct (including

signal sequence) is as follows (SEQ ID NO: 196):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGF BRII-1* fragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGA ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT wo WO 2021/247604 PCT/US2021/035285

(Linker)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAG (Human TGF BRII-2" fragment)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACO GATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCA CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAA GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTAC CGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA

AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGO AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGC AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCA AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

Additionally, an IL-21/TF/IL-15 construct was made linking the IL-21 sequence

to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF

construct to the N-terminus coding region of IL-15 (Figure 41). The nucleic acid

sequence of the IL-21/TF/IL-15 construct (including leader sequence) is as follows (SEQ

ID NO: 192):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC (Human IL-21)

AGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA

ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGO ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG AAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCO CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCA CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA

CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human I IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCO

Example 22: Secretion of TGFBRIIIL-15RaSu and IL-21/TF/IL-15 fusion proteins

The TGF3RII/IL-15RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a

pMSGV-1 modified retrovirus expression vector (as described in Hughes et al., Hum wo 2021/247604 WO PCT/US2021/035285

Gene Ther 16:457-72, 2005, herein incorporated by reference), and the expression vector

was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1

cells resulted in secretion of the soluble IL-21/TF/IL-15:TGFBRII/IL-15RaSt protein

complex (referred to as 21t15-TGFRs; Figure 42 and Figure 43). The 21t15-TGFRs

complex was purified from CHO-K1 cell culture supernatant using anti-TF antibody

affinity chromatography and other chromatography methods.

The amino acid sequence of the TGF3RII/LL-15RaSu construct (including signal

peptide sequence) is as follows (SEQ ID NO: 195):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGF/BRII-1* fragment)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD PGETFFMCSCSSDECNDNIFSEEYNTSNPD (Linker)

GGGGSGGGGSGGGGS (Human TGF,BRII-2"*fragment)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the mature IL-21/TF/IL-15 fusion protein (including

signal peptide sequence) is as follows (SEQ ID NO: 191):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21) wo 2021/247604 WO PCT/US2021/035285

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSC QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate a mature form that may be soluble or secreted.

Example 23: Purification of 21t15-TGFRs by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare AKTATM

Avant protein purification system. The flow rate was 4 mL/min for all steps except the

elution step, which was 2 mL/min.

Cell culture harvest of 21t15-TGFRs was adjusted to pH 7.4 with 1M Tris base

and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes

of PBS. After loading the sample, the column was washed with 5 column volumes PBS,

followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280

nm was collected and then the sample was then neutralized to pH 7.5-8.0 by adding 1M

Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon

centrifugal filters with a 30 KDa molecular weight cutoff. Figure 44 shows that the

21t15-TGFRs complex binds anti-TF antibody affinity column, wherein TF is a 21t15-

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

TGFRs binding partner. The buffer-exchanged protein sample is stored at 2-8°C for

further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6

column volumes 0. 1M glycine, pH 2.5. The column was then neutralized using 10

column volumes PBS, 0.05% sodium azide, and stored at 2-8°C.

Example 24: Size exclusion chromatography of 21t15-TGFRs

A GE Healthcare Superdex 200 Increase 10/300 GL gel filtration column was

connected to a GE Healthcare AKTATM Avant protein purification system. The column

was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A

capillary loop was used to inject 200 uL of 1 mg/mL of 21t15-TGFRs complex onto the

column. The injection was then chased with 1.25 column volumes of PBS. The SEC

chromatograph was shown in Figure 45. There were two protein peaks, likely

representing a monomer and dimer forms of 21t15-TGFRs.

Example 25: SDS-PAGE of 21t15-TGFRs To determine the purity and protein molecular weight, the purified 21t15-TGFRs

complex protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDS-

PAGE under reduced conditions. The gel was stained with InstantBlueTM for about 30

min, followed by destaining overnight in purified water. Figure 46 shows an example

SDS gel of anti-TF antibody affinity purified 21t15-TGFRs, with bands at 39.08 kDa and

53 kDa

Glycosylation of 21t15-TGFRs in CHO cells was confirmed using the Protein

Deglycosylation Mix II kit (New England Biolabs) and the manufacturer's instructions.

Deglycosylation reduces the molecular weight of 21t15-TGFRs, as seen in lane 4 of

Figure 46.

Example 26: Recombinant protein quantitation of 21t15-TGFRs complexes

The 21t15-TGFRs complex was detected and quantified using standard sandwich

ELISA methods (Figures 47-50). Anti-human tissue factor antibody served as the

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

capture antibody and biotinylated anti-human IL-21, IL-15, or TGFßRII served as the

detection antibody. Tissue factor was also detected using an anti-human tissue factor

capture antibody (143), and anti-human tissue factor antibody detection. The 143/ anti-

TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 27: Immunostimulatory capacity of the 21t15-TGFRs complex

To assess the IL-15 - immunostimulatory activity of the 21t15-TGFRs complexes,

increasing concentrations of 21t15-TGFRs was added to 32DB cells (104 cell/well) in 200

uL IMDM:10% FBS media and cells were incubated for 3 days at 37°C. On the fourth

day, WST-1 proliferation reagent (10 uL/well) then was added and after 4 hours,

absorbance was measured at 450 nm to determine cell proliferation based on cleavage of

WST-1 to a soluble formazan dye. Bioactivity of the human recombinant IL-15 was

assessed as a positive control. As shown in Figure 51, 21t15-TGFRs demonstrated IL-

15-dependent 32D cell proliferation. The 21t15-TGFRs complex was reduced

compared to that of human recombinant IL-15, possibly due to the linkage of IL-21 and

tissue factor to the IL-15 domain.

Additionally, HEK-Blue TGFß reporter cells (hkb-tgfb, InvivoGen) were

used to measure the ability of 21t15-TGFRs to block TGFß1 activity (Figure 52).

Increasing concentrations of 21t15-TGFRs were mixed with 0.1 nM of TGFß1

and added to HEK-Blue TGFB reporter cells (2.5x104 cell/well) in 200 uL

IMDM:109 heat-inactivated FBS media. Cells were incubated overnight at

37°C. The next day, 20 ul of induced HEK-Blue TGFß reporter cell supernatant

was added to 180 ul of QUANTI-Blue (InvivoGen) and incubated for 1-3 hours at

37°C. 21t15-TGFRs activity was assessed by measuring absorbance at 620 nm.

Human recombinant TGFßRII/Fc activity was assessed as a positive control.

These results demonstrate that TGFßRII domain of the 21t15-TGFRs complex

retains its ability to trap TGFB1. The ability of 21t15-TGFRs to block TGFB1 activity

was reduced compared to that of human recombinant TGF3RII/Fc, possibly due to the

linkage of TGFßRII to the IL-15Ra sushi domain.

Example 28: Induction of cytokine-induced memory-like NK cells by the 21t15-

TGFRs complex Cytokine-induced memory-like NK cells can be induced ex vivo following

overnight stimulation of purified NK cells with saturating amounts of cytokines. These

memory-like properties can be measured through expression of IL-2 receptor a (IL-2Ra,

CD25), CD69 (and other activation markers), and increased IFN-y production. To

evaluate the ability of 21t15-TGFRs complexes to promote generation of cytokine-

induced memory-like NK cells, purified human NK cells (>95% CD56+) were stimulated

for 14-18 hours with 1 nM to 100 nM of the 21t15-TGFRs complex. Cell-surface CD25

and CD 69 expression and intracellular IFN-y levels were assessed by antibody-staining

and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells

were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The

purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were

counted and resuspended in 0.2 X 106/mL in a 96 well flat bottom plate in 0.2 mL of

complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mix-

cytokines of hIL-21 (50 ng/ml) (Biolegend) and hIL-15 (50 ng/ml) (NCI) or with 1 nM,

10 nM, or 100 nM 21t15-TGFRs complex overnight at 37°, 5% CO2 for 14-18 hrs. The

cells were then harvested and surface stained for CD56-BV421, CD16-BV510, CD25-

PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were

washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (1X PBS

(Hyclone) with 0.5% BSA (EMD Millipore) and 0,001% sodium azide (Sigma)). After

two washes, cells were analyzed using a BD FACSCelestaTM flow cytometer. (Plotted

Data-Mean Fluorescence Intensity; Figure 53 and Figure 54).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells

were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The

purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

WO wo 2021/247604 PCT/US2021/035285

BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were

counted and resuspended in 0.2 X 106/ml in a 96 well flat bottom plate in 0.2 mL of

complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mix-

cytokines of hIL-21 (50 ng/ml) (Biolegend) and hIL-15 (50 ng/ml) (NCI) or with 1 nM,

10 nM, or 100 nM 21t15-TGFRs complex overnight at 37°, 5% CO2 for 14-18 hrs. The

cells were then treated with 10 ug/ml of Brefeldin A (Sigma) and 1X of Monensin

(eBioscience) for 4 hrs. Cells were harvested and surface stained for CD56-BV421,

CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After

staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS

buffer 1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide

(Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed

(1500 RPM for 5 minutes at room temperature) with 1x permeabilized buffer

(eBioscience) and stained for intracellular IFN-y- PE (Biolegend) for 30 minutes at room

temperature. Cells were washed once again with 1x permeabilized buffer and then

washed with FACS buffer. Cell pellets were resuspended in 300 uls of FACS Buffer and

analyzed using a BD FACSCelestaTM flow cytometer. (Plotted % of IFN-y Positive

Cells; Figure 55).

Example 29: In vitro cytotoxicity of NK cells against human tumor cells

K562 (CellTrace violet labelled), human myelogenous leukemia cells, were

incubated with purified human NK cells (using StemCell human NK cell purification kit

(E:T ratio; 2:1)) in the presence of increasing concentrations of the 21t15-TGFRs

complex. After 20 hours, the cultures were harvested, stained with propidium iodide

(PI), and assessed by flow cytometry. As shown in Figure 56, the 21t15-TGFRs complex

induced human NK cytotoxicity against K562, as compared to control.

wo WO 2021/247604 PCT/US2021/035285

Example 30: Creation of an IL-21/TF mutant/IL-15 DNA construct and resulting

fusion protein complex with TGFBRII/IL-15RaSu

In a non-limiting example, an IL-21/TF mutant/IL-15 DNA construct was made

by linking IL-21 directly to the N-terminus coding region of a tissue factor 219 mutant,

and further linking the IL-21/TF mutant to the N-terminus coding region of IL-15.

The nucleic acid sequence of the IL-21/TF mutant/IL-15 construct (including

signal peptide sequence) is as follows (SEQ ID NO: 324, shaded nucleotides are mutant

and the mutant codons are underlined):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCC GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT

CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor 219 mutants)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAC ACCAACTTO GCGACAGCTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGCTTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC GGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGGCGCGGAATAA CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGGGCGGAATA

CACAGCTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGT CACA TTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGT wo WO 2021/247604 PCT/US2021/035285

ACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAA CGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAA GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAA GCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTT GCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTG AGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the IL-21/TF mutant/IL-15 construct (including

signal peptide sequence) is as follows (SEQ ID NO: 325, substituted residues are

shaded):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS EFLERFKSLLOKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKC

FYTTDTEC ALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

WO wo 2021/247604 PCT/US2021/035285

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/TF mutant/IL-15 DNA construct may be

combined with an TGF3RII/IL-15RaSu DNA construct, transfected into cells using a

retroviral vector as described above, and expressed as IL-21/TF mutant/IL-15 and

TGFBRII/IL-15RaSu fusion proteins. The IL-15RaSu domain of the TGF3RII/IL-

15RaSu fusion protein binds to the IL-15 domain of the IL-21/TF mutant/IL-15 fusion

protein to create an IL-21/TF mutant/IL-15:TGFBRII/IL-15RaSucomplex

Example 31: Creation of IL-21/IL-15RaSu and TGFBRII/TF/IL-15 DNA constructs

and the resulting fusion protein complex

In a non-limiting example, an IL-21/IL-15RaSu DNA construct was made by

linking IL-21 directly to the IL-15RaSu subunit sequence. The nucleic acid sequence of

the IL-21/IL-15RaSu construct (including signal sequence) is as follows (SEQ ID NO:

214):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT

CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCC GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTT6 AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA

AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTO (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the IL-21/IL-15RaSu construct (including signal

peptide sequence) is as follows (SEQ ID NO: 213):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

[TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/IL-15RaSu DNA construct may be combined

with a TGFBRII/TF/IL-15 DNA construct, transfected into a retroviral vector as

described above, and expressed as IL-21/IL-15RaSu and TGF3RII/TF/IL-15 fusion

proteins. The IL-15RaSu domain of the IL-21/IL-15RaSu fusion protein binds to the IL-

15 domain of the TGF3RII/TF/IL-15 fusion protein to create a TGF6RII/TF/IL-15:IL-

21/IL-15RaSu complex.

The TGFBRII/TF/IL-15RaSu DNA construct was created by linking the TGFßRII

sequence to the N-terminus coding region of human tissue factor 219 form, and then wo WO 2021/247604 PCT/US2021/035285 linking the TGF3RII/TF construct to the N-terminus coding region of IL-15. As described above, a single-chain version of TGFßRII (TGFBRII-linker-TGFBRII) was used. The nucleic acid sequence of the TGF3RII/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 239):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGF BRII-1* fragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACO GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTAT ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA

GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTG AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (Linker)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGF BRII-2" fragment)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACO ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACC ATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCA CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAAT GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTAC

ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGO AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGO

ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT wo 2021/247604 WO PCT/US2021/035285

ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCA CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCA TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAA GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG

CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGA0 (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAAT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC

TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT7 AGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCA GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTT GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the TGF3RII/TF/IL-15 fusion protein (including

signal peptide) is as follows (SEQ ID NO: 238):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGF,BRII-1* fragment)

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker)

GGGGSGGGGSGGGGS wo 2021/247604 WO PCT/US2021/035285

(Human TGF.BRII-2"^fragment)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD PGETFFMCSCSSDECNDNIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY TNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYV ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 32. Production of an Exemplary Single-Chain Chimeric Polypeptides

An exemplary single-chain chimeric polypeptide including a first target-binding

domain that is an anti-CD3 scFv, a soluble human tissue factor domain, and a second

target-binding domain that is an anti-CD28 scFv was generated

(aCD3scFv/TF/aCD28scFv) (Figure 57). The nucleic acid and amino acid sequences of

this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide

(aCD3scFv/TF/aCD28scFv) (SEQ ID NO: 158)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCT ATTCC (aCD3 light chain variable region)

WO wo 2021/247604 PCT/US2021/035285

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGT GAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACT GGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCA GCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCA CAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTA CAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACT ATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCT ATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCT CGAAATCAATCGT (Linker)

GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGC ( CC33 heavy chain variable region)

CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGC CTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAA CTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAA TGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATAT CAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCC ACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTT AACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCAC AACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCAC TACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGO (Human tissue factor 219 form)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTC PACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCA AAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCC CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTAT AGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCA CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGT CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG wo WO 2021/247604 PCT/US2021/035285

CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (aCD28 light chain variable region)

GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCC GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCC GTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCA GTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCA GTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAA GTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAA CCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACT CCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACT TTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTA ITCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTAC ATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTAC

TGGGGACGGGGCACAACACTGACCGTGAGCAGC (Linker)

GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCC (aCD28 light chain variable region)

GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGO GGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCA GGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCAT TGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACC TGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCA GCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAG GCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAG- TACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTT GTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGAC GTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGAC AAAGAGG

Exemplary Single-Chain Chimeric Polypeptide (aCD3scFv/TF/aCD28scFv) (SEQ

ID NO: 157)

(Signal peptide)

MKWVTFISLLFLFSSAYS (aCD3 light chain variable region)

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDT SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLE] NR (Linker) wo 2021/247604 WO PCT/US2021/035285

GGGGSGGGGSGGGGS (aCD3 heavy chain variable region)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSS (Human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP) FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW

KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE ( CCD28 light chain variable region)

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGS INPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNY

WGRGTTLTVSS (Linker)

GGGGSGGGGSGGGGS (aCD28 heavy chain variable region)

DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLA DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLA

SGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR SGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR A second exemplary single-chain chimeric polypeptide including a first target-

binding domain that is an anti-CD28 scFv, a soluble human tissue factor domain, and a

second target-binding domain that is an anti-CD3 scFv was generated

(aCD28scFv/TF/aCD3scFv) (Figure 57). The nucleic acid and amino acid sequences of

this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide

(aCD28scFv/TF/aCD3scFv) (SEQ ID NO: 326)

(Signal peptide)

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ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCT ACAGC (aCD28 light chain variable region)

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCC GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCC

TGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCA GTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCA ATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAA CCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTC CCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTC GACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAAC TGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACT `CTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATT TCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTG

GGGCCGGGGAACTACTTTAACAGTGAGCTCC (Linker)

GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAG GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGC (aCD28 heavy chain variable region)

GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGA GAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTT GAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTT ACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACT CAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACC TCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTT TCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTT TGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAG TTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAG

ACCAAGCGG ACCAAGCGG (Human tissue factor 219 form)

AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCO AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCC ACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTT ACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTT ATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTC7 ATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTA

ACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAA CAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCA CCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTAT CTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCA CAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAAC CAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACA ATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTG

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ATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCA TATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCA ACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGC/ ACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCA AGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTG GAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAC GAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAG

(aCD3 light chain variable region)

CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGT AAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAAG GAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACT GGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACC GGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAG AAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAAC/

AGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATT AGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATT ACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTC GAGATTAATCGT (Linker)

GGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGC (aCD3 heavy chain variable region)

CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCT CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCT TCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAAT TCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAAT GCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATe AACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCA AACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCA

CCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTT CCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTT AACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCAT ACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC ACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC

Exemplary Single-Chain Chimeric Polypeptide (aCD28scFv/TF/aCD3scFv) (SEQ

ID NO: 327)

(Signal peptide)

MKWVTFISLLFLFSSAYS (aCD28 light chain variable region) wo WO 2021/247604 PCT/US2021/035285

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGS VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGS INPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNY INPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNY WGRGTTLTVSS (Linker)

GGGGSGGGGSGGGGS (aCD28 heavy chain variable region)

DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYS TSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (Human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL CTNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (aCD3 light chain variable region)

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDT QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYD' SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLE NR (Linker)

GGGGSGGGGSGGGGS (aCD3 heavy chain variable region)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEV GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSS

The nucleic acid encoding aCD3scFv/TF/aCD28scFv was cloned into a modified

retrovirus expression vectors as described previously (Hughes et al., Hum Gene Ther

16:457-72, 2005). The expression vector encoding aCD3scFv/TF/aCD28scFv was

transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells

allowed for secretion of the soluble aCD3scFv/TF/aCD28scFv single-chain chimeric

591

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polypeptide (referred to as 3t28), which can be purified by anti-TF Ab affinity and other

chromatography methods.

An anti-tissue factor affinity column was used to purify the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide. The anti-tissue factor

affinity column was connected to a GE Healthcare AKTA Avant system. A flow rate of 4

mL/min was used for all steps except the elution step, which was 2 mL/min.

Cell culture harvest including aCD3scFv/TF/aCD28scFv single-chain chimeric

polypeptide was adjusted to pH 7.4 with 1M Tris base and loaded onto the anti-TF

antibody affinity column (described above) which was equilibrated with 5 column

volumes of PBS. After sample loading, the column was washed with 5 column volumes

PBS, followed by elution with 6 column volumes 0.1 M acetic acid, pH 2.9. An A280

elution peak was collected and then neutralized to pH 7.5-8.0 by adding 1 M Tris base.

The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal

filters with a 30 kDa molecular weight cutoff. The data in Figure 58 show that the anti-

tissue factor affinity column can bind the aCD3scFv/TF/aCD28scFv single-chain

chimeric polypeptide, which contains a human soluble tissue factor domain. The buffer-

exchanged protein sample was stored at 2-8 °C for further biochemical analysis and

biological activity testing.

After each elution, the anti-tissue factor affinity column was stripped using 6

column volumes of 0.1 M glycine, pH 2.5. The column was then neutralized using 10

column volumes of PBS, 0.05% NaN3, and stored at 2-8 °C.

Analytical size exclusion chromatography (SEC) was performed on the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide using a Superdex 200

Increase 10/300 GL gel filtration column (from GE Healthcare) connected to an AKTA

Avant system (from GE Healthcare). The column was equilibrated with 2 column

volumes of PBS. A flow rate of 0.8 mL/min was used. Two hundred uL of

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide (1 mg/mL) was injected

onto the column using a capillary loop. After injection of the single-chain chimeric

polypeptide, 1.25 column volumes of PBS were flowed into the column. The SEC

chromatograph is shown in Figure 59. The data show that there are 3 protein peaks,

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likely representing a monomer and dimer or other different forms of the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide.

To determine the purity and protein molecular weight of the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide, the purified

aCD3scFv/TF/aCD28scFv protein sample from anti-tissue factor affinity column was

analyzed by standard sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-

Tris gel) electrophoresis (SDS-PAGE) method under reduced conditions. The gel was

stained with InstantBlue for about 30 minutes and destained overnight with purified

water. Figure 60 shows the SDS gel of the aCD3scFv/TF/aCD28scFv single-chain

chimeric polypeptide purified using an anti-tissue factor affinity column. The results

show that the purified aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide has

the expected molecular weight (72 kDa) in reduced SDS gel.

Example 33. Functional Characterization of aCD3scFv/TF/aCD28scFvS Single-

Chain Chimeric Polypeptide

ELISA-based methods confirmed the formation of the aCD3scFv/TF/aCD28scFv

single-chain chimeric polypeptide. The aCD3scFv/TF/aCD28scFv single-chain

chimeric polypeptide was detected using an anti-TF antibody (143)/anti-TH antibody-

specific ELISA with a capture antibody, anti-human tissue factor antibody (143), and a

detection antibody, anti-TF antibody(Figure 61). A purified tissue factor protein with a

similar concentration was used as a control.

A further in vitro experiment was performed to determine whether the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide is capable of activating

human peripheral blood mononuclear cells (PBMCs). Fresh human leukocytes were

obtained from the blood bank and peripheral blood mononuclear cells (PBMC) were

isolated using density gradient Histopaque (Sigma). The cells were counted and

resuspended in 0.2 X 106/mL in a 96-well flat bottom plate in 0.2 mL of complete media

(RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies),

penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and

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10% FBS (Hyclone)). The cells were stimulated with aCD3scFv/TF/aCD28scFv single-

chain chimeric polypeptide from 0.01 nM to 1000 nM for 3 days at 37 °C, 5% CO2. After

72 hours, the cells were harvested and surface stained for CD4-488, CD8-PerCP Cy5.5,

CD25-BV421,CD69-APCFire750, CD62L-PE Cy7, and CD44-PE specific antibodies

(Biolegend) for 30 minutes. After surface staining, the cells were washed (1500 RPM for

5 minutes at room temperature) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA

(EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were

resuspended in 300 uL of FACS buffer and analyzed by Flow Cytometry (Celesta-BD

Bioscience). The data in Figures 62 and 63 show that the aCD3scFv/TF/aCD28scFv

single-chain chimeric polypeptide is able to stimulate both CD8+ and CD4+ T-cells.

A further experiment was performed, in which PBMCs isolated from blood using

Histopaque (Sigma) were counted and resuspended in 0.2 X 106/mL in a 96-well flat

bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2

mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies),

streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were

then stimulated with the aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide

from 0.01 nM to 1000 nM for 3 days at 37 °C, 5% CO2. After 72 hours, the cells were

harvested and surface stained for CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69-

APCFire750, CD62L-PE Cy7, and CD44-PE (Biolegend) for 30 minutes. After surface

staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS

buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide

(Sigma)). After two washes, the cells were resuspended in 300 uL of FACS buffer and

analyzed by Flow Cytometry (Celesta-BD Bioscience). The data again show that the

aCD3scFv/TF/aCD28scFv single-chain chimeric polypeptide was able to stimulate

activation of CD4+ T cells (Figure 64).

Example 34: Creation of an IL-7/IL-15RaSu DNA construct

In a non-limiting example, an IL-7/IL-15RoSu DNA construct was created (see

Figure 65). The human IL-7 sequence, human IL-15RaSu sequence, human IL-15

sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-7 sequence to the IL-15RaSu sequence. The final IL-7/IL-15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-7/IL-

15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 206):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC (Human IL-7)

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTA GGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATT GCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAG GAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAAT GAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAA GAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAA

CAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCT CAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCT GGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGG GGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGA ACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATA CAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATA AAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (Human IL-15R a sushi domain)

ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTC ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA CGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAG

The second chimeric polypeptide of IL-7/IL-15RaSu construct (including signal

peptide sequence) is as follows (SEQ ID NO: 205):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDA) KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKO FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 35: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (Figure 66) by

linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and

further linking the IL-21/TF construct with the N-terminus coding region of IL-15.

The nucleic acid sequence encoding the first chimeric polypeptide of IL-

21/TF/IL-15 construct (including leader sequence), synthesized by Genewiz, is as follows

(SEQ ID NO: 202):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC

(Human IL-21 fragment)

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATT CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATT TTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGO TCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGA AGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATO AGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATG-

TATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAA TATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAA wo WO 2021/247604 PCT/US2021/035285

GACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACO GACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACC ACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCAT ACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCAT CAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC CAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (Human Tissue Factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCA ACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTO ACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCT ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTA ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTA ACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAA CACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAA GCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGO

ACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTA CTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA CCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA CAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAAG ACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAAC AACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACT AACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACT ATATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACT TTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACT AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCA AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCA GCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTA GAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-21/TF/IL-15 construct including leader

sequence is SEQ ID NO: 201:

(Signal peptide)

MGVKVLFALICIAVAEA (SEQ ID NO: 328) wo 2021/247604 WO PCT/US2021/035285

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTT SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETN, TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSS QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR

E (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN DASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVOMFIN TS

Example 36: Secretion of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The IL-7/IL-15RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a

pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene

Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of a soluble IL-21/TF/IL-15:IL-7/IL-15RaSu protein

complex (referred to as 21t15-7s; Figures 67 and Figure 68). The 21t15-7s protein was

purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity

chromatography and size exclusion chromatography resulting in soluble (non-aggregated)

protein complexes consisting of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from the intact

polypeptide to generate the mature form that may be soluble or secreted.

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Example 37: Purification of 21t15-7s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE HealthcareTM AKTA

Avant protein purification system. The flow rate was 4 mL/min for all steps except the

elution step, which was 2 mL/min.

Cell culture harvest of 21t15-7s was adjusted to pH 7.4 with 1M Tris base and

loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of

PBS. After loading the sample, the column was washed with 5 column volumes PBS,

followed by elution with 6 column volumes 0. 1M acetic acid, pH 2.9. Absorbance at 280

nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding 1M Tris

base. The neutralized sample was then buffer exchanged into PBS using Amicon®

centrifugal filters with a 30 KDa molecular weight cutoff. The buffer-exchanged protein

sample was stored at 2-8°C for further biochemical analysis and biological activity

testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6

column volumes 0. 1M glycine, pH 2.5. The column was then neutralized using 10

column volumes PBS, 0.05% sodium azide and stored at 2-8 °C.

Example 38: Size exclusion chromatography

A GE Healthcare Superdex 200 Increase 10/300 GL gel filtration column was

connected to a GE Healthcare AKTATM Avant protein purification system. The column

was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A

capillary loop was used to inject 200uL of 1 mg/mL of 7t15-21 scomplex onto the

column. The injection was chased with 1.25 column volumes of PBS.

Example 39: SDS-PAGE of 21t15-7s and 21t15-TGFRs

To determine the purity and protein molecular weight, the purified 21t15-7s or

21t15-TGFRs protein sample were analyzed using 4-12% NuPage Bis-Tris protein gel

SDS-PAGE. The gel will be stained with InstantBlueTM for about 30 min, followed by

destaining overnight in purified water.

Example 40: Glycosylation of 21t15-7s and 21t15-TGFRs in CHO-K1 cells

Glycosylation of 21t15-7s in CHO-K1 cells or 21t15-TGFRs in CHO-K1 cells

were confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs),

according to the manufacturer's instructions.

Example 41: Recombinant protein quantitation of 21t15-7s and 21t15-TGFRs

complexes

The 21t15-7s complex or the 21t15-TGFRs complex were detected and quantified

using standard sandwich ELISA methods. Anti-human tissue factor antibody (IgG1)

served as the capture antibody and biotinylated anti-human IL-21, IL-15, or IL-7

antibody (21t15-7s) or biotinylated anti-human IL-21, IL-15, or TGF-BRII antibody

(21t15-TGFRs) served as the detection antibody. Tissue factor in purified 21t15-7s or

21t15-TGFRs protein complexes was detected using an anti-human tissue factor capture

antibody, and anti-human tissue factor antibody (IgG1) detection antibody. The anti-TF

antibody ELISA will be compared to purified tissue factor at similar concentrations.

Example 42: Creation of an IL-21/IL-15RaSu DNA construct

In a non-limiting example, an IL-21/IL-15RaSu DNA construct was created. The

human IL-21 sequence and human IL-15RaSu sequence were obtained from the UniProt

website and DNA for these sequences was synthesized by Genewiz. A DNA construct

was made linking the IL-21 sequence to the IL-15RaSu sequence. The final IL-21/IL-

15RaSu DNA construct sequence was synthesized by Genewiz. See Figure 69.

Example 43: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an IL-7/TF/IL-15 construct was made by linking the

IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking

the IL-7/TF construct with the N-terminus coding region of IL-15. See Figure 70.

Example 44: Creation of an IL-21/IL-15Ra Sushi DNA construct

In a non-limiting example, a second chimeric polypeptide of IL-21/IL-15RaSu

was generated. The human IL-21 and human IL-15Ra sushi sequences were obtained

from the UniProt website and DNA for these sequences was synthesized by Genewiz. A

DNA construct was made linking the IL-21 sequence to the L-15Ra sushi sequence.

The final IL-21/IL-15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-21/IL-

15RaSu domain (including leader sequence), synthesized by Genewiz, is as follows

(SEQ ID NO: 214):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC GAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC0

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAG GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC GTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The second chimeric polypeptide of IL-21/IL-15Ra sushi domain (including

leader sequence) is as follows (SEQ ID NO: 213):

(Signal Sequence) wo WO 2021/247604 PCT/US2021/035285

MKWVTFISLLFLFSSAYS (Human IL-21)

GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER

FKSLLQKMIHQHLSSRTHGSEDS (HumanIL-15Ra sushidomain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR

Example 45: Creation of an IL-7/TF/IL-15 DNA construct In a non-limiting example, an exemplary first chimeric polypeptide of IL- -

7/TF/IL-15 was made by linking the IL-7 sequence to the N-terminus coding region of

tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding

region of IL-15. The nucleic acid sequence encoding the first chimeric polypeptide of

IL-7/TF/IL-15 (including leader sequence), synthesized by Genewiz, is as follows (SEQ

ID NO: 210):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C

(Human IL-7 fragment)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(Human Tissue Factor 219) wo 2021/247604 WO PCT/US2021/035285

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACT CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAAC GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTG AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTA AGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGC GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA CATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), is as

follows (SEQ ID NO: 209).

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKE]

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(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNI TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFE KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG JNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN DASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

Example 46: Secretion of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins

The IL-21/IL-15RaSu and IL-7/TF/IL-15 DNA constructs were cloned into a

pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene

Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of a soluble IL-7/TF/IL-15:IL-21/IL-15RaSu protein

complex (referred to as 7t15-21s). The 7t15-21s protein was purified from CHO-K1 cell

culture supernatant using anti-TF antibody (IgG1) affinity chromatography and size

exclusion chromatography resulting in soluble (non-aggregated) protein complexes

consisting of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins. See Figure 71 and

Figure 72.

Example 47: Expansion capacity of primary natural killer (NK) cells by 7t15-21s

complex + anti-TF IgG1 antibody

To assess the 7t15-21s complex's ability to expand primary natural killer (NK)

cells, 7t15-21s complex and 7t15-21s complex + anti-TF IgG1 antibody are added to NK

cells obtained from samples of fresh human leukocytes. Cells are stimulated with 50nM

of 7t15-21s complex with or without 25 nM of anti-TF IgG1 or anti-TF IgG4 antibody at

37° and 5% CO2. Cells are maintained at concentration at 0.5 X 106/mL not exceeding 2.0

WO wo 2021/247604 PCT/US2021/035285

X 106/mL by counting every 48-72 hours and media is replenished with fresh stimulator.

Cells stimulated with 7t15-21s complex or anti-TF IgG1 antibody or anti-TFIgG4

antibody or anti-TF IgG4 + 7t15-21s complex are maintained up to day 5. Expansion of

primary NK cells upon incubation with 21t15-7s complex + anti-TF IgG1 antibody is

observed.

Example 48: Activation of expanded NK cells by the 7t15-21s complex + anti-TF

IgG1 antibody

Primary NK cells are induced ex vivo following overnight stimulation of purified

NK cells with 7t15-21s complex + anti-TF IgG1 antibody. Fresh human leukocytes are

obtained from a blood bank and CD56+ NK cells are isolated with the RosetteSep/human

NK cell reagent (StemCell Technologies). The purity of NK cells is >80% and is

confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies

(BioLegend). Cells are counted and resuspended in 1 X 106/mL in a 24 well flat bottom

plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L-

glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies),

streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life

Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)).

Cells are stimulated with 50 nM of 7t15-21s with or without 25 nM of anti-TF IgG1

antibody at 37° and 5% CO2. Cells are counted every 48-72 hours and maintained at a

concentration of 0.5 x 106/mL to 2.0 x 106/mL until day 14. Media is periodically

replenished with fresh stimulator. Cells are harvested and surface stained at day 3 for

CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies

(Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). The activation

marker CD25 MFI are observed to increase with 7t15-21s complex + anti-TF IgG1

antibody stimulation, but not 7t15-21s complex stimulation. The activation marker CD69

MFI is observed to increase with both 7t15-21s complex + anti-TF IgG1 antibody and

with 7t15-21s complex, alone.

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Example 49: Increase in Glucose Metabolism in NK Cells Using 18t15-12s

A set of experiments was performed to determine the effect of the construct of

18t15-12s on oxygen consumption rate and extracellular acidification rate (ECAR) on

NK cells purified from human blood.

In these experiments, fresh human leukocytes were obtained from the blood bank

from two different human donors and NK cells were isolated via negative selection using

the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells

was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific

antibodies (BioLegend). The cells were counted and resuspended in 2 X 106/mL in 24-

well, flat-bottom plates in 1 mL of complete media (RPMI 1640 (Gibco) supplemented

with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life

Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid

(Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies) and 10%

FBS (Hyclone)). The cells were stimulated with either (1) media alone, (2) 100 nM

18t15-12s, or (3) mixture of single cytokines recombinant human IL-12 (0.25 ug),

recombinant human IL-15 (1.25 ug), and recombinant human IL-18 (1.25 ug) overnight

at 37 °C, 5% CO2. On the next day, the cells were harvested and extracellular flux assays

on expanded NK cells were performed using a XFp Analyzer (Seahorse Bioscience). The

harvested cells washed and plated 2.0 X 105 cells/well in at least duplicate for

extracellular flux analysis of OCR (Oxygen Consumption Rate) and ECAR (Extracellular

Acidification Rate). The glycolysis stress tests were performed in Seahorse Media

contain 2 mM of glutamine. The following were used during the assay: 10 mM glucose;

100 nM oligomycin; and 100 mM 2-deoxy-D-glycose (2DG).

The data show that the 18t15-12s results in significantly increased oxygen

consumption rate (Figure 73) and extracellular acidification rate (ECAR) as compared to

the same cells activated with a combination of recombinant human IL-12, recombinant

human IL-15, and recombinant human IL-18 (Figure 74).

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Example 50: 7t15-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising of anti-CD16scFv/IL-

15RaSu/IL-21 and IL-7/TF/IL-15 fusion proteins. The human IL-7 and IL-21 sequences

were obtained from the UniProt website and DNA for these sequences was synthesized

by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N-

terminus coding region of tissue factor 219 followed by the N-terminus coding region of

IL-15.

The nucleic acid and protein sequences of a construct comprising IL-7 linked to

the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown

below.

The nucleic acid sequence of the IL-7/TF/IL-15 construct (including signal

peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC (Human IL-7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT

ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA wo 2021/247604 WO PCT/US2021/035285

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACAC AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAAT? CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT CAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of IL-7/TF/IL-15 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS MKWVTFISLLFLFSSAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN

EGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKO SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY, FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW

KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by linking the anti-CD16scFv sequence to the N-

terminus coding region of IL-15RaSu chain followed by the N-terminus coding region of

IL-21 which was synthesized by Genewiz. The nucleic acid and protein sequences of a

construct comprising the anti-CD16scFv linked to the N-terminus of IL-15RaSu chain

followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16SscFv/IL-15 RaSu/IL-21 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC ((Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC

GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTO CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACT. GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA wo WO 2021/247604 PCT/US2021/035285

TCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTC6 CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGO CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGO CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACA

CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTT6 AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA

AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCO CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RaSu/IL-21 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Anti-human CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGI RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKI NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL

TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQ SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

(Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSA QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RaSu/IL-21 and IL-7/TF/IL-15 constructs were cloned

into a modified retrovirus expression vectors as described previously (Hughes MS, Yu

YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from

a patient with a marked antitumor response conveys highly active T-cell effector

functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected

into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for

formation and secretion of the soluble IL-7/TF/IL-15:anti-CD16scFv/IL-15RaSu/IL-21

protein complex (referred to as 7t15-16s21; Figure 75 and Figure 76), which can be

purified by anti-TF IgG1 antibody-based affinity and other chromatography methods.

Binding of 7t15-16s21 to CHO cells expressing human CD16b

CHO cells were transfected with human CD16b in a pMC plasmid and selected

with 10 ug/mL of blasticidin for 10 days. The CHO cells stably expressing CD16b were

stained with 1.2 ug/mL of 7t15-16s21, containing anti-human CD16 scFv or 18t15-12s,

which does not contain anti-human CD16 scFv, as a negative control, and then stained

with biotinylated anti-human tissue factor and PE conjugated streptavidin. Only anti-

human CD16scFv containing 7t15-16s21 stained the cells as shown in Figure 77A.

18t15-12s did not stain the CHO cells expressing human CD16b as showed in Figure

77B.

Detection of IL-15, IL-21, and IL-7 in 7t15-16s21 using ELISA

A 96-well plate was coated with 100 uL (8 ug/mL) of anti-TF IgG1 in R5

(coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were

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washed 3 times and blocked with 100 uL of 1% BSA in PBS. Serial dilution of 7t15-

16s21 (at a 1:3 ratio) were added to the wells, and incubated at RT for 60 min. Following

3 washes, 50 ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 500

ng/mL of biotinylated-anti-IL-21 antibody (13-7218-81, R&D Systems), or 500 ng/mL of

biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added to the wells and

incubated at RT for 60 min. The plate was washed 3 times, and incubated with 0.25

ug/mL of HRP-SA (Jackson ImmunoResearch) at 100 uL per well for 30 min at RT,

followed by 4 washes and incubation with 100 ul of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 78A-78C, the IL-15, IL-21, and

IL-7 domains in 7t15-16s21 were detected by the individual antibodies.

The IL-15 in 7t15-16s21 promotes IL-2RB and common y chain containing 32DB cell

proliferation

To analyze the activity of IL-15 in 7t15-16s21, the IL-15 activity of 7t15-16s21

was compared to recombinant IL-15 using 32DB cells that express IL2R and common Y

chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32DB

cells were washed 5 times with IMDM-10% FBS and seeded in the wells at X 104

cells/well. Serially-diluted 7t15-16s21 or IL-15 were added to the cells (Figure 79).

Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was

detected by adding 10 ul of WST1 to each well on day 3 and incubating for an additional

3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by

analyzing the amount of formazan dye produced. As shown in Figure 79, 7t15-16s21 and

IL-15 promoted 32DB cell proliferation, with the EC50 of 7t15-16s21 and IL-15 being

172.2 pM and 16.63 pM, respectively.

Purification elution chromatograph of 7t15-16s21 from anti-TF antibody affinity column

7t15-16s21 harvested from cell culture was loaded onto the anti-TF antibody

affinity column equilibrated with 5 column volumes of PBS. The column was then

washed with 5 column volumes of PBS, followed by elution with 6 column volumes of

0. 1M acetic acid (pH 2.9). A280 elution peak was collected and neutralized to pH 7.5-8.0

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with 1M Tris base. The neutralized sample was buffer exchanged into PBS using

Amicon centrifugal filters with a 30 KDa molecular weight cutoff. Figure 80 is a line

graph showing the chromatographic profile of 7t15-16s21 protein containing cell culture

supernatant following binding and elution on anti-TF antibody resin. As shown in Figure

80, the anti-TF antibody affinity column bound 7t15-16s21 which contains TF. The

buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses

and biological activity tests. After each elution, the anti-TF antibody affinity column was

stripped using 6 column volumes of 0. 1M glycine (pH 2.5). The column was then

neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for

storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA

Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which

was 2 mL/min.

Analytical size exclusion chromatography (SEC) analysis of 7t15-16s21

To perform size exclusion chromatography (SEC) analysis for 7t15-16s21, a

Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) connected to an

AKTA Avant system (GE Healthcare) was used. The column was equilibrated with 2

column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 7t15-16s21

in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed

by SEC. As shown in Figure 81, the SEC results showed two protein peaks for 7t15-

16s21.

Example 51: TGFRt15-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising anti-human CD16scFv/IL-

15RaSu/IL21 and TGFB Receptor II/TF/IL-15 fusion proteins (Figure 82 and 83). The

human TGFB Receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were

obtained from the UniProt website and DNA for these sequences was synthesized by

Genewiz. Specifically, a construct was made linking two TGFß Receptor II sequences

with a G4S(3) linker to generate a single chain version of TGFß Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N- terminus coding region of IL-15. -

The nucleic acid and protein sequences of a construct comprising two TGFß

Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus

of IL-15 are shown below.

The nucleic acid sequence of the two TGFB Receptor II/TF/IL-15 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC (Two Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGAC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTC. GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA

CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATO ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTG' AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA IGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGG AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCC GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATO TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

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AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT IACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTT CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGO CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCG7 AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAAT' CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT' TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTO TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGF Receptor II/TF/IL-15 fusion protein (including

the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN

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NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching anti-human CD16scFv directly linking to

the N-terminus coding region of IL-15RaSu chain followed by the N-terminus coding

region of IL-21 which was synthesized by Genewiz. The nucleic acid and protein

sequences of a construct comprising the anti-human CD16scFv linked to the N-terminus

of IL-15RaSu followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16scFv/IL-15 RaSu/IL-21 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCC' ACTCC

(Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGG GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG

CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGF wo 2021/247604 WO PCT/US2021/035285

CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAA0 CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGG. CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCC GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA GGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTC CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGO CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCC ACCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCT7 AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA

)ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAG AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RaSu/IL-21 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Anti-human (CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKI TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR

AEDTAVYYCARGRSLLFDYWGQGTLVTVSR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT

[TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RaSu/IL-21 anti-CD16scFv/IL-15RqSu/IL-21 and TGFR/TF/IL-15 constructs were

cloned into a modified retrovirus expression vectors as described previously (Hughes

MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene

derived from a patient with a marked antitumor response conveys highly active T-cell

effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of the soluble TGFR/TF/IL-15:CD16scFv/IL-

15RaSu/IL-21 protein complex (referred to as TGFRt15-16s21), which can be purified

by anti-TF IgG1-based affinity and other chromatography methods.

Interaction between TGFRt15-16s21 and CHO cells expressing human CD16b

CHO cells were transfected with human CD16b in a pMC plasmid and selected

with 10 ug/mL of blasticidin for10 days. Cells stably expressing CD16b were stained

with 1.2 ug/mL of TGFRt15-16s21, containing anti-human CD16 scFv, or 7t15-21s, not

containing anti-human CD16 scFv, as a negative control, and with biotinylated anti-

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human tissue factor antibody and PE conjugated streptavidin. As shown in Figures 84A

and 84B, TGFRt15-16s21, which contains anti-human CD16scFv, showed positive

binding, while 7t15-21s did not show binding.

Effect of TGFRt15-16s21 on TGFB1 activity in HEK-Blue TGFB cells

To evaluate the activity of TGFBRII in TGFRt15-16s21, the effect of TGFRt15-

16s21 on the activity of TGF31 in HEK-Blue TGFß cells was analyzed. HEK-Blue TGFß

cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the

testing medium (DMEM, 10% heat-inactivated FCS, 1x glutamine, 1x anti-anti, and 2x

glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 ul cells were added to

each well (2.5 x 104 cells/well) and followed with 50 uL 0. 1nM TGFß1 (R&D systems).

TGFRt15-16s21 or TGFR-Fc (R&D Systems) prepared at a 1:3 serial dilution was then

added to the plate to reach a total volume of 200 uL. After 24 hrs of incubation at 37°,

40 uL of induced HEK-Blue TGFß cell supernatant was added to 160 uL pre-warmed

QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3

hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655

nM. The IC50 of each protein sample was calculated with GraphPad Prism 7.04. The

IC50 of TGFRt15-16s21 and TGFR-Fc were 9127 pM and 460.6 pM respectively. These

results showed that the TGFßRII domain in TGFRt15-16s21 was able to block the

activity of TGFB-1 in HEK-Blue TGFß cells.

The IL-15 in TGFRt15-16s21 promotes IL-2RB and common chain containing 32DB cell

proliferation

To analyze the activity of IL-15 in TGFRt15-16s21, the IL-15 activity of

TGFRt15-16s21 was compared to recombinant IL-15 using 32DB cells that express

IL2R and commony chain, and evaluating their effects on promoting cell proliferation.

IL-15 dependent 32DB cells were washed 5 times with IMDM-10% FBS and seeded in

the wells at 2 X 104 cells/well. Serially-diluted TGFRt15-16s21 or IL-15 were added to

the cells (Figure 86). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell

proliferation was detected by adding 10 uL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. The data are shown in Figure 85. As shown in Figure 86, TGFRt15-16s21 and IL-15 promoted 32DB cell proliferation, with the EC50 of TGFRt15-16s21 and IL-15 being 51298 pM and 10.63 pM, respectively.

Detection of IL-15, IL-21, and TGFBRII in TGFRt15-16s21 using ELISA

A 96-well plate was coated with 100 uL (8 ug/mL) of anti-TF IgG1 in R5

(coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were

washed 3 times and blocked with 100 uL of 1% BSA in PBS. TGFRt15-16s21 serially

diluted at a 1:3 ratio was added and incubated at RT for 60 min. Following three washes,

50 ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL of

biotinylated-anti-IL-21 antibody (13-7218-81, R&D Systems), or 200 ng/mL of

biotinylated-anti-TGFBRII antibody (BAF241, R&D Systems) was applied per well, and

incubated at RT for 60 min. Following three washes, incubation with 0.25 ug/mL of

HRP-SA (Jackson ImmunoResearch at 100 uL per well for 30 min at RT was carried out,

followed by 4 washes and incubation with 100 uL of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 87A-87C, the IL-15, IL-21, and

TGFßRII domains in TGFRt15-16s21 were detected by the respective antibodies.

Purification elution chromatograph of TGFRt15-16s21 using anti-TF antibody affinity

column

TGFRt15-16s21 harvested from cell culture was loaded onto the anti-TF antibody

affinity column equilibrated with 5 column volumes of PBS. After sample loading, the

column was washed with 5 column volumes of PBS, followed by elution with 6 column

volumes of 1M acetic acid (pH 2.9). A280 elution peak was collected and then

neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer

exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight

cutoff. As shown in Figure 88, the anti-TF antibody affinity column bound to TGFRt15-

16s21, which contains tissue factor as a fusion partner. The buffer-exchanged protein

WO wo 2021/247604 PCT/US2021/035285

sample was stored at 2-8 °C for further biochemical analyses and biological activity tests.

After each elution, the anti-TF antibody affinity column was stripped using 6 column

volumes of .1M glycine (pH 2.5). The column was then neutralized using 5 column

volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF

antibody affinity column was connected to a GE Healthcare AKTA Avant system. The

flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE of TGFRt15-16s21

To determine the purity and molecular weight of the TGFRt15-16s21 protein,

protein sample purified with anti-TF antibody affinity column was analyzed by sodium

dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-

PAGE) under reduced condition. After electrophoresis, the gel was stained with

InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRt15-16s21 protein undergoes glycosylation after

translation in CHO cells, a deglycosylation experiment was conducted using the Protein

Deglycosylation Mix II kit from New England Biolabs according to the manufacturer's

instructions. Figure 89 shows results from the reduced SDS-PAGE analysis of the

sample in non-deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow

outline) state. The results showed that the TGFRt15-16s21 protein is glycosylated when

expressed in CHO cells. After deglycosylation, the purified sample showed expected

molecular weights (69 kDa and 48 kDa) in the reduced SDS gel. Lane M was loaded with

10uL of SeeBlue Plus2 Prestained Standard.

Example 52: 7t15-7s fusion protein generation and characterization

A fusion protein complex was generated comprising IL-7/TF/IL-15 and IL-7/IL-

15RaSu fusion proteins (Figure 90 and Figure 91). The human IL-7, tissue factor 219,

and IL-15 sequences were obtained from the UniProt website and DNA for these

sequences was synthesized by Genewiz. Specifically, a construct was made linking the

IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-

terminus coding region of IL-15.

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The nucleic acid and protein sequences of a construct comprising IL-7 linked to

the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown

below.

The nucleic acid sequence of 7t15 construct (including signal peptide sequence) is

as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT ATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGG

ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG TCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTG ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGO ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACAC' GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC wo WO 2021/247604 PCT/US2021/035285 PCT/US2021/035285

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCO TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leader sequence) is

as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKE

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKO FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTI ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQJ KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLOSFVHIVG LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by linking the IL-7 sequence to the N-terminus coding

region of IL-15RaSu chain which was synthesized by Genewiz. The nucleic acid and

protein sequences of a construct comprising the IL-7 linked to the N-terminus of IL-

15RaSu chain are shown below.

The nucleic acid sequence of 7s construct (including signal peptide sequence) is

as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAA TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA

GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGG0 GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTC AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of 7s fusion protein (including the leader sequence) is as

follows: follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(Human IL7)

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CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human IL-15R a sushi domain)

[TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR The IL-7/TF/IL-15 and IL-7/IL-15RaSu constructs were cloned into a modified

retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME,

Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a

marked antitumor response conveys highly active T-cell effector functions. Hum Gene

Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells.

Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion

of the soluble IL-7/TF/IL-15:IL-7/IL-15RaSu protein complex referred to as 7t15-7s,

which can be purified by anti-TF antibody IgG1 affinity and other chromatography

methods.

Purification elution chromatograph of 7t15-7s using anti-TF antibody affinity column

7t15-7s harvested from cell culture was loaded onto the anti-TF antibody affinity

column equilibrated with 5 column volumes of PBS. After sample loading, the column

was washed with 5 column volumes of PBS, followed by elution with 6 column volumes

of 0. 1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH

7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS

using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in

Figure 92, the anti-TF antibody affinity column bound to 7t15-7s which contains tissue

factor (TF) as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C

for further biochemical analyses and biological activity tests. After each elution, the anti-

TF antibody affinity column was stripped using 6 column volumes of 0. 1M glycine (pH

2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column

volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps

except the elution step, which was 2 mL/min.

Immunostimulation of 7t15-7s in C57BL/6 mice

7t15-7s is a multi-chain polypeptide (a type A multi-chain polypeptide described

herein) that includes the first polypeptide that is a soluble fusion of human IL-7, human

tissue factor 219 fragment and human IL-15 (7t15), and the second polypeptide that is a

soluble fusion of human IL-7 and sushi domain of human IL-15 receptor alpha chain (7s).

CHO cells were co-transfected with the IL7-TF-IL-15 (7t15) and IL7-IL-15Ra

sushi domain (7s) vectors. The 7t15-7s complex was purified from the transfected CHO

cell culture supernatant. The IL-7, IL-15 and tissue factor (TF) components were

demonstrated in the complex by ELISA as shown in Figure 93. A humanized anti-TF

antibody monoclonal antibody (anti-TF IgG1) was used as the capture antibody to

determine TF in 7t15-7s, and biotinylated anti-human IL-15 antibody (R&D systems) and

biotinylated anti-human IL-7 antibody (R&D Systems) were used as the detection

antibodies to respectively detect IL-15 and IL-7 in 7t15-7s, followed by peroxidase

conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics

IVD, Inc.).

7t15-7s was subcutaneously injected into C57BL/6 mice at 10 mg/kg to determine

the immunostimulatory activity of 7t15-7s in vivo. C57BL/6 mice subcutaneously

treated with PBS were used as control. The mouse spleens were collected and weighed

day 4 post treatment. Single splenocytes suspensions were prepared, and with

fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 antibodies and the

percentage of CD4+ T cells, CD8+ T cells, and NK cells was analyzed by flow cytometry.

The results showed that 7t15-7s was effective at expanding splenocytes based on spleen

weight (Figure 94A) and specifically, the percentages of CD8+ T cells and NK cells were

higher compared to control-treated mice (Figure 94B).

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Example 53: TGFRt15-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGFß Receptor II/IL-

15RaSu and TGFß Receptor II/TF/IL-15 fusion proteins (Figure 95 and Figure 96). The

human TGFB Receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were

obtained from the UniProt website and DNA for these sequences was synthesized by

Genewiz. Specifically, a construct was made linking two TGFB Receptor II sequences

with a G4S(3) linker to generate a single chain version of TGFB Receptor II and then

directly linking to the N-terminus coding region of tissue factor 219 followed by the N-

terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGFB

Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus

of IL-15 are shown below.

The nucleic acid sequence of the two TGFß Receptor II/TF/IL-15 construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Two Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGA0 GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG wo WO 2021/247604 PCT/US2021/035285

CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCO CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATC

TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC TACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTG ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAG ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT ACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAAT? CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC "TGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTO TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFß Receptor II/TF/IL-15 fusion protein (including

the leader sequence) is as follows: wo WO 2021/247604 PCT/US2021/035285

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS

TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC eSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFT FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI ESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVG LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS MFINTS

Constructs were also made by attaching two TGFB Receptor II directly to the IL- -

15RaSu chain which was synthesized by Genewiz. The nucleic acid and protein

sequences of a construct comprising the TGFB Receptor II linked to the N-terminus of

IL-15RaSu are shown below.

The nucleic acid sequence of the TGFß Receptor II/IL-15 RaSu construct

(including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC

(Two human TGFB Receptor II fragments)

629

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ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAA/ CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGA AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA AACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGA, CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATC TTAGCGAGGAATACAATACCAGCAACCCCGAC TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the two TGFß Receptor II/IL-15RaSu construct

(including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Two human TGFB Receptor II extra-cellular domains)

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDI WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD CNDNIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The TGF3R/IL-15RaSu and TGFBR/TF/IL-15 constructs were cloned into a

modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a

patient with a marked antitumor response conveys highly active T-cell effector functions.

Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-

K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation

and secretion of the soluble TGFBR/TF/IL-15:TGFBR/IL-15RaSu protein complex

(referred to as TGFRt15-TGFRs), which can be purified by anti-TF IgG1 affinity and

other chromatography methods.

Effect of TGFRt15-TGFRs on TGFB1 activity in HEK-Blue TGFB cells

To evaluate the activity of TGFßRII in TGFRt15-TGFRs, the effect of TGFRt15-

16s21 on the activity of TGFß1 in HEK-Blue TGFß cells was analyzed. HEK-Blue

TGFB cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the

testing medium (DMEM, 10% heat-inactivated FCS, 1x glutamine, 1x anti-anti, and 2x

glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 uL cells were added to

each well (2.5 X 104 cells/well) and followed with 50 uL 0. 1nM TGFß1 (R&D systems).

TGFRt15-16s21 or TGFR-Fc (R&D Systems) prepared at a 1:3 serial dilution was then

added to the plate to reach a total volume of 200 uL. After 24hrs of incubation at 37°C,

40 uL of induced HEK-Blue TGFß cell supernatant was added to 160 uL pre-warmed

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QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3

hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655

nM (Figure 97). The IC50 of each protein sample was calculated with GraphPad Prism

7.04. The IC50 of TGFRt15-TGFRs and TGFR-Fc were 216.9 pM and 460.6 pM

respectively. These results showed that the TGFßRII domain in TGFRt15-TGFRs was

able to block the activity of TGFB1 in HEK-Blue TGFB cells.

The IL-15 in TGFRt15-TGFRs promotes IL-2RB and common y chain containing 32DB

cell proliferation

To evaluate the activity of IL-15 in TGFRt15-TGFRs, the IL-15 activity of

TGFRt15-TGFRs was compared to recombinant IL-15 using 32DB cells that express

IL2RB and common chain, and evaluating their effects on promoting cell proliferation.

IL-15 dependent 32D cells were washed 5 times with IMDM-10% FBS and seeded in

the wells at 2 X 104 cells/well. Serially-diluted TGFRt15-TGFRs or IL-15 were added to

the cells (Figure 98). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell

proliferation was detected by adding 10 uL of WST1 to each well on day 3 and

incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450

nm was measured by analyzing the amount of formazan dye produced. As shown in

Figure 98, TGFR115-TGFRs and IL-15 promoted 32DB cell proliferation, with the EC50

of TGFRt15-16s21 and IL-15 being 1901 pM and 10.63 pM, respectively.

Detection of IL-15 and TGFBRII domains in TGFRt15-TGFRs with corresponding

antibodies using ELISA

A 96-well plate was coated with 100 uL (8 ug/mL) of anti-TF IgG1 in R5

(coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were

washed 3 times and blocked with 100 uL of 1% BSA in PBS. TGFRt15-TGFRs was

added at a 1:3 serial dilution, and incubated at RT for 60 min. After 3 washes, 50 ng/mL

of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), or 200 ng/mL of

biotinylated-anti-TGFbRII antibody (BAF241, R&D Systems) was added to the wells

and incubated at RT for 60 min. Next the plates were washed 3 times, and 0.25 ug/mL of

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HRP-SA (Jackson ImmunoResearch) at 100 uL per well was added and incubated for 30

min at RT, followed by 4 washes and incubation with 100 uL of ABTS for 2 mins at RT.

Absorbance at 405 nm was read. As shown in Figure 99A and 99B, the IL-15 and

TGFßRII domains in TGFRt15-TGFRs were detected by the individual antibodies.

Purification elution chromatograph of TGFRt15-TGFRs from anti-TF antibody affinity

column

TGFRt15-TGFRs harvested from cell culture was loaded onto the anti-TF

antibody affinity column equilibrated with 5 column volumes of PBS. After sample

loading, the column was washed with 5 column volumes of PBS, followed by elution

with 6 column volumes of 0. 1M acetic acid (pH 2.9). A280 elution peak was collected

and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then

buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular

weight cutoff. As shown in Figure 100, the anti-TF antibody affinity column bound to

TGFRt15-TGFRs which contains TF as a fusion partner. The buffer-exchanged protein

sample was stored at 2-8 °C for further biochemical analyses and biological activity tests.

After each elution, the anti-TF antibody affinity column was stripped using 6 column

volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column

volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF

antibody affinity column was connected to a GE Healthcare AKTA Avant system. The

flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Analytical size exclusion chromatography (SEC) analysis of TGFRt15-TGFRs

A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare)

was connected to an AKTA Avant system (from GE Healthcare). The column was

equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample

containing TGFRt15-TGFRs in PBS was injected into the Superdex 200 column using a

capillary loop, and analyzed by SEC. The SEC chromatograph of the sample is shown in

Figure 101. The SEC results showed four protein peaks for TGFRt15-TGFRs.

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Reduced SDS-PAGE analysis of TGFR+15-TGFRs

To determine the purity and molecular weight of the TGFRt15-TGFRs protein,

protein sample purified with anti-TF antibody affinity column was analyzed by sodium

dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-

PAGE) method under reduced condition. After electrophoresis, the gel was stained with

InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRt15-TGFRs protein undergoes glycosylation after

translation in CHO cells, a deglycosylation experiment was conducted using the Protein

Deglycosylation Mix II kit from New England Biolabs and the manufacturer's

instructions. Figure 102 shows the reduced SDS-PAGE analysis of the sample in non-

deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state.

The results showed that the TGFR115-TGFRs protein is glycosylated when expressed in

CHO cells. After deglycosylation, the purified sample showed expected molecular

weights (69 kDa and 39 kDa) in the reduced SDS gel. Lane M was loaded with 10 ul of

SeeBlue Plus2 Prestained Standard.

Immunostimulatory activity of TGFRt15-TGFRs in C57BL/61 mice

TGFRt15-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide

described herein) that includes a first polypeptide that is a soluble fusion of two TGFßRII

domains, human tissue factor 219 fragment and human IL-15, and the second polypeptide

that is a soluble fusion of two TGFBRII domains and sushi domain of human IL-15

receptor alpha chain.

Wild type C57BL/6 mice were treated subcutaneously with either control solution

or with TGFRt15-TGFRs at a dosage of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg. Four

days after treatment, spleen weight and the percentages of various immune cell types

present in the spleen were evaluated. As shown in Figure 103A, the spleen weight in

mice treated with TGFRt15-TGFRs increased with increasing dosage of TGFRt15-

TGFRs. Moreover, the spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and 10

mg/kg of TGFRt15-TGFRs were higher as compared to mice treated with the control

solution, respectively. In addition, the percentages of CD4+ T cells, CD8+ T cells, NK

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cells, and CD19+ B cells present in the spleen of control-treated and TGFRt15-TGFRs-

treated mice were evaluated. As shown in Figure 103B, in the spleens of mice treated

with TGFRt15-TGFRs, the percentages of CD8+ T cells and NK cells both increased with

increasing dosage of TGFRt15-TGFRs. Specifically, the percentages of CD8+ T cells

were higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs

compared to control-treated mice, and the percentages of NK cells were higher in mice

treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared

to control-treated mice. These results demonstrate that TGFRt15-TGFRs is able to

stimulate immune cells in the spleen, in particular CD8+ T cells and NK cells.

The pharmacokinetics of TGFRt15-TGFRs molecules were evaluated in wild type

C57BL/6 mice. The mice were treated subcutaneously with TGFRt15-TGFRs at a

dosage of 3 mg/kg. The mouse blood was drained from tail vein at various time points

and the serum was prepared. The TGFRt15-TGFRs concentrations in mouse serum was

determined with ELISA (capture: anti-human tissue factor antibody; detection:

biotinylated anti-human TGFB receptor antibody and followed by peroxidase conjugated

streptavidin and ABTS substrate). The results showed that the half-life of TGFRt15-

TGFRs was 12.66 hours in C57BL/6 mice.

The mouse splenocytes were prepared in order to evaluate the immunostimulatory

activity of TGFRt15-TGFRs over time in mice. As shown in Figure 104A, the spleen

weight in mice treated with TGFRt15-TGFRs increased 48 hours posttreatment and

continued to increase over time. In addition, the percentages of CD4+ T cells, CD8+ T

cells, NK cells, and CD19+ B cells present in the spleen of control-treated and TGFRt15-

TGFRs-treated mice were evaluated. As shown in Figure 104B, in the spleens of mice

treated with TGFRt15-TGFRs, the percentages of CD8+ T cells and NK cells both

increased at 48 hours after treatment and were higher and higher overtime after the single

dose treatment. These results further demonstrate that TGFRt15-TGFRs is able to

stimulate immune cells in the spleen, in particular CD8+ T cells and NK cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression

of splenocytes and cytotoxicity potential based on granzyme B expression were evaluated

in splenocytes isolated from mice following a single dose (3 mg/kg) of TGFRt15-TGFRs.

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As shown in Figure 105A and 105B, in the spleens of mice treated with TGFRt15-

TGFRs, the expression of Ki67 and granzyme B by NK cells increased at 24 hours after

treatment and its expression of CD8+ T cells and NK cells both increased at 48 hours and

later time points after the single dose treatment. These results demonstrate that

TGFRt15-TGFRs not only increases the numbers of CD8+ T cells and NK cells but also

enhance the cytotoxicity of these cells. The single dose treatment of TGFRt15-TGFRs

led CD8+ T cells and NK cells to proliferate for at least 4 days.

The cytotoxicity of the splenocytes from TGFRt15-TGFRs-treated mice against

tumor cells was also evaluated. Mouse Moloney leukemia cells (Yac-1) were labeled

with CellTrace Violet and were used as tumor target cells. Splenocytes were prepared

from TGFRt15-TGFRs (3 mg/kg)-treated mouse spleens at various time points post

treatment and were used as effector cells. The target cells were mixed with effector cells

at an E:T ratio = 10:1 and incubated at 37°C for 20 hours. Target cell viability was

assessed by analysis of propidium iodide positive, violet-labeled Yac-1 cells using flow

cytometry. Percentage of Yac-1 tumor inhibition was calculated using the formula, (1-

[viable Yac-1 cell number in experimental sample]/[viable Yac-1 cell number in the

sample without splenocytes]) X 100. As shown in Figure 106, splenocytes from

TGFRt15-TGFRs-treated mice had stronger cytotoxicity against Yac-1 cells than the

control mouse splenocytes.

Tumor size analysis in response to chemotherapy and/or TGFR15-TGFRs

Pancreatic cancer cells (SW1990, ATCC CRL-2172) were subcutaneously (s.c.)

injected into C57BL/6 scid mice (The Jackson Laboratory, 001913, 2x106 cells/mouse, in

100uL HBSS) to establish the pancreatic cancer mouse model. Two weeks after tumor

cell injection, chemotherapy was initiated in these mice intraperitoneally with a

combination of Abraxane (Celgene, 68817-134, 5 mg/kg, i.p.) and Gemcitabine (Sigma

Aldrich, G6423, 40 mg/kg, i.p.), followed by immunotherapy with TGFRt15-TGFRs (3

mg/kg, s.c.) in 2 days. The procedure above was considered one treatment cycle and was

repeated for another 3 cycles (1 cycle/week). Control groups were set up as the SW1990-

injected mice that received PBS, chemotherapy (Gemcitabine and Abraxane), or

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TGFRt15-TGFRs alone. Along with the treatment cycles, tumor size of each animal was

measured and recorded every other day, until the termination of the experiment 2 months

after the SW 1990 cells were injected. Measurement of the tumor volumes were analyzed

by group and the results indicated that the animals receiving a combination of

chemotherapy and TGFRt15-TGFRs had significantly smaller tumors comparing to the

PBS group, whereas neither chemotherapy nor TGFRt15-TGFRs therapy alone work as

sufficiently as the combination (Figure 107).

In vitro senescent B16F10 melanoma model

Next, in vitro killing of senescent B16F10 melanoma cells by activated mouse

NK cells was evaluated. B16F10 senescence cells (B16F10-SNC) cells were labelled

with CellTrace violet and incubated for 16 hrs with different E:T ratio of in vitro 2t2-

activated mouse NK cells (isolated from spleen of C57BL/6 mice injected with

TGFRt15-TGFRs10 mg/kg for 4 days). The cells were trypsinized, washed and

resuspended in complete media containing propidium iodide (PI) solution. The

cytotoxicity was assessed by flow cytometry (Figure 108).

Example 54: 7t15-21s137L (long version) fusion protein creation and

characterization

A fusion protein complex was generated comprising of IL-21/IL-

15RaSu/CD137L and IL-7/TF/IL-15 fusion proteins (Figure 109 and Figure 110).

Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding

region of tissue factor 219 followed by the N-terminus coding region of IL-15. The

nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-

terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7t15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

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(Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGC GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAA0 TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA

AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGC

AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTT ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGO ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAA ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT CATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGO AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

ACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCO TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG wo WO 2021/247604 PCT/US2021/035285

AGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leader sequence) is

as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN

KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL

ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYV ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVO LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid and protein sequences of the 21s137L are shown below. The

nucleic acid sequence of the 21s137L construct (including signal peptide sequence) is as

follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

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CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT CCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCAT ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGG GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGA' AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTC AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAG CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC TGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGA4 ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC

GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGG GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC GGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGG TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT ACTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCC GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG CAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21s137L fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCE QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF

QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPI QKAQLKSANTGNNERINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R sushi domain) (HumanIL-15Rasushidomain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The IL-21/IL-15RaSu/CD137L and IL-7/TF/IL-15 constructs were cloned into a

modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a

patient with a marked antitumor response conveys highly active T-cell effector functions.

Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-

K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation

and secretion of the soluble IL-7/TF/IL-15: IL-21/IL-15RaSu/CD137L protein complex

(referred to as 7t15-21s137L), which can be purified by anti-TF antibody IgG1 affinity

and other chromatography methods.

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Purification elution chromatograph of 7t15-21s137L using anti-TF antibody affinity

column

7t15-21s137L harvested from cell culture was loaded onto the anti-TF antibody

affinity column equilibrated with 5 column volumes of PBS. After sample loading, the

column was washed with 5 column volumes of PBS, followed by elution with 6 column

volumes of 0. 1M acetic acid (pH 2.9). A280 elution peak was collected and then

neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer

exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight

cutoff. As shown in Figure 111, the anti-TF antibody affinity column bound to 7t15-

21s137L which contains TF as a fusion partner. The buffer-exchanged protein sample

was stored at 2-8 °C for further biochemical analyses and biological activity tests. After

each elution, the anti-TF antibody affinity column was stripped using 6 column volumes

of 0. 1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of

PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity

column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4

mL/min for all steps except for the elution step, which was 2 mL/min. Figure 112 shows

the analytical SEC profile of 7t15-21s137L.

Example 55: 7t15-21s137L (short version) fusion protein generation and

characterization

A fusion protein complex was generated comprising of IL-21/IL-

15RaSu/CD137L and IL-7/TF/IL-15 fusion proteins. Specifically, a construct was made

linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed

by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a

construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with

the N-terminus of IL-15 are shown below.

The nucleic acid sequence of 7t15 construct (including signal peptide sequence) is

as follows:

(Signal peptide)

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ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGC ATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC `GCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAAC AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT ACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTO ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA CAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGO ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTT

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGA AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT TATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC

TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT wo WO 2021/247604 PCT/US2021/035285

TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leader sequence) is

as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL EAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP) TNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY) ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE

(Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS MFINTS

The nucleic acid and protein sequences of the 21s137L (short version) are shown

below. The nucleic acid sequence of 21s137L (short version) construct (including signal

peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT

ACTCC wo 2021/247604 WO PCT/US2021/035285

(Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT GTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCC GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC

AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGC GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATO CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTC ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137 Ligand short version)

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTO GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTG GTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACC GTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACC CAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACA CAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACA AAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACT CGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTA GAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGC GAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGC GCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGAC TGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACC GTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCC GTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCA

GGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCA0 GGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCAC ACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCT TGGGACTCTTCCGGGTGACCCCCGAAATC

The amino acid sequence of the 21s137L (short version) construct (including

signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF

QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPJ QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R sushi domain) (HumanIL-15Rasushidomain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA1 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137 Ligand short version)

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL TVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEI

The IL-21/IL-15RaSu/CD137L (short version) and IL-7/TF/IL-15 constructs were

cloned into a modified retrovirus expression vectors as described previously (Hughes

MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene

derived from a patient with a marked antitumor response conveys highly active T-cell

effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of the soluble IL-7/TF/IL-15: IL-21/IL-

15RaSu/CD137L protein complex (referred to as 7t15-21s137L (short version)), which

can be purified by anti-TF antibody IgG1 affinity and other chromatography methods.

Binding of 7t15-21s137L (short version) to CD137 (4.1BB)

On day 1, a 96-well plate was coated with 100 uL (2.5 ug/mL) of GAH IgG Fc

(G-102-C, R&D Systems) in R5 (coating buffer) or R5 only and incubated at 4°C,

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overnight. On day 2, the plates were washed three times and blocked with 300 uL of 1%

BSA in PBS at 37°C for 2 hrs. 10 ng/mL of 4.1BB/Fc (838-4B, R&D Systems) was

added at 100 uL/well and incubated for 2 hrs at RT. After three washes, the 7t15-

21s137L or 7t15-21s serially diluted at a 1/3 ratio (starting at 10 nM), and incubated at

4°C overnight. On day 3, following 3 washes, 300 ng/mL of biotinylated-anti-hTF

antibody (BAF2339, R&D Systems) was added at 100 uL per well and incubated at RT

for 2 hrs. The plate was then washed three times and incubated with 0.25 ug/mL of HRP-

SA (Jackson ImmuneResearch) at 100 uL per well for 30 min, followed by 3 washes and

incubation with 100 uL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As

shown in Figure 113, 7t15-21s137L (short version) showed significant interaction with

4. 1BB/Fc (blue line) as compared to 7t15-21s.

Detection of IL-15, IL-21, and IL-7 in 7t15-21s137L (short version) with ELISA

A 96-well plate was coated with 100 uL (8 ug/mL) of anti-TF antibody IgG1 in

R5 (coating buffer) and incubated at RT for 2 hrs. The plates were washed 3 times and

blocked with 100 uL of 1% BSA in PBS. 7t15-21s137L (short version), serially diluted

at a 1:3 ratio was added, and incubated at RT for 60 min. After three washes, 50 ng/mL

of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL of

biotinylated-anti-IL21 antibody (13-7218-81, R&D Systems), or 500 ng/mL of

biotinylated-anti-IL7 antibody (506602, R&D Systems) was added to the wells and

incubated at RT for 60 min. After three washes and incubation with 0.25 ug/mL of HRP-

SA (Jackson ImmunoResearch) at 100 uL per well was carried out for 30 min at RT,

followed by four washes and incubation with 100 uL of ABTS for 2 mins at RT.

Absorbance was read at 405 nm. As shown in Figures 114A-114C, the IL-15, IL-21, and

IL-7 domains in 7t15-21s137L (short version) were detected by the respective antibodies.

The IL-15 in 7t15-1s137L (short version) promotes IL2RaBy containing CTLL2 cell

proliferation

To evaluate the IL-15 activity of 7t15-21s137L (short version), 7t15-21s137L

(short version) was compared with recombinant IL-15 in promoting proliferation of

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IL2Rapy expressing CTLL2 cells. IL-15-dependent CTLL2 cells were washed 5 times

with IMDM-10% FBS and seeded to the wells at 2 X 104 cells/well. Serially diluted 7t15-

21s137L (short version) or IL-15 were added to the cells (Figure 115). Cells were

incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by

adding 10 uL of WST1 to each well on day 3 and incubated for an additional 3 hours in a

CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by

measuring the absorbance at 450 nm. As shown in Figure 115, 7t15-21s137L (short

version) and IL-15 promoted CTLL2 cell proliferation. The EC50 of 7t15-21s137L (short

version) and IL-15 was 55.91 pM and 6.22 pM. respectively.

The IL-21 in 7t15-1s137L (short version) promotes IL21R containing B9 cell

proliferation

To evaluate the IL-21 activity of 7t15-21s137L (short version), 7t15-21s137L

(short version) was compared with recombinant IL-21 in promoting proliferation of IL-

21R expressing B9 cells. IL-21R containing B9 cells were washed 5 times with RPMI-

10% FBS and seeded to the wells at 1 X 104 cells/well. Serially diluted 7t15-21s137L (short

version) or IL-21 were added to the cells (Figure 116). Cells were incubated in a CO2

incubator at 37°C for 5 days. Cell proliferation was detected by adding 10 uL of WST1 to

each well on day 5 and incubated for an additional 4 hours in a CO2 incubator at 37°C. The

amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm.

As shown in Figure 116, 7t15-21s137L (short version) and IL-21 promoted B9 cell

proliferation. The EC50 of 7t15-21s137L (short version) and IL-21 was 104.1 nM and

72.55 nM. respectively.

Example 56: 7t15-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGFB Receptor II/IL-

15RaSu and IL-7/TF/IL-15 fusion proteins (Figure 117 and Figure 118). The human TGFß

Receptor II (Ile24-Asp159), tissue factor 219, IL-15, and IL-7 sequences were obtained

from the UniProt website and DNA for these sequences was synthesized by Genewiz.

Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding

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region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic

acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of

tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7t15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAA TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGG

ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGO ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGG CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACAC' AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC 649 wo WO 2021/247604 PCT/US2021/035285 PCT/US2021/035285

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCO TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leader sequence) is

as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKE

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKO FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTI ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQJ KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLOSFVHIVG LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVO MFINTS

Constructs were also made by attaching two TGFß Receptor II directly to the IL-

15RaSu chain which was synthesized by Genewiz. The nucleic acid and protein

sequences of a construct comprising the TGFB Receptor II linked to the N-terminus of

IL-15RaSu are shown IL-15RSu are shown below. below.

The nucleic acid sequence of the TGFRs construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGAC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC ACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATO GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA AAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCC GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC IGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACO CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCC AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATC TTAGCGAGGAATACAATACCAGCAACCCCGAC TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

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AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of TGFRs fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDI CNDNIIFSEEYNTSNPD (Human IL-15Rasushidomain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

Effect of 7t15-TGFRs on TGFB1 activity in HEK-Blue TGFB cells

To evaluate the activity of TGFßR in 7t15-TGFRs, the effect of 7t15-TGFRs on

the activity of TGFß1 in HEK-Blue TGFß cells was analyzed. HEK-Blue TGFB cells

(Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing

medium (DMEM, 10% heat-inactivated FCS, 1x glutamine, 1x anti-anti, and 2x

glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 uL cells were added to

each well (2.5 X 104 cells/well) and followed with 50 uL 0. 1nM TGFB1 (R&D systems).

7t15-TGFRs or TGFR-Fc (R&D Systems) prepared at a1:3 serial dilution was then added

to the plate to reach a total volume of 200 uL. After 24hrs of incubation at 37°, 40 uL

of induced HEK-Blue TGFB cell supernatant was added to 160 uL pre-warmed

QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3

hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. The data are shown in Figure 119. The IC50 of each protein sample was calculated with GraphPad Prism 7.04. The IC50 of 7t15-TGFRs and TGFR-Fc were 1142 pM and

558.6 pM respectively. These results showed that the TGFßR in 7t15-TGFRs was able to

block the activity of TGFß1 in HEK-Blue TGFß cells.

Detection of IL-15, TGFBRII, and IL-7 in 7t15-TGFRs with ELISA

A 96-well plate was coated with 100 uL (8 ug/mL) of anti-TF antibody IgG1 in

R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were

washed three times and blocked with 100 uL of 1% BSA in PBS. Serial dilution of 7t15-

TGFRs (1:3 ratio) was added, and incubated at RT for 60 mins. After 3 washes, 50

ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 200 ng/mL of

biotinylated-anti-TGFbRII antibody (BAF241, R&D Systems), or 500 ng/mL of

biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added and incubated at RT

for 60 min. Following three washes, incubation with 0.25 ug/mL of HRP-SA (Jackson

ImmunoResearch) at 100 uL per well was carried out for 30 min at RT, followed by 4

washes and incubation with 100 uL of ABTS for 2 mins at RT. Absorbance was read at

405 nm. As shown in Figures 120A-120C, the IL-15, TGFR, and IL-7 in 7t15-TGFRs

were detected by the respective antibodies.

The IL-15 in 7t15-TGFRs promotes IL-2RB and common y chain containing 32DB cell

proliferation

To evaluate the activity of IL-15 in 7t15-TGFRs, 7t15-TGFRs was compared to

recombinant IL-15 using 32DB cells that express IL2RB and common Y chain, and

evaluating their effects on promoting cell proliferation. IL-15 dependent 32DB cells were

washed 5 times with IMDM-10% FBS and seeded in the wells at 2 X 104 cells/well.

Serially-diluted 7t15-TGFRs or IL-15 were added to the cells (Figure 121). Cells were

incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by

adding 10 uL of WST1 to each well on day 3 and incubating for an additional 3 hours in

a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by

measuring the absorbance at 450 nm. As shown in Figure 121, 7t15-TGFRs and IL-15

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promoted 32DB cell proliferation, with the EC50 of 7t15-TGFRs and IL-15 being 126 nM

and 16.63 pM, respectively.

Purification elution chromatograph of 7t15-TGFRs using anti-TF antibody affinity column

7t15-TGFRs harvested from cell culture was loaded onto the anti-TF antibody

affinity column equilibrated with 5 column volumes of PBS. After sample loading, the

column was washed with 5 column volumes of PBS, followed by elution with 6 column

volumes of 0. 1M acetic acid (pH 2.9). A280 elution peak was collected and then

neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer

exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight

cutoff. As shown in Figure 122, the anti-TF antibody affinity column can bind 7t15-

TGFRs which contains TF as a fusion partner of 7t15-TGFRs. The buffer-exchanged

protein sample was stored at 2-8 °C for further biochemical analyses and biological

activity tests. After each elution, the anti-TF antibody affinity column was stripped using

6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5

column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF

antibody affinity column was connected to a GE Healthcare AKTA Avant system. The

flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of 7t15-TGFRs

To determine the purity and molecular weight of the protein, 7t15-TGFRs protein

sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl

sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE)

method under reduced condition. After electrophoresis, the gel was stained with

InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the 7t15-TGFRs protein undergoes glycosylation after translation in

CHO cells, a deglycosylation experiment was conducted using the Protein

Deglycosylation Mix II kit from New England Biolabs and the manufacturer's

instructions. Figure 123 shows reduced SDS-PAGE analysis of the sample in non-

deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state.

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These results showed that the protein is glycosylated when it is expressed in CHO cells.

After deglycosylation, the purified sample showed expected molecular weights (55 kDa

and 39 kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2

Prestained Standard.

Characterization of 7t15-TGFRs

7t15-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide

described herein) that includes the first polypeptide that is a soluble fusion of human IL-7,

human tissue factor 219 fragment and human IL-15 (7t15), and the second polypeptide that

is a soluble fusion of single chain two TGFßRII domains and sushi domain of human IL-

15 receptor alpha chain (TGFRs).

CHO cells were co-transfected with 7t15 and TGFRs vectors. The 7t15-TGFRs

complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL-

15, TGFB receptor and tissue factor (TF) components were demonstrated in the complex

by ELISA as shown in Figure 124. A humanized anti-TF antibody monoclonal antibody

(anti-TF antibody IgG1) was used as the capture antibody to determine TF in 7t15-

TGFRs, and biotinylated antibodies against human IL-15 (R&D systems), human IL-7

(Biolegend), anti-TGFB receptor (R&D Systems) were used as the detection antibodies to

respectively determine IL-7, IL-15 and TGFB receptor in 7t15-TGFRs. Peroxidase

conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics

IVD, Inc.) were then used to detect the bound biotinylated antibodies. The results were

analyzed by ELISA (Figure 124).

In vivo characterization of 7t15-TGFRs in C57BL/6 mice

To determine the immunostimulatory activity of 7t15-TGFRs in vivo, C57BL/6

mice were subcutaneously treated with control solution (PBS) or 7t15-TGFRs at 0.3, 1, 3

and 10 mg/kg. The treated mice were euthanized. The mouse spleens were collected and

weighed day 4 post treatment. Single splenocyte suspensions were prepared and stained

with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 antibodies and the

percentage of CD4+ T cells, CD8+ T cells, and NK cells was analyzed by flow cytometry.

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The results showed that 7t15-TGFRs was effective at expanding splenocytes based on

spleen weight (Figure 125A), especially at 1-10 mg/kg. The percentages of CD8+ T cells

and NK cells were higher compared to control-treated mice (Figure 125B) at all doses

tested.

CD44 Expression of CD4+ and CD8+ T cells

It has been known that IL-15 induces CD44 expression on T cells and

development of memory T cells. CD44 expression of CD4+ and CD8+ T cells in the

7t15-TGFRs treated mice were assessed. C57BL/6 mice were subcutaneously treated

with 7t15-TGFRs. The splenocytes were stained with fluorochrome-conjugated anti-

CD4, anti-CD8 and anti-CD44 monoclonal antibodies for immunocyte subsets. The

percentages of CD4*CD44high T cells of total CD4+ T cells and CD8*CD44high T cells of

total CD8+ T cells were analyzed by flow cytometry. As shown in Figures 126A and

126B, 7t15-TGFRs significantly activated CD4+ and CD8+ T cells to differentiate into

memory T cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression

of splenocytes and cytotoxicity potential based on granzyme B expression of the

splenocytes induced by 7t15-TGFRs after the single dose treatment of mouse were also

evaluated. C57BL/6 mice were subcutaneously treated with 7t15-TGFRs at 3 mg/kg. The

treated mice were euthanized and the splenocytes were prepared. The prepared

splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-

NK1.1 (NK) antibodies for immunocyte subsets and then intracellularly stained with anti-

Ki67 antibody for cell proliferation and anti-granzyme B antibody for cytotoxic marker.

The mean fluorescent intensity (MFI) of Ki67 and granzyme B of corresponding

immunocyte subsets was analyzed by flow cytometry. As shown in Figures 127A and

127B, in the spleens of mice treated with 7t15-TGFRs, the expression of Ki67 and

granzyme B by CD8+ T cells and NK cells increased compared with PBS control

treatment. These results demonstrate that 7t15-TGFRs is not only to increase numbers of

CD8+ T cells and NK cells but also enhance potential cytotoxicity of these cells.

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Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also

evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor

target cells. The splenocytes were prepared from 7t15-TGFRs-treated mice and used as

effector cells. The target cells were mixed with effector cells at E:T ratio = 10:1 in

RPMI-10 medium with or without 7t15-TGFRs at 100 nM and incubated at 37°C for 20

hours. Target Yac-1 cell inhibition was assessed by analysis of viable violet-labeled Yac-1

cells using flow cytometry. Percentage of Yac-1 inhibition was calculated using a

formula, (1-viable Yac-1 cell number in experimental sample/viable Yac-1 cell number in

the sample without splenocytes) X 100. As shown in Figure 128, 7t15-TGFRs-treated

mouse splenocytes had stronger cytotoxicity against Yac-1 cells than the control mouse

splenocytes and addition of 7t15-TGFRs during cytotoxic assay further enhanced

cytotoxicity of splenocytes against Yac-1 target cells.

Example 57: TGFRt15-21s137L fusion protein generation and characterization

A fusion protein complex was generated comprising IL-21/IL-15RaSu/CD137L

and TGFß Receptor II/TF/IL-15 fusion proteins (Figure 129 and Figure 130). The human

TGFB Receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained

from the UniProt website and DNA for these sequences was synthesized by Genewiz.

Specifically, a construct was made linking two TGFB Receptor II sequences with a

G4S(3) linker to generate a single chain version of TGFB Receptor II and then directly

linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus

coding region of IL-15.

The nucleic acid sequence of the TGFRt15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC

GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA wo 2021/247604 WO PCT/US2021/035285

GTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTAT ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA GTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATC GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT

TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG ACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAG ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAG

AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15) wo WO 2021/247604 PCT/US2021/035285

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTO CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT7 TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT

AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVESYPAGNVESTGSAGEPLYENSPEFTPYL YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLOSFVHIVG LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS wo 2021/247604 WO PCT/US2021/035285

The nucleic acid and protein sequences of the 21s137L are shown below. The

nucleic acid sequence of the 21s137L construct (including signal peptide sequence) is as

follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCC7 ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT AGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA GCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATO AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT

CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA

CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGO ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC

CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGA ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC GGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCC AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGC AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA wo WO 2021/247604 PCT/US2021/035285

GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCC< GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21s137L fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCI QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS EFLERFKSLLQKMIHOHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The IL-21/IL-15RaSu/CD137L and TGFR/TF/IL-15 constructs were cloned into

a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a

patient with a marked antitumor response conveys highly active T-cell effector functions.

661

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Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-

K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation

and secretion of the soluble TGFR/TF/IL-15: IL-21/IL-15RaSu/CD137L protein complex

(referred to as TGFRt15-21s137L), which can be purified by anti-TF antibody IgG1

affinity and other chromatography methods.

Purification elution chromatograph of TGFR115-21s137L using anti-TF antibody affinity

column

TGFRt15-21s137L harvest from cell culture was loaded onto the anti-TF antibody

affinity column equilibrated with 5 column volumes of PBS. After sample loading, the

column was washed with 5 column volumes of PBS, followed by elution with 6 column

volumes of 0. 1M acetic acid (pH 2.9). A280 elution peak was collected and then

neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then buffer

exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight

cutoff. As shown in Figure 131, the anti-TF antibody affinity column bound to TGFRt15-

21s137L which contains TF as a fusion partner of TGFRt15-21s137L. The buffer-

exchanged protein sample was stored at 2-8 °C for further biochemical analyses and

biological activity tests. After each elution, the anti-TF antibody affinity column was

stripped using 6 column volumes of 0. 1M glycine (pH 2.5). The column was then

neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for

storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA

Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which

was 2 mL/min.

Example 58: TGFRt15-TGFRs21 fusion protein generation and characterization

A fusion protein complex was generated comprising of TGFB Receptor II/IL-

15Ra.Su/IL-21 and TGFß Receptor II/TF/IL-15 fusion proteins (Figure 132 and Figure

133). The human TGF Receptor II (Ile24-Asp159), tissue factor 219, IL-21, and IL-15

sequences were obtained from the UniProt website and DNA for these sequences was

synthesized by Genewiz. Specifically, a construct was made linking two TGFB Receptor

WO wo 2021/247604 PCT/US2021/035285

II sequences with a G4S(3) linker to generate a single chain version of TGFB Receptor II

and then directly linking to the N-terminus coding region of tissue factor 219 followed by

the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF

Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus

of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTG

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCC< GTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCG CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG

CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACO TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC

(Human Tissue Factor 219) wo WO 2021/247604 PCT/US2021/035285

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT IACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAG ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTT ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGO CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCG7 TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAAT' CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTO TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT7 TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG

GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTO

The amino acid sequence of TGFRt15 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN wo 2021/247604 WO PCT/US2021/035285

NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPY, FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGFß Receptor II directly to the IL-

15RaSu chain, followed by the N-terminus coding region of IL-21, which was

synthesized by Genewiz. The nucleic acid and protein sequences of a construct

comprising the TGFB Receptor II linked to the N-terminus of IL-15RaSu following with

the N-terminus of IL-21 are shown below.

The nucleic acid sequence of the TGFRs21 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACO ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC TCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT

GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC wo 2021/247604 WO PCT/US2021/035285

ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCO GTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGC CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCG GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA eGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG

AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT CCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCO CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of TGFRs21 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK

GETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN ENDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCE QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPI QKAQLKSANTGNNERINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into a

modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a

patient with a marked antitumor response conveys highly active T-cell effector functions.

Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-

K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation

and secretion of the soluble TGFR/TF/IL-15:TGFR/IL-15RaSu/IL-21 protein complex

(referred to as TGFRt15-TGFRs21), which can be purified by anti-TF antibody IgG1

affinity and other chromatography methods.

667

Purification elution chromatograph of TGFRt15-TGFRs21 using anti-TF antibody

affinity column

TGFRt15-TGFRs21 harvested from cell culture was loaded onto the anti-TF

antibody affinity column equilibrated with 5 column volumes of PBS. After sample

loading, the column was washed with 5 column volumes of PBS, followed by elution

with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected

and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample was then

buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular

weight cutoff. As shown in Figure 134, the anti-TF antibody affinity column bound to

TGFRt15-TGFRs21 which contains TF as a fusion partner. The buffer-exchanged protein

sample was stored at 2-8 °C for further biochemical analyses and biological activity tests.

After each elution, the anti-TF antibody affinity column was stripped using 6 column

volumes of 0.1M glycine (pH 2 2.5). The column was then neutralized using 5 column

volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF

antibody affinity column was connected to a GE Healthcare AKTA Avant system. The

flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of TGFR115-TGFRs21

To determine the purity and molecular weight of the protein, TGFRt15-TGFRs21

protein sample purified with anti-TF antibody affinity column was analyzed by sodium

dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-

PAGE) method under reduced condition. After electrophoresis, the gel was stained with

InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRt15-TGFRs21 protein undergoes glycosylation after

translation in CHO cells, a deglycosylation experiment was conducted using the Protein

Deglycosylation Mix II kit from New England Biolabs and the manufacturer's

instructions. Figure 135 shows the reduced SDS-PAGE analysis of the sample in non-

deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state.

It is clear that the protein is glycosylated when it is expressed in CHO cells. After

deglycosylation, the purified sample showed expected molecular weights (69 kDa and 55

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained

Standard.

Immunostimulation of TGFRt15-TGFRs21 in C57BL/6 mice

TGFRt15-TGFRs21 is a multi-chain polypeptide (a type A multi-chain

polypeptide described herein) that includes the first polypeptide that is a soluble fusion of

single chain two TGFßRII domains, human tissue factor 219 fragment and human IL-15

(TGFRt15), and the second polypeptide that is a soluble fusion of single chain two

TGFßRII domains, sushi domain of human IL-15 receptor alpha chain and human IL-21

(TGFRs21).

CHO cells were co-transfected with TGFRt15 and TGFRs21 vectors. The

TGFRt15-TGFRs21 complex was purified from the transfected CHO cell culture

supernatant. The TGFB receptor, IL-15, IL-21 and tissue factor (TF) components were

demonstrated in the complex by ELISA as shown in Figure 136. A humanized anti-TF

monoclonal antibody (anti-TF IgG1) was used as the capture antibody to determine TF in

TGFRt15-TGFRs21, biotinylated anti-human IL-15 antibody (R&D systems),

biotinylated anti-human TGFB receptor antibody (R&D systems, and biotinylated anti-

human IL-21 antibody (R&D Systems) were used as the detection antibodies to

respectively determine IL-15, TGFß receptor, and IL-21 in TGFRt15-TGFRs21. For

detection, peroxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS

were used.

Wild type C57BL/6 mice were treated subcutaneously with either control solution

(PBS) or with TGFRt15-TGFRs21 at 3 mg/kg. Four days after treatment, spleen weight

and the percentages of various immune cell types present in the spleen were evaluated.

As shown in Figure 137A, the percentages of CD4+ T cells, CD8+ T cells, and NK cells

present in the spleen of control-treated and TGFRt15-TGFRs21-treated mice were

evaluated. The dynamic proliferation of immune cells based on Ki67 expression after

TGFRt15-TGFRs21 treatment was also evaluated. The splenocytes were stained with

fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies and then

intracellularly stained with anti-Ki67 antibody. The percentage of CD4+ T cells, CD8+ T

WO wo 2021/247604 PCT/US2021/035285

cells, and NK cells and the mean fluorescent intensity (MFI) of Ki67 of corresponding

immunocyte subsets were analyzed by flow cytometry (Figures 137A and 137B).

Furthermore, cytotoxicity potential based on granzyme B expression of the splenocytes

induced by TGFRt15-TGFRs21 after the single dose treatment of mouse was also

evaluated. As shown in Figure 138, in the spleens of mice treated with TGFRt15-

TGFRs21, the expression of granzyme B by NK cells increased after treatment. The

splenocytes from TGFRt15-TGFRs21-treated mice were stained with fluorochrome-

conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies and then intracellularly

stained with anti-granzyme B antibody. The mean fluorescent intensity (MFI) of

granzyme B of corresponding immunocyte subsets was analyzed by flow cytometry

(Figure 138).

As shown in Figure 137A, in the spleens of mice treated with TGFRt15-

TGFRs21, the percentages of CD8+ T cells and NK cells both increased on day 4 after a

single TGFRt15-TGFRs21 treatment. These results demonstrate that TGFRt15-

TGFRs21 is able to induce immune cells to proliferate in mouse spleen, in particular

CD8+ T cells and NK cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also

evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor

target cells. The splenocytes were prepared from TGFRt15-TGFRs21-treated mice and

used as effector cells. The target cells were mixed with effector cells at E:T ratio = 10:1

in RPMI-10 medium with or without TGFRt15-TGFRs21 at 100 nM and incubated at

37°C for 24 hours. Target Yac-1 cell inhibition was assessed by analysis of viable violet-

labeled Yac-1 cells using flow cytometry. Percentage of Yac-1 inhibition was calculated

using a formula, (1-[viable Yac-1 cell number in experimental sample]/[viable Yac-1 cell

number in the sample without splenocytes]) X 100. As shown in Figure 139, TGFRt15-

TGFRs21-treated mouse splenocytes had stronger cytotoxicity against Yac-1 cells than

the control mouse cells in the presence of TGFRt15-TGFRs21 during cytotoxic assay

(Figure 139).

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Example 59: TGFRt15-TGFRs16 fusion protein generation

A fusion protein complex was generated comprising of TGFß Receptor II/IL-

15RaSu/ anti-CD16scFv and TGFß Receptor II/TF/IL-15 fusion proteins (Figure 140 and

Figure 141). The human TGFß Receptor II (Ile24-Asp159), tissue factor 219, and IL-15

sequences were obtained from the UniProt website and DNA for these sequences was

synthesized by Genewiz. Specifically, a construct was made linking two TGFB Receptor

II sequences with a G4S(3) linker to generate a single chain version of TGFß Receptor II

and then directly linking to the N-terminus coding region of tissue factor 219 followed by

the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGFB

Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus

of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFB Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA

CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG wo 2021/247604 WO PCT/US2021/035285

CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCO CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGG AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATC GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT

TTAGCGAGGAATACAATACCAGCAACCCCGAC TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT ACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTG ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAG ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT STATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACAC GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA ACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTG AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTO TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leader

sequence) is as follows: wo 2021/247604 WO PCT/US2021/035285

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGFB Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI

TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD CNDNIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFT TNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY) ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS ESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGFB Receptor II directly to the IL- -

15RaSu chain, followed by the anti-CD16scFv sequence, which was synthesized by

Genewiz. The nucleic acid and protein sequences of a construct comprising the TGFB

Receptor II linked to the N-terminus of IL-15RaSu following with the anti-CD16scFv

sequence are shown below.

The nucleic acid sequence of the TGFRs16 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC

WO wo 2021/247604 PCT/US2021/035285

(Human TGF B Receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGAC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTC. GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAA. CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCAC CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC

GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TAGCGAGGAATACAATACCAGCAACCCCGAC TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGT ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTC. AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGAC TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACA/ ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACC CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTO CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG wo WO 2021/247604 PCT/US2021/035285

CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGG4 CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCC2 GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGT

GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

The amino acid sequence of TGFRs16 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGF B Receptor 1 II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKE TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK GETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVI PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE

CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Anti-human CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYG WNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/anti-CD16scFv TGFR/IL-15RqSu/anti-CD16scFv and TGFR/TF/IL-15 constructs were

cloned into a modified retrovirus expression vectors as described previously (Hughes

MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene

derived from a patient with a marked antitumor response conveys highly active T-cell

effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were

transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells

allowed for formation and secretion of the soluble TGFR/TF/IL-15:TGFR/IL-

15RaSu/anti-CD16scFv protein complex (referred to as TGFRt15-TGFRs16), which can

be purified by anti-TF IgG1 affinity and other chromatography methods.

Example 60: The TGFRt15-TGFRs137L fusion protein generation

A fusion protein complex was generated comprising of TGFB Receptor II/IL-

15RaSu/ CD137L and TGFß Receptor II/TF/IL-15 fusion proteins (Figure 142 and

Figure 143). The human TGFß Receptor II (Ile24-Asp159), tissue factor 219, CD137L,

and IL-15 sequences were obtained from the UniProt website and DNA for these

sequences was synthesized by Genewiz. Specifically, a construct was made linking two

TGFß Receptor II sequences with a G4S(3) linker to generate a single chain version of

TGFß Receptor II and then directly linking to the N-terminus coding region of tissue

factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGFB

Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus

of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFBReceptor II fragments)

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ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTC GTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAC AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCC AACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCO CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTO TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT ACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA CAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA

CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACAC GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC wo WO 2021/247604 PCT/US2021/035285

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGA (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAA AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGF B Receptor II)

PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKE PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVI PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN MIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV. NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (HumanTissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP) FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

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NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV MFINTS

Constructs were also made by attaching two TGFß Receptor II directly to the IL-

15RaSu chain, followed by a (G4S)3 linker and the CD137L sequence, which was

synthesized by Genewiz. The nucleic acid and protein sequences of a construct

comprising the TGFB Receptor II linked to the N-terminus of IL-15RaSu following with

a (G4S)3 linker and the CD137L sequence are shown below.

The nucleic acid sequence of the TGFRs137L construct (including signal peptide

sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCC7 ACTCC (Human TGFBReceptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG TGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCO GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA GTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCG GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACC CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTG

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA 679 wo 2021/247604 WO PCT/US2021/035285

GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTO ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGO CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCC CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGA ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCC AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCG. GCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGC GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTO GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of TGFRs137L fusion protein (including the leader

sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human TGF B Receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVI

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INDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to

generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/CD137L TGFR/IL-15RuSu/CD137L and TGFR/TF/IL-15 constructs were cloned into

a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a

patient with a marked antitumor response conveys highly active T-cell effector functions.

Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into

CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for

formation and secretion of the soluble TGFR/TF/IL-15:TGFR/IL-15RaSu/CD137L

protein complex (referred to as TGFRt15-TGFRs137L), which can be purified by anti-TF

IgG1 affinity and other chromatography methods.

Example 61. Production and characterization of the Exemplary Single-Chain

Chimeric Polypeptide 2t2

An exemplary single-chain chimeric polypeptide including a first target-binding

domain that binds to an IL-2 receptor, a soluble human tissue factor domain, and a

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second target-binding domain that binds to an IL-2 receptor was generated (IL-2/TF/IL-2;

referred to as 2t2) (Figure 144). The nucleic acid and amino acid sequences of this

single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide (IL-2/TF/IL-

2) (SEQ ID NO: 164)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (First IL-2 fragment)

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCAT TTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACO TTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACC CCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCAC CGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAG CGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAG

GTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAAT CAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTO ATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTT ATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTT GGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC (Human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT TACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTG ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGO ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC wo WO 2021/247604 PCT/US2021/035285

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGA (Second IL-2 fragment)

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCAT GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGC. TACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAAT TTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCC CAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACA CAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACA GAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAG TGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAAT TGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAAT CAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTC ATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGAT GGATTACCTTTTGTCAAAGCATCATCTCAACACTAAC GGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT

Exemplary Single-Chain Chimeric Polypeptide (IL-2/TF/IL-2) (SEQ ID NO: 163)

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-2)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE, TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFCQSIISTLT

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP) FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE (Human IL-2)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE, TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFCQSISTLT YADETATIVEFLNRWITFCQSIISTLT wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285

The nucleic acid encoding IL-2/TF/IL-2 was cloned into a modified retrovirus

expression vector as described previously (Hughes et al., Hum Gene Ther 16:457-72,

2005). The expression vector encoding IL-2/TF/IL-2 was transfected into CHO-K1 cells.

Expression of the expression vector in CHO-K1 cells allowed for secretion of the soluble

IL-2/TF/IL-2 single-chain chimeric polypeptide (referred to as 2t2), which can be

purified by anti-TF antibody affinity and other chromatography methods.

IL-2 and 2t2 promoted IL-2RB and common chain containing 32DB cell proliferation in

a similar manner

To evaluate the IL-2 activity of 2t2, 2t2 was compared with recombinant IL-2 for

promoting proliferation of 32DB cells that express IL-2RB and common Y chain. IL-2

dependent 32DB cells were washed 5 times with IMDM-10% FBS and seeded to the

wells at 2 X 104 cells/well. Serial dilutions of 2t2 or IL-2 were added to the cells (Figure

145). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was

detected by adding 10 ul of WST1 to each well on day 3 and incubating for an additional

3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed

by measuring the absorbance at 450 nm. As shown in Figure 145, 2t2 and IL-2 activated

32DB cells in a similar manner The EC50 of 212 and IL-2 was 158.1 pM and 140 pM.

respectively.

2t2 showed improved ability to promote IL-2RaBy containing CTLL-2 cell proliferation

as compared to IL-2

To evaluate the IL-2 activity of 2t2, 2t2 was compared with recombinant IL-2 for

promoting proliferation of CTLL-2 cells that express IL-2Ra, IL-2RB and common Y

chain. IL-2 dependent CTLL-2 cells were washed 5 times with IMDM-10% FBS and

seeded to the wells at 2 X 104 cells/well. Serial dilutions of 2t2 or IL-2 were added to the

cells (Figure 146). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell

proliferation was detected by adding 10 ul of WST1 to each well in the day 3 and

incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan

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dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure

146, 2t2 promoted CTLL-2 cell proliferation 4-5-fold stronger than IL-2. The EC50 of

2t2 was 123.2 pM and IL-2 was 548.2 pM.

2t2 suppressed the increase of the high fat-induced hyperglycemia in ApoE- mice

Six-week-old female ApoE- mice (Jackson Lab) were fed with standard chow

diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein,

and 15% starch (TD88137, Harlan Laboratories) and maintained in the standard

conditions. At week 7, mice fed with high fat diet were randomly assigned into the

control group and treatment group. Mice then received either 2t2 (treatment group) or

PBS (chow diet group and control group) per subcutaneous injection at a dosage of 3

mg/kg. Three days post dosing, the mice were fasted overnight, and blood samples were

collected through retro-orbital venous plexus puncture. Overnight fasting glucose levels

were measured using a OneTouch Glucometer. As shown in Figure 147, the results

showed that 2t2 injection effectively suppresses the increase of glucose levels in ApoE-

mice.

2t2 significantly upregulate the ratio of CD4+CD25+FoxP3+1 T regulatory (Treg) cells in

blood lymphocytes

Six-week-old female ApoE- mice (Jackson Lab) were fed with standard chow

diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein,

and 15% starch (TD88137, Harlan Laboratories) and maintained in the standard

conditions. At week 7, mice fed with the high fat diet were randomly assigned into

control group and treatment group. Mice then received either 2t2 (treatment group) or

PBS (chow diet group and control group) per subcutaneous injection at a dosage of 3

mg/kg. Three days after the dosing, overnight fasting blood samples were collected

through retro-orbital venous plexus puncture and incubated with ACK lysing buffer

(Thermo Fisher Scientific) at 37°C for 5 minutes. Samples were then resuspended in

FACS buffer (1 X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium

azide (Sigma)) and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies

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(BioLegend) for 30 minutes. Surface-stained samples were further fixed and

premetallized with Fix/Perm buffer (BioLegend) and intracellular stained with PE-anti-

Foxp3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer

followed by centrifugation at 1500 RPM for 5 minutes at room temperature. The cells

were analyzed by flow cytometry (Celesta-BD Bioscience). As shown in Figure 148, 2t2

treatment significantly increased Treg populations in blood lymphocytes (3.5%+0.32)

compared to the untreated groups (0.4%+0.16 for chow diet group and 0.46%=0.09 for

high fat diet group).

Purification elution chromatograph of 2t2 from anti-TF antibody affinity column

2t2 harvested from cell culture was loaded onto the anti-TF antibody affinity

column equilibrated with 5 column volumes of PBS. After sample loading, the column

was washed with 5 column volumes of PBS, followed by elution with 6 column volumes

of 0. 1M acetic acid, pH 2.9. A280 elution peak was collected and then neutralized to pH

7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS

using Amicon centrifugal filters with a 30 kDa molecular weight cutoff. As shown in

Figure 149, the anti-TF antibody affinity column bound to 2t2 which contains TF as a

fusion domain. The buffer-exchanged protein sample was stored at 2-8 °C for further

biochemical analyses and biological activity tests. After each elution, the anti-TF

antibody affinity column was stripped using 6 column volumes of 1M glycine, pH 2.5.

The column was then neutralized using 5 column volumes of PBS, and 7 column

volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected

to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps

except for the elution step, which was 2 mL/min.

Analytical size exclusion chromatography (SEC) analysis of 2t2

To analyze 2t2 using analytical size exclusion chromatography (SEC), a Superdex

200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an

AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column

volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 2t2 in PBS was

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injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The

SEC chromatograph of the sample is shown in Figure 150. The SEC results indicated two

protein peaks for 2t2.

Reduced SDS-PAGE of 2t2

To determine the purity and molecular weight of the protein, 2t2 protein sample

purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate

polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method

under reduced condition. After electrophoresis, the gel was stained with InstantBlue for

about 30 min, followed by destaining overnight in purified water.

To verify that the 2t2 protein undergoes glycosylation after translation in CHO

cells, a deglycosylation experiment was conducted using the Protein Deglycosylation

Mix II kit from New England Biolabs according to the manufacturer's instructions.

Figures 151A and 151B show the reduced SDS-PAGE analysis of the sample in non-

deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state.

The results show that the 2t2 protein is glycosylated when expressed in CHO cells. After

deglycosylation, the purified sample ran with expected molecular weights (56 kDa) in

reduced SDS gel. Lane M was loaded with 10 uL of SeeBlue Plus2 Prestained Standard.

In vivo characterization of 2t2

2t2 was subcutaneously injected into C57BL/6 mice at various doses to determine

the immunostimulatory activity of 2t2 in vivo. Mice were subcutaneously treated with

control solution (PBS) or 2t2 at 0.1, 0.4, 2 and 10 mg/kg. The treated mice were

euthanized day 3 post treatment. The mouse spleens were collected and weighed day 3

post treatment. Single splenocyte suspensions were prepared, and the prepared

splenocytes were stained for CD4+ T cells, CD8+ T cells and NK cells (with

fluorochrome-conjugated anti-CD4, -CD8, and -NK1.1 antibodies), and analyzed by

flow cytometry. The results showed that 2t2 was effective at expanding splenocytes

based on spleen weight (Figure 152A) especially at 0.1-10 mg/kg. The percentage of

CD8+ T cells were higher compared to control-treated mice (Figure 152B) at 2 and 10

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mg/kg. The percentage of NK cells were higher compared to control-treated mice

(Figure 152B) at all doses tested.

It has been known that IL-2 upregulates CD25 expression by immunocytes. We

therefore accessed CD25 expression of CD4+ T cells, CD8+ T cells and NK cells in the

2t2 treated mice. C57BL/6 mice were subcutaneously treated with 2t2 as described in the

paragraph above. The splenocytes were stained with fluorochrome-conjugated anti-CD4,

-CD8, CD25 and NK1.1 monoclonal antibodies. The CD25 expression (MFI) of

splenocyte subsets was analyzed by flow cytometry. As shown in Figure 153, at the doses

and time points tested, 2t2 significantly upregulated CD25 expression by CD4+ T cells

but not CD8+ T cells or NK cells.

The pharmacokinetics of 2t2 in C57BL/6 mice were also investigated. 2t2 was

subcutaneously injected into C57BL/6 mice at 1 mg/kg. The mouse blood was drawn

from tail vein at various time points as shown in Figure 154 and the serum was prepared.

2t2 concentrations were determined with ELISA (Capture: anti-tissue factor antibody;

Detection: biotinylated anti-human IL-2 antibody followed by SA-HRP and ABTS

substrate). The half-life of 2t2 was 1.83 hours calculated with PK Solutions 2.0 (Summit

Research Services).

2t2 attenuated the formation of high fat-induced atherosclerotic plaques in ApoE- mice

Six-week-old female ApoE- mice (The Jackson Laboratory) were fed with

standard chow diet or high diet fat (21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5%

casein, and 15% starch) (TD88137, Harlan Laboratories) and maintained in the standard

conditions. At week 7, mice fed with high fat diet (HFD) were randomly assigned into

control group and treatment group. Mice were then administrated either 2t2 (treatment

group) or PBS (chow diet group and control group) subcutaneously at a dosage of 3

mg/kg weekly for 4 weeks. At week 12, all mice were euthanized by isoflurane. Aortas

were collected, opened longitudinally and stained with Sudan IV solution (0.5%) using en

face method. The percentage of plaque area (red color as shown in Figure 155A) relative

to total aorta area was then quantified with Image J software. Figure 155A shows a

representative view of atherosclerotic plaques from each group. Figure 155B shows the

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results of quantitative analysis of atherosclerotic plaques of each group. The percentage

of plaque areas in control group (HF Diet) was much higher than the treatment group

(HFD+2t2), being 10.28% VS 4.68 %.

2t2 suppresses the progression of type 2 diabetes.

Male BKS.Cg-Dock7" +/+ Leprdb/J (db/db (Jackson Lab)) mice were fed with

standard chow diet and received drinking water ad libitum. At the age of six weeks, mice

were randomly assigned into control group and treatment group. The treatment group

received 2t2 by subcutaneous injection at 3 mg/kg bi-weekly, while control group

received vehicle (PBS) only. Overnight fasting glucose levels were measure weekly using

a OneTouch Glucometer. The results showed that 2t2 effectively suppressed the increase

of glucose levels in BKS.Cg-Dock7" +/+ Leprdb/J mice (Figure 156).

2t2 significantly upregulates the ratio of CD4+CD25+FoxP3+ T regulatory cells in blood

lymphocytes after the first injection

Male BKS.Cg-Dock7m +/+ Leprdb/J (db/db) (The Jackson Laboratory) mice were

fed with standard chow diet and received drinking water ad libitum. At the age of six

weeks, mice were randomly assigned into control group and treatment group. The

treatment group received 2t2 by subcutaneous injection at 3 mg/kg bi-weekly, while the

control group received vehicle (PBS) only. Four days after the first drug injection,

overnight fasting blood samples were collected and incubated with ACK lysing buffer

(Thermo Fisher Scientific) at 37°C for 5 minutes. Samples were then resuspended in

FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium

azide (Sigma)) and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies

(BioLegend) for 30 minutes. Surface-stained samples were further fixed and

premetallized with Fix/Perm buffer (BioLegend) and intracellular stained with PE-anti-

Foxp3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer

and were analyzed by flow cytometry (Celesta-BD Bioscience). The percentage of

CD4*CD25*FoxP3* Tregs in blood lymphocytes were measured. As shown in Figure

157, the results showed that 2t2 significantly upregulated the ratio of Tregs in blood

lymphocytes (* p<0.05).

Example 62. Production and characterization of the Exemplary Single-Chain

Chimeric Polypeptide 15t15

A second exemplary single-chain chimeric polypeptide including a first target-

binding domain that binds to an IL-15 receptor, a soluble human tissue factor domain,

and a second target-binding domain that binds to an IL-15 receptor was generated (IL-

15/TF/IL-15; referred to at 15t15) (Figure 158). The nucleic acid and amino acid

sequences of this single-chain chimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide (IL-

15/TF/IL-15) (SEQ ID NO: 170)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (First IL-15 fragment)

AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATO CAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTA

GCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTC7 TTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCAT TAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCA AGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCT TCGTGCACATCGTGCAGATGTTCATCAACACTAGO

(Human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTC ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGA.

ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGO wo WO 2021/247604 PCT/US2021/035285

ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTT CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGO ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC ACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACA

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACAG GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTG AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT IGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Second IL-15 fragment)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT AGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTO TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAC GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Exemplary Single-Chain Chimeric Polypeptide (IL-15/TF/IL-15) (SEQ ID NO: 169)

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVO LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVO MFINTS

(Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE

KGEFRE 691

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(Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ LESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid encoding IL-15/TF/IL-15 was cloned into a modified retrovirus

expression vector as described previously (Hughes et al., Hum Gene Ther 16:457-72,

2005). The expression vector encoding IL-15/TF/IL-15 was transfected into CHO-K1

cells. Expression of the expression vector in CHO-K1 cells allowed for secretion of the

soluble IL-15/TF/IL-15 single-chain chimeric polypeptide (referred to as 15t15), which

can be purified by anti-TF antibody affinity and other chromatography methods.

15t15 promotes IL-2RB and common ychain containing 32DB cell proliferation

IL-15 activity of 15t15 was compared with recombinant IL-15 in IL2R and

common chain expressed 32D cells. IL-15 dependent 32DB cells were washed five

times with IMDM-10% FBS and seeded to the wells at 2 X 104 cells/well. Serial dilutions

of 15t15 or IL-15 were added to the cells (Figure 159). Cells were incubated in a CO2

incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 ul of WST1 to

each well in the day 3 and incubating for an additional 3 hours in a CO2 incubator at

37°C. The amount of formazan dye produced was analyzed by measuring the absorbance

at 450 nm. As shown in Figure 159, 15t15 promoted 32DB cell proliferation less

efficiently as compared to IL-15. The EC50 of 15t15 and IL-15 was 161.4 pM and 1.6

pM. respectively.

Purification elution chromatograph of 15t15 from anti-TF antibody affinity column

15t15 harvested from cell culture was loaded onto the anti-TF antibody affinity

column equilibrated with 5 column volumes of PBS. After sample loading, the column

was washed with 5 column volumes of PBS, followed by elution with 6 column volumes

of 0. 1M acetic acid, pH 2.9. A280 elution peak was collected and then neutralized to pH

7.5-8.0 with 1M Tris base. The neutralized sample was then buffer exchanged into PBS

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using Amicon centrifugal filters with a 30 kDa molecular weight cutoff. As shown in

Figure 160, the anti-TF antibody affinity column bound to 15t15 which contains TF as a

fusion domain. The buffer-exchanged protein sample was stored at 2-8 °C for further

biochemical analyses and biological activity tests. After each elution, the anti-TF

antibody affinity column was stripped using 6 column volumes of 1M glycine, pH 2.5.

The column was then neutralized using 5 column volumes of PBS, and 7 column

volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected

to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps

except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE of 15t15

To determine the purity and molecular weight of the protein, 15t15 protein sample

purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate

polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method

under reduced condition. After electrophoresis, the gel was stained with InstantBlue for

about 30 min, followed by destaining overnight in purified water.

To verify that the 15t15 protein undergoes glycosylation after translation in CHO

cells, a deglycosylation experiment was conducted using the Protein Deglycosylation

Mix II kit from New England Biolabs and the manufacturer's instructions. Figures 161A

and 161B show the reduced SDS-PAGE analysis of the sample in non-deglycosylated

(lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results

showed that the 15t15 protein is glycosylated when expressed in CHO cells. After

deglycosylation, the purified sample ran with expected molecular weights (50 kDa) in

reduced SDS gel. Lane M was loaded with 10 uL of SeeBlue Plus2 Prestained Standard.

Example 63: Stimulation of NK cells in vitro

A set of experiments was performed to assess the changes in surface phenotype of

NK cells after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s + anti-TF antibody.

In these experiments, fresh human leukocytes were obtained from the blood bank and

CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell

Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-

BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The

cells were counted and resuspended at 0.2 X 106/mL in a 96-well flat-bottom plate in 0.2

mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine

(Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin

(Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with:

18t15-12s (100 nM); 18t15-12s16 (100 nM); a mixture of single cytokines rhIL-15 (50

ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL) (Peprotech);

7t15-21s + anti-TF antibody (100 nM-50 nM); 7t15-21s (100 nM); or anti-TF antibody

(50nM) at 37 °C and 5% CO2 for 16 hours. The next day, the cells were harvested and

surface stained for 30 minutes with CD56, CD16, CD25, CD69, CD27, CD62L, NKp30,

and NKp44 specific antibodies. After surface staining, the cells were washed (1500 RPM

for 5 minutes at room temperature) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA

(EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were

analyzed by flow cytometry (Celesta-BD Bioscience). Figure 162A and 162B shows that

overnight incubation of purified NK cells with 18t15-12s, 18t15-12s16, and 7t15-21s +

anti-TF antibody resulted in an increase in the percentage of cells expressing CD25,

CD69, NKp44, and NKp30 activation markers and a decrease in the percentage of cells

expressing CD62L. All activation marker data is from CD56+ gated lymphocytes.

A set of experiments was performed to assess changes in the surface phenotype of

lymphocyte populations after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s. In

these experiments, fresh human leukocytes were obtained from the blood bank.

Peripheral blood lymphocytes were isolated with the Ficoll-PAQUE Plus (GE

Healthcare) density gradient media. The cells were counted and resuspended at 0.2 X

106/mL in a 96-well flat-bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco)

supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo

Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)).

The cells were stimulated with: 18t15-12s (100 nM); 18t15-12s16 (100 nM), a mixture of

single cytokines rhIL-15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and

rhIL-12 (10 ng/mL) (Peprotech); 7t15-21s (100 nM) + anti-TF antibody (50 nM); 7t15-

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21s (100 nM); or anti-TF antibody (50 nM) at 37 °C and 5% CO2 for 16 hours. The next

day, the cells were harvested and surface stained for 30 minutes for CD4 or CD8,

CD62L, and CD69 specific antibodies. After surface staining, cells were washed (1500

RPM for 5 minutes at room temperature) in FACS buffer (1X PBS (Hyclone) with 0.5%

BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells

were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 163 shows that

overnight incubation of purified lymphocyte populations (CD4 and CD8 T cells) with

18t15-12s, 18t15-12s16, or 7t15-21s + anti-TF antibody resulted in an increase in the

percentage of CD8 and CD4 T cells expressing CD69. Additionally, incubation with

7t15-21s + anti-TF antibody resulted in an increase in the percentage of CD8 and CD4 T

cells expressing CD62L (Figure 163).

A set of experiments was performed to determine the effect of 18t15-12s on the

extracellular acidification rate (ECAR) of NK cells purified from human blood. ECAR

can be used to measure glycolysis. Glycolysis is the intracellular biochemical conversion

of one molecule of glucose into two molecules of pyruvate with the concurrent

generation of two molecules of ATP. An increase in glycolysis was indicated by an

increase in ECAR measured by a Seahorse XF96 Analyzer. In these experiments, fresh

human leukocytes were obtained from the blood bank and CD56+ NK cells were isolated

with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK

cells was >70% and confirmed by staining for CD56-BV421, CD16-BV510, CD25-PE,

and CD69-APCFire750 antibodies (BioLegend). The cells were counted and

resuspended in 0.2 X 106/mL in a 96-well flat-bottom plate in 0.2 mL of complete media

(RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies),

penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and

10% FBS (Hyclone)). The cells were stimulated with either a mixture of single cytokines

hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D), and hIL-15 (50 ng/mL)

(NCI) or 18t15-12s (100 nM) at 37 °C and 5% CO2 for 14-18 hours. The next day, the

cells were harvested and washed two times in Seahorse media. The cells (2 x 105

cells/well) were seeded in 96-well flux plates that were coated with 10 uL of poly-L-

lysine (Sigma). NK cells were adhered to plates for 30 minutes prior to the assay.

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Glucose, oligomycin, and 2DG solutions were prepared at 10x concentration in buffered

Seahorse medium and injected in port A, B, and C of the calibration plate. ECAR

readings were taken every 6.5-7 minutes and ECAR results represent the average

readings over 80 minutes or average readings at each timepoint. Figure 164 shows

overnight stimulation of NK cells with 18t15-12s resulted in increased basal ECAR

levels. The addition of glucose and oligomycin further showed enhanced glycolysis and

glycolytic capacity, respectively, of NK cells stimulated with 18t15-12s overnight (Figure

164). NK cells treated overnight with media alone or a mixture of IL12, IL18, and IL-15

were used for comparison (Figure 164).

A set of experiments was performed to determine the increase in phospho-STAT4

and phospho-STATS levels in NK cells after stimulation with 18t15-12s. In these

experiments, fresh human leukocytes were obtained from the blood bank and CD56+ NK

cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies).

The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

BV510, CD25-PE, and CD69-APCFire750 specific antibodies (BioLegend). The cells

were counted and resuspended in 0,05 X 106/mL in a 96-well flat-bottom plate in 0.1 mL

of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). The cells were stimulated with hIL-12 (10

ng/mL) (Biolegend) or hIL-15 (50 ng/mL) (NCI) (Single cytokines), or 18t15-12s (100

nM) at 37 °C and 5% CO2 for 90 minutes. Unstimulated NK cells (US) were used as a

control. The cells were harvested and fixed in paraformaldehyde (Sigma) to a final

concentration of 1.6% Plates were incubated in the dark at room temperature for 10

minutes. FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001%

sodium azide (Sigma)) (100 uL) was added and cells were transferred to 96-well "V"

bottom plate. The cells were washed for 1500 RPM for 5 minutes at room temperature.

The cell pellet was mixed with 100 uL chilled methanol by gently pipetting up and down,

and cells were incubated for 30 minutes at 4 °C. The cells were mixed with 100 mL of

FACS buffer and washed for 1500 RPM for 5 minutes at room temperature. The cell

pellets were mixed with 50 mL of FACS buffer containing 4 mL of pSTAT4 (BD

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Bioscience) and pSTAT5 antibodies (BD Bioscience) followed by incubation for 30

minutes at room temperature in the dark. The cells were mixed with 100 mL of FACS

buffer and washed for 1500 RPM for 5 minutes at room temperature. The cell pellets

were mixed with 50 mL of FACS buffer and cells were analyzed by flow cytometry

(Celesta-BD Bioscience). Figure 165 shows that incubation of NK cells with 18t15-12s

induced an increase in pSTAT4 and pSTAT5 (plotted data, normalized fold-change).

A set of experiments was performed to determine the effect of 18t15-12s or a

mixture of cytokines (e.g., IL12, IL18, and IL-15) on oxygen consumption rate (OCR)

and extracellular acidification rate (ECAR) on NK cells purified from human blood.

OCR and ECAR were measured by a Seahorse XF96 Analyzer. In these experiments,

fresh human NK cells were isolated from human leukocytes via negative selection using

the RosetteSep/human NK cell reagent (StemCell Technologies). Freshly purified NK

cells were stimulated overnight (16 h) with either 18t15-12s (100nM) or a mixture of

rhIL12 (10 ng/mL), rhIL18 0 ng/mL), and rhIL-15 (50 ng/mL) cytokines as a control.

The next day, the cells were washed, counted, and equal numbers of cells were plated in

buffered Seahorse media. Glucose, oligomycin, and 2DG solutions were prepared at 10x

concentration in buffered Seahorse medium and injected in port A, B, and C of the

calibration plate. Figure 166 shows OCR (left) and ECAR (right) data from two

individual donors. Overnight stimulation of NK cells with 18t15-12s resulted in an

increase in basal ECAR and OCR levels. Addition of glucose and oligomycin further

showed enhanced glycolysis and glycolytic capacity, respectively, of NK cells stimulated

with 18t15-12s overnight. NK cells treated overnight with media alone or a mixture of

IL12, IL18, and IL-15 were used for comparison.

Example 64: Stimulation of NK cells in vivo by 2t2 and/or TGFRt15-TGFRs

A set of experiments was performed to determine the effect of the 2t2 construct

on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were

subcutaneously treated with control solution (PBS) or 2t2 at 0.1, 0.4, 2, and 10 mg/kg.

Treated mice were euthanized 3 days post-treatment. Spleen weight was measured and

single splenocyte suspensions were prepared. Splenocytes suspensions were stained with wo 2021/247604 WO PCT/US2021/035285 conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. The percentage of

CD4+ T cells, CD8+ T cells, and NK cells, and CD25 expression on lymphocyte subsets

were analyzed by flow cytometry. Figure 167A shows that 2t2 was effective at

expanding splenocytes based on spleen weight especially at a dose level of 0.1-10 mg/kg.

Following treatment, the percentage of CD8+ T cells were higher in 2t2-treated mice

compared to control-treated mice at 2 and 10 mg/kg (Figure 167B). The percentage of

NK cells were also higher in 2t2-treated mice compared to control-treated mice at all

doses of 2t2 tested (Figure 167B). Additionally, 2t2 significantly upregulated CD25

expression by CD4+ T cells, but not CD8+ T cells and NK cells following treatment at 0.4

to10 mg/kg (Figure 167C).

A set of experiments was performed to determine the effect of the TGFRt15-

TGFRs construct on immune stimulation in C57BL/6 mice In these experiments,

C57BL/6 mice were subcutaneously treated with control solution (PBS) or TGFRt15-

TGFRs at 0.3, 1, 3, and 10 mg/kg. The treated mice were euthanized 4 days post-

treatment. Spleen weight was measured and single splenocyte suspensions were

prepared. The splenocytes suspensions were stained with conjugated anti-CD4, anti-

CD8, and anti-NK1.1 (NK) antibodies. The percentage of CD4+ T cells, CD8+ T cells,

and NK cells were analyzed by flow cytometry. Figure 168A shows that spleen weight in

mice treated with TGFRt15-TGFRs increased with increasing dosage of TGFRt15-

TGFRs. Additionally, spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and 10

mg/kg of TGFRt15-TGFRs were higher as compared to mice treated with the control

solution. Figure 168B shows that the percentages of CD8+ T cells and NK cells both

increased with increasing dosage of TGFRt15-TGFRs. Specifically, the percentages of

CD8+ T cells were higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of

TGFRt15-TGFRs compared to control-treated mice, and the percentages of NK cells

were higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of

TGFRt15-TGFRs compared to control-treated mice.

A set of experiments was performed to determine the effect of the TGFRt15-

TGFRs construct or 2t2 construct on immune stimulation in ApoE- mice fed with a

Western diet. In these experiments, 6-week old female B6. 129P2-ApoEtmlUnc/ mice

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(Jackson Laboratory) were fed with a Western diet containing 21% fat, 0.15%

cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Envigo

Laboratories). After 8-weeks of the Western diet, the mice were injected subcutaneously

with TGFRt15-TGFRs or 2t2 at 3 mg/kg. Three days post treatment, mice were fasted

for 16 hours and then blood samples were collected through retro-orbital venous plexus

puncture. The blood was mixed with 10 uL 0.5 M EDTA, and 20 uL blood was taken for

lymphocyte subsets analysis. The red blood cells were lysed with ACK (0.15 M NH4Cl,

1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) and the lymphocytes were stained with

anti-mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at 4 °C in FACS

staining buffer (1% BSA in PBS). The cells were washed once and analyzed with a BD

FACS Celesta. For Treg staining, ACK treated blood lymphocytes were stained with

anti-mouse CD4 and anti-mouse CD25 antibodies for 30 minutes at 4 °C in FACS

staining buffer. The cells were washed once and resuspended in

fixation/permeabilization working solution and incubated at room temperature for 60

minutes. The cells were washed once and resuspended in permeabilization buffer. The

samples were centrifuged at 300-400 X g for 5 minutes at room temperature and the

supernatant was then discarded. The cell pellet was resuspended in residual volume and

the volume adjusted to about 100 uL with 1 X permeabilization buffer. Anti-Foxp3

antibody was added to the cells, and the cells were incubated for 30 minutes at room

temperature. Permeabilization buffer (200 uL) was added to the cells, and the cells were

centrifuged at 300-400 X g for 5 minutes at room temperature. The cells were

resuspended in flow cytometry staining buffer and analyzed on a flow cytometer. Figures

169B-169C show that treatment with TGFRt15-TGFRs and 2t2 increased the percentage

of NK cells and CD8+ T cells in ApoE- mice fed with Western diet. Figure 169A shows

that treatment with 2t2 also increased the percentage of Treg cells.

Example 65: Induction of proliferation of immune cells in vivo

A set of experiments was performed to determine the effect of the 2t2 construct

on immune cell stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were

subcutaneously treated with control solution (PBS) or 2t2 at 0.1, 0.4, 2, and 10 mg/kg.

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Treated mice were euthanized 3 days post-treatment. Spleen weight was measured and

single splenocyte suspensions were prepared. The splenocyte suspensions were stained

with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. The percentage

of CD4+ T cells, CD8+ T cells, and NK cells were analyzed by flow cytometry. Figure

170A shows that 2t2 treatment was effective at expanding splenocytes based on spleen

weight especially at 0.1-10 mg/kg. The percentage of CD8+ T cells was higher compared

to control-treated mice at 2 and 10 mg/kg (Figure 170B). Additionally, the percentage of

NK cells was higher compared to control-treated mice at all doses of 2t2 tested (Figure

170B). These results demonstrate that 2t2 treatment was able to induce proliferation of

CD8+ T cells and NK cells in C57BL/6 mice.

A set of experiments was performed to determine the effect of the TGFRt15-

TGFRs construct on immune stimulation in C57BL/6 mice. In these experiments,

C57BL/6 mice were subcutaneously treated with control solution (PBS) or TGFRt15-

TGFRs at 0.1, 0.3, 1, 3, and 10 mg/kg. The treated mice were euthanized 4 days post-

treatment. Spleen weight was measured and splenocyte suspensions were prepared. The

splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and anti-

NK1.1 (NK) antibodies. The cells were additionally stained for proliferation marker

Ki67. Figure 171A shows that spleen weight in mice treated with TGFRt15-TGFRs

increased with increasing dosage of TGFRt15-TGFRs. Additionally, spleen weight in

mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs was higher as

compared to mice treated with just the control solution. The percentages of CD8+ T cells

and NK cells both increased with increasing dosage of TGFRt15-TGFRs (Figure 171B).

Finally, TGFRt15-TGFRs significantly upregulated expression of cell proliferation

marker Ki67 in both CD8+ T cells and NK cells at all doses of TGFRt15-TGFRs tested.

These results demonstrate that TGFRt15-TGFRs treatment induced proliferation of both

CD8+ T cells and NK cells in C57BL/61 mice.

A set of experiments was performed to determine the effect of the TGFRt15-

TGFRs construct or the 2t2 construct on immune stimulation in ApoE* mice fed with a

Western diet. In these experiments, 6-week old female B6.129P2-ApoEtmlUx mice

(Jackson Laboratory) were fed with a Western diet containing 21% fat, 0.15%

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cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Envigo

Laboratories). After 8-week of the Western diet, the mice were injected subcutaneously

with TGFRt15-TGFRs or 2t2 at 3 mg/kg. Three days post-treatment, the mice were

fasted for 16 hours and then blood samples were collected through retro-orbital venous

plexus puncture. The blood was mixed with 10 uL 0.5 M EDTA and 20 uL blood was taken for

lymphocyte subsets analysis. The red blood cells were lysed with ACK (0.15 M NH4Cl, 1.0

mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) and the lymphocytes were stained with anti-

mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at 4 °C in FACS staining

buffer (1% BSA in PBS). The cells were washed once and resuspended in Fixation

Buffer (BioLegend Cat# 420801) for 20 minutes at room temperature. The cells were

centrifuged at 350 x ; for 5 minutes, the fixed cells were resuspended in Intracellular

Staining Permeabilization Wash Buffer (BioLegend Cat# 421002) and then centrifuged at

350 X g for 5 minutes. The cells were then stained with anti-Ki67 antibody for 20

minutes at RT. The cells were washed twice with Intracellular Staining Permeabilization

Wash Buffer and centrifuged at 350 X g for 5 minutes. The cells were then resuspended

in FACS staining buffer. Lymphocyte subsets were analyzed with a BD FACS Celesta.

As described in Figure 172A, treatment of ApoE- mice with TGFRt15-TGFRs induced

proliferation (Ki67-positive staining) in NK and CD8+ T cells. Additionally, Figure

172B shows treatment of ApoE- mice with 2t2 also induced proliferation (Ki67-positive

staining) in NK and CD8+ T cells.

A set of experiments was performed to determine the effect 7t15-21s + anti-TF

antibody-expanded NK cells in NSG mice following treatment with 7t15-21s, TGFRt15-

TGFRs, and 2t2. In these experiments, fresh human leukocytes were obtained from the

blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell

reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by

staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies

(BioLegend). The cells were counted and resuspended in 2 X 106/mL in a 24-well flat-

bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM

L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies),

streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were

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stimulated with: 7t15-21s (100 nM) and anti-TF antibody (50 nM) for 15 days. After

every 2 days, the cells were resuspended at 2 X 106/mL with fresh media containing 100

nM 7t15-21s and 50 nM of anti-TF antibody. As the volume of the cultures increased,

the cells were transferred to higher volume flasks. The cells were counted using trypan

blue to access the fold-expansion. 7t15-21s + anti-TF antibody-expanded NK cells were

washed three times in warm HBSS Buffer (Hyclone) at 1000 RPM for 10 minutes at

room temperature. The 7t15-21s + anti-TF antibody-expanded-NK cells were

resuspended in 10 X 106/0.2 mL HBSS buffer and injected intravenously into the tail vein

of NSG mice (NOD scid common gamma mouse) (Jackson Laboratories). The

transferred NK cells were supported every 48 hours with either 7t15-21s (10 ng/dose,

i.p.), TGFRt15-TGFRs (10 ng/dose, i.p.) or 2t2 (10 ng/dose, i.p.) for up to 21 days.

Engraftment and persistence of the human 7t15-21s + anti-TF antibody-expanded NK

cells were measured every week in blood staining for hCD45, mCD45, hCD56, hCD3,

and hCD16 antibodies by flow cytometry (Celesta-BD Bioscience) (Data represent 3

mice per group). Figure 173 indicates that treatment of mice bearing adoptively-

transferred 7t15-21s + anti-TF antibody-expanded NK cells with 7t15-21s-, TGFRt15-

TGFRs-, or 2t2-induced expansion and persistence of the adoptively transferred NK cells

compared to control treated mice.

Example 66: NK-mediated cytotoxicity following treatment with single-chain

constructs or multi-chain constructs

A set of experiments was performed to determine if treatment of NK cells with

TGFRt15-TGFRs enhanced cytotoxicity of NK cells. In these experiments, Human

Daudi B lymphoma cells were labeled with CellTrace Violet (CTV) and used as tumor

target cells. Mouse NK effector cells were isolated with NK1.1-positive selection using a

magnetic cell sorting method (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 days

post TGFRt15-TGFRs subcutaneous treatment at 3 mg/kg. Human NK effector cells

were isolated from peripheral blood mononuclear cells derived from human blood buffy

coats with the RosetteSep/human NK cell reagent (Stemcell Technologies). The target

cells (Human Daudi B lymphoma cells) were mixed with effector cells (either mouse NK wo 2021/247604 WO PCT/US2021/035285 effector cells or human NK effector cells) in the presence of 50 nM TGFRt15-TGFRs or in the absence of TGFRt15-TGFRs (control) and incubated at 37 °C for 44 hours for mouse NK cells and for 20 hours for human NK cells. Target cell (Daudi) viability was assessed by analysis of propidium iodide-positive, CTV-labeled cells using flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1- viable tumor cell number in experimental sample/viable tumor cell number in the sample without NK cells) X 100. Figure 174 shows that mouse (Figure 174A) and human (Figure

174B) NK cells had significantly stronger cytotoxicity against Daudi B cells following

NK cell activation with TGFRt15-TGFRs than in the absence of TGFRt15-TGFRs

activation.

A set of experiments was performed to determine antibody-dependent cellular

cytotoxicity (ADCC) of mouse and human NK cells following treatment with TGFRt15-

TGFRs. In these experiments, human Daudi B lymphoma cells were labeled with

CellTrace Violet (CTV) and used as tumor target cells. Mouse NK effector cells were

isolated with NK1.1-positive selection using a magnetic cell sorting method (Miltenyi

Biotec) of C57BL/6 female mouse spleens 4 days post-TGFRt15-TGFRs subcutaneous

treatment at 3 mg/kg. Human NK effector cells were isolated from peripheral blood

mononuclear cells derived from human blood buffy coats with the RosetteSep/human NK

cell reagent (Stemcell Technologies). The target cells (Daudi B cells) were mixed with

effector cells (either mouse NK effector cells or human NK effector cells) in the presence

of anti-CD20 antibody (10 nM Rituximab, Genentech) and in the presence of 50 nM

TGFRt15-TGFRs, or in the absence of TGFRt15-TGFRs (control) and incubated at 37 °C

for 44 hours for mouse NK cells and for 20 hours for human NK cells. The Daudi B cells

express the CD20 targets for the anti-CD20 antibody. Target cell viability was assessed

after incubation by analysis of propidium iodide-positive, CTV-labeled target cells using

flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1-

viable tumor cell number in experimental sample/viable tumor cell number in the sample

without NK cells) X 100. Figure 175 shows that mouse NK cells (Figure 175A) and

human NK cells (Figure 175B) had stronger ADCC activity against Daudi B cells

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following NK cell activation with TGFRt15-TGFRs than in the absence of TGFRt15-

TGFRs activation.

A set of experiments was performed to determine cytotoxicity of TGFRt15-

TGFRs-activated mouse NK cells towards senescent B16F10 melanoma cells. In these

experiments, mouse NK cells were activated in vivo by injecting C57BL/6 mice with 10

mg/kg of TGFRt15-TGFRs for 4 days followed by isolation of splenic NK cells. The NK

cells were then expanded in vitro for 7 days in the presence of 100 nM 2t2. The B16F10

senescent target cells (B16F10-SNC) were labelled with CellTrace Violet (CTV) and

incubated at different Effector: Target (E:T) ratios with the activated mouse NK effector

cells for 16 hours. The cells were trypsinized, washed, and resuspended in complete

media containing propidium iodide (PI) solution. The cytotoxicity of the TGFRt15-

TGFRs/2t2-activated NK cells against the senescent cell targets was accessed by flow

cytometry based on PI staining of the CTV-labeled cells. The findings demonstrate that

in vivo activation of NK cells with TGFRt15-TGFRs followed by in vitro expansion and

activation with 2t2 resulted in increased killing of senescent melanoma tumor cells by the

NK cells (Figure 176).

Example 67: Treatment of Cancer, Diabetes, and Atherosclerosis

A set of experiments was performed to assess antitumor activity of TGFRt15-

TGFRs plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a

melanoma mouse model. In these experiments, C57BL/6 mice were subcutaneously

injected with 0.5 X 106 B16F10 melanoma cells. The mice were treated with three doses

of chemotherapy docetaxel (10 mg/kg) (DTX) on day 1, day 4, and day 7, followed by

treatment with single dose of combination immunotherapy TGFRt15-TGFRs (3 mg/kg) +

anti-TRP1 antibody TA99 (200 ug) on day 9. Figure 177A shows a schematic of the

treatement regimen. Tumor growth was monitored by caliper measurement, and tumor

volume was calculated using the formula V = (L X W2)/2, where L is the largest tumor

diameter and W is the perpendicular tumor diameter. Figure 177B shows that treatment

with DTX + TGFRt15-TGFRs + TA99 significantly reduced tumor growth compared to

saline control and DTX treatment groups (N=10, p <0.001, Multiple t test analyses).

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To assess immune cell subsets in the B16F10 tumor model, peripheral blood

analysis was performed. In these experiments, C57BL/6 mice were injected with B16F10

cells and treated with DTX, DTX + TGFRt15-TGFRs + TA99, or saline. Blood was

drawn from the submandibular vein of B16F10 tumor-bearing mice on days 2, 5, and 8

post-immunotherapy for the DTX + TGFRt15-TGFRs + TA99 group and day 11 post-

tumor injection for the DTX and saline groups. RBCs were lysed in ACK lysis buffer

and the lymphocytes were washed and stained with anti-NK1.1, anti-CD8, and anti-CD4

antibodies. The cells were analyzed by flow cytometry (Celesta-BD Bioscience).

Figures 177C-177E show that DTX + TGFRt15-TGFRs + TA99 treatment induced an

increase in the percentage of NK cells and CD8+ T cells in the tumors compared to the

saline and DTX treatment groups.

On day 17, total RNA was extracted from tumors of mice treated with saline,

DTX or DTX + TGFRt15-TGFRs + TA99 using Trizol. Total RNA (1 ug) was used for

cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time

PCR was carried out with CFX96 Detection System (Bio-Rad) using FAM-labeled

predesigned primers for senescence cell markers, (F) p21 (G) DPP4 and (H) IL6. The

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in expression levels. The expression of each target mRNA relative to 18S

rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Cttarget Cti8s. The data is

presented as fold-change as compared to saline control. Figure 177F-177H show that

DTX treatment induced an increase in senescent tumor cells that were subsequently

reduced following treatment with TGFRt15-TGFRs + TA99 immunotherapy.

A set of experiments was performed to investigate amelioration of Western diet-

induced hyperglycemia in ApoE- mice by 2t2. In these experiments, 6-week old female

129P2-ApoEtmlUnc/ mice (Jackson Laboratory) were fed with a Western diet

containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch

(TD88137, Envigo Laboratories). After 8-weeks of the Western diet, the mice were

injected subcutaneously with TGFRt15-TGFRs or 2t2 at 3 mg/kg. Three days post-

treatment, the mice were fasted for 16 hours and then blood samples were collected

through retro-orbital venous plexus puncture. Blood glucose was detected with a glucose

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meter (OneTouch UltraMini) and GenUltimated test strips using a drop of fresh blood.

As shown in Figure 178A, 2t2 treatment significantly reduced hyperglycemia induced by

the Western diet (p<0.04). The plasma insulin and resistin levels were analyzed with

Mouse Rat Metabolic Array by Eve Technologies. HOMA-IR was calculated using the

following formula: homeostatic model assessment-insulin resistance = Glucose (mg/dL)

* Insulin (mU/mL)/405. As shown in Figure 178B, both 2t2 and TGFRt15-TGFRs

treatment reduced insulin resistance compared to the untreated group. Both 2t2 (p<0.02)

and TGFRt15-TGFRs (p<0.05) reduced resistin levels significantly compared to the

untreated group as shown in Figure 178C, which may relate to the reduced insulin

resistance induced by 2t2 and TGFRt15-TGFRs (Figure F3B).

Example 68: Induction of differentiation of NK cells into cytokine-induced memory

like NK cells

A set of experiments was performed to assess the differentiation of NK cells into

cytokine-induced memory like NK Cells (CIMK-NK Cells) after stimulation with 18t15-

12s. In these experiments, fresh human leukocytes were obtained from the blood bank

and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell

Technologies). The purity of NK cells was >90% and confirmed by staining with CD56-

BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The

cells were counted and resuspended in X 106/mL in a 24-well flat-bottom plate in 2 mL

of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)). The cells were unstimulated ("No Spike") or

stimulated with 18t15-12s (100 nM) or a mixture of single cytokines including rhIL-15

(50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL)

(Peprotech) ("single cytokines") at 37 °C and 5% CO2 for 16 hrs. The next day, the cells

were harvested, and washed two times with warm complete media at 1000 RPM for 10

minutes at room temperature. The cells were resuspended at 2 X 106/mL in a 24-well flat-

bottom plate in 2 mL of complete media with rhIL-15 (1 ng/mL). After every 2 days,

half of the medium was replaced with fresh complete media containing rhIL-15.

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To assess the change in memory phenotype of NK cells at day 7, the cells were

stained with antibodies to cell-surface CD56, CD16, CD27, CD62L, NKp30, and NKp44

(BioLegend). After surface staining, the cells were washed (1500 RPM for 5 minutes at

room temperature) in FACS buffer (1X) PBS (Hyclone) with 0.5% BSA (EMD Millipore)

and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow

cytometry (Celesta-BD Bioscience). Figure 179 shows that incubation of NK cells with

18t15-12s resulted in an increase in the percentage of CD16*CD56+ NK cells expressing

CD27, CD62L, and NKp44, and an increase in the levels (MFI) of NKp30 in

CD16*CD56+ NK cells.

Example 69. Upregulation of CD44 memory T cells

C57BL/6 mice were subcutaneously treated with TGFRt15-TGFRs or 2t2. The

treated mice were euthanized and the single splenocyte suspensions were prepared 4 days

(TGFRt15-TGFRs) or 3 days (2t2) following the treatment. The prepared splenocytes

were stained with fluorochrome-conjugated anti-CD4, anti-CD8 and anti-CD44

antibodies and the percentages of CD44high T cells in CD4+ T cells or CD8+ T cells were

analyzed by flow cytometry. The results show that TGFRt15-TGFRs and 2t2

upregulated expression of the memory marker CD44 on CD4+ and CD8+ T cells (Figures

180). These findings indicate that TGFRt15-TGFRs and 2t2 molecules were able to

induce mouse T cells to differentiate into memory T cells.

Example 70: Improvement of the texture and/or appearance and/or hair

To examine the effect of 2t2 on hair regrowth, dorsal hair of C57BL6/J mice

(Jackson Laboratory) was first shortened with clippers followed by application of

depilatory cream (Nair) to the shaved region for a period of 30 seconds before wiping

clean. After 4 hours, 2t2 (3 mg/kg, single dose), low dose recombinant IL-2 (25000 IU, 5

consecutive days, 1 dose/day), or PBS were administered subcutaneously. The mice

were monitored for skin pigmentation related to hair regrowth and pictures were taken

and analyzed using the Image J software. Figure 181A shows skin pigmentation 10 days

after depilation in PBS-, 2t2-, or IL-2-treated mice. Figure 181B shows the percent

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pigmentation in each group of mice 10 days post-treatment as analyzed using the Image J

software. The results showed that treatment of mice with 2t2 or IL-2 promoted hair

regrowth following depilation compared to PBS-treated mice.

Dorsal hair of C57BL6/J mice (Jackson Laboratory) was first shortened with

clippers before applying depilatory cream (Nair) to the shaved region for a period of

exactly 30 seconds before wiping clean. After 4 hours, 2t2 (3 mg/kg, single dose), low

dose recombinant IL-2 (25000 IU, 5 consecutive days, 1 dose/day) or PBS were

administered subcutaneously. The mice were monitored for skin pigmentation related to

hair regrowth and pictures were taken and analyzed using Image J software. Figure 182

shows skin pigmentation 14 days after depilation in PBS-, 2t2-, or IL-2-treated mice.

The results showed that treatment of mice with 2t2 or IL-2 promoted hair regrowth

following depilation compared to the PBS-treated mice.

Example 71: Tissue factor coagulation assays following treatment with single-chain

or multi-chain chimeric polypeptides

A set of experiments was performed to assess blood coagulation following

treatment with single-chain or multi-chain chimeric polypeptides. To initiate the blood

coagulation cascade pathway, tissue factor (TF) binds to Factor VIIa (FVIIa) to form a

TF/FVIIa complex. The TF/FVIIa complex then binds Factor X (FX) and converts FX to

FXa.

Factor VIIa (FVIIa) activity Assay

One assay to measure blood coagulation involves measuring Factor VIIa (FVIIa)

activity. This type of assay requires the presence of tissue factor and calcium. The

TF/FVIIa complex activity can be measured by a small substrate or by a natural protein

substrate, for example, Factor X (FX). When FX is used as a substrate, phospholipids are

also required for TF/FVIIa activity. In this assay, FVIIa activity is determined with

FVIIa-specific chromogenic substrate S-2288 (Diapharma, West Chester, OH). The

color change of the S-2288 substrate can be measured spectrophotometrically and is

proportional to the proteolytic activity of FVIIa (e.g., the TF/FVIIa complex).

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In these experiments, the FVIIa activity of the following groups were compared:

the 219-amino acid extracellular domain of tissue factor domain (TF219), a multi-chain

chimeric polypeptide with a wild-type tissue factor domain, and a multi-chain chimeric

polypeptide with a mutant tissue factor domain. The chimeric polypeptides containing

mutant tissue factor molecules were constructed with mutations to the TF domain at

amino acid sites: Lys20, Ile22, Asp58, Arg135, and Phe140.

In order to assess activity of FVIIa, FVIIa, and TF219 or a TF219 -containing multi-

chain chimeric polypeptide were mixed at an equal molar concentration (10 nM) in all

wells of a 96-well ELISA plate in a total volume of 70 uL. After incubation for 10

minutes at 37 °C, 10 uL of 8 mM S-2288 substrate was added to start the reaction. The

incubation was then kept at 37 °C for 20 minutes. Finally, color change was monitored

by reading absorbance at 405 nm. The OD values of different TF/VIIa complexes are

shown in Table 1 and Table 2. Table 1 shows a comparison of TF219, 21t15-21s wild-type

(WT) and 21t15-21s mutant (Mut). Table 2 shows a comparison of TF219, 21t15-TGFRs

wild-type (WT), and 21t15-TGFRs mutant (Mut). These data show that TF219-containing

multi-chain chimeric polypeptides (e.g., 21t15-21s-WT, 21t15-21s-Mut, 21t15-TGFRS-

WT, and 21t15-TGFRS-Mut) have lower FVIIa activity than TF219 when the

chromogenic S-2288 was used as a substrate. Notably, the multi-chain chimeric

polypeptides containing TF219 mutations showed much lower FVIIa activity when

compared to multi-chain chimeric polypeptides containing wild type TF219.

Table 1. FVIIa activity

Molecule OD value at 405 nm

TF219 0.307 0.307

21t15/21S-WT 21t15/21S-WT 0.136 0.136

21t15/21S-Mut 0.095

WT: wild type of TF219, Mut: TF219 containing mutations.

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Table 2. FVIIa activity

Molecule OD value at 405 nm

TF219 0.345

21t15/TGFRS-WT 0.227

21t15/TGFRS-Mut 0.100

WT: wild type of TF219, Mut: TF219 containing mutations.

Factor X (FX) Activation Assay

An additional assay to measure blood coagulation involves measuring activation

of Factor X (FX). Briefly, TF/VIIa activates blood coagulation Factor X (FX) to Factor

Xa (FXa) in the presence of calcium and phospholipids. TF243, which contains the

transmembrane domain of TF, has much higher activity in activating FX to FXa than

TF219, which does not contain the transmembrane domain. TF/VIIa dependent activation

of FX is determined by measuring FXa activity using an FXa-specific chromogenic

substrate S-2765 (Diapharma, West Chester, OH). The color change of S-2765 can be

monitored spectrophotometrically and is proportional to the proteolytic activity of FXa.

In these experiments, FX activation with a multi-chain chimeric polypeptide

(18t15-12s, mouse (m)21t15, 21t15-TGFRs, and 21t15-7s) was compared with a positive

control (Innovin) or TF219. TF219 (or TF219-containing multi-chain chimeric

polypeptides)/FVIIa complexes were mixed at an equal molar concentration (0.1 nM

each) in a volume of 50 uL in round bottom wells of a 96-well ELISA plate, after which

10 uL of 180 nM FX was added. After 15 minutes of incubation at 37 °C, during which

time FX was converted to FXa, 8 uL of 0.5 M EDTA (which chelates calcium and thus

terminates FX activation by TF/VIIa) was added to each well to stop FX activation.

Next, 10 uL of 3.2 mM S-2765 substrate was added to the reaction mixture.

Immediately, the plate absorbance was measured at 405 nm and was recorded as the

absorbance at time 0. The plate was then incubated for 10-20 minutes at 37 °C. The

color change was monitored by reading absorbance at 405 nm following the incubation.

Results of FX activation as measured by FXa activity using chromogenic substrate S-

2765 are shown in Figure 183. In this experiment, Innovin, which is a commercial

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prothrombin reagent containing lipidated recombinant human TF243, was used as a

positive control for FX activation. Innovin was reconstituted with purified water to about

10 nM of TF243. Next, 0.1 nM TF/VIIa complex was made by mixing an equal volume of

0.2nM of FVIIa with 0.2 nM of Innovin. Innovin demonstrated very potent FX activation

activity, while TF219 and TF219-containing multi-chain chimeric polypeptides had very

low FX activation activity, confirming that TF219 is not active in a TF/FVIIa complex for

activating natural substrate FX in vivo.

Prothrombin Time Test

A third assay to measure blood coagulation is the prothrombin time (PT) test,

which measures blood clotting activity. Here, the PT test was performed using

commercially available normal human plasma (Ci-Trol Coagulation Control, Level I).

For a standard PT test, clot reactions were initiated by addition of Innovin, a lipidated

recombinant human TF243, in the presence of calcium. Clotting time was monitored and

reported by STart PT analyzer (Diagnostica Stago, Parsippany, N.J.). PT assays were

started by injecting 0.2 mL of various dilutions of Innovin diluted in PT assay buffer (50

mM Tris-HCl, pH 7.5, 14.6 mM CaCl2, 0.1% BSA) into cuvettes containing 0.1 mL of

normal human plasma prewarmed at 37 °C. In the PT assay, shorter PT time (clotting

time) indicates a higher TF-dependent clotting activity while longer PT (clotting time)

means lower TF-dependent clotting activity.

As seen in Figure 184, addition of different amounts of Innovin (e.g., Innovin

reconstituted with purified water equivalent to 10 nM of lipidated recombinant human

TF243 was considered to be 100% Innovin) to the PT assay demonstrated a dose-response

relationship, where lower concentrations of TF243 resulted in a longer PT time (lower

clotting activity). For example, 0.001% Innovin had a PT time greater than 110 seconds,

which was almost the same as buffer alone.

In another experiment, the PT test was conducted on TF219 and multi-chain

chimeric polypeptides including: 18t15-12s, 7t15-21s, 21t15-TGFRs-WT, and 21t15-

TGFRs-Mut. Figure 185 show that TF219 and TF219-containing multi-chain chimeric

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polypeptides (at a concentration of 100 nM) had prolonged PT times indicating extremely

low or no clotting activity.

Studies were also conducted to evaluate whether incubating the multi-chain

chimeric polypeptides in the presence of other cells carrying receptors for the cytokine

components of the multi-chain chimeric polypeptide (32DB or human PBMCs) would

affect the clotting time in the PT assay. To examine whether cells that express IL-15

receptor (32DB cells) or IL-15 and IL-21 receptors (PBMCs) would bind IL-15 -

containing multi-chain chimeric polypeptides to mimic natural TF as a cellular FVIIa

receptor, TF219-containing multi-chain chimeric polypeptides (at a concentration of 100

nM for each molecule) were diluted in the PT assay buffer and preincubated with 32DB

cells (at 2 X 105 cells/mL) or PBMC (at 1 X 105 cells/mL) for 20-30 minutes at room

temperature. The PT assay was then conducted as described above. Figures 186 and 187

shows that TF219 and TF219-containing multi-chain chimeric polypeptides mixed with

32DB cells (Figure 186) or PBMC (Figure 187) at a final concentration of 100 nM had

prolonged PT times similar to 0.001-0.01% Innovin (equivalent to 0.1 pM to 1.0 pM of

TF243). Expressed in percentage of relative TF243 activity, TF219-containing multi-chain

chimeric polypeptides had 100,000 to 1,000,000 times lower TF dependent clotting

activity when compared to Innovin. This demonstrated that TF219-containing multi-chain

chimeric polypeptides had extremely low or no TF-dependent clotting activity, even

while the molecules were bound to an intact cell membrane surface, such as 32DB or

PBMCs.

Example 72: Characterization of 7t15-21s137L (long version)

The nucleic acid sequence of the 7t15 construct (including signal peptide

sequence) is as follows (SEQ ID NO: 210):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTO C (Human IL7) wo WO 2021/247604 PCT/US2021/035285

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCO TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGA

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATO AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACA CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACO ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACA CTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGO TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTC AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACA CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA STTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAG GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

ACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAG AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTO AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT wo WO 2021/247604 PCT/US2021/035285

GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA CATTGTCCAGATGTTCATCAATACCTCC CATTGTCCAGATGTTCATCAATACCTCO

The amino acid sequence of 7t15 fusion protein (including the leader sequence) is

as follows (SEQ ID NO: 209):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL7)

CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM

FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG

QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG

DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN DASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFN TS TS The nucleic acid sequence of the 21s137L construct (including signal peptide

sequence) is as follows (SEQ ID NO: 331):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG AGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC

CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG wo 2021/247604 WO PCT/US2021/035285

GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTO GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGO AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA

GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAG GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC

GTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S). 3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

GCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG CCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGF GCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA TCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT GGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21s137L fusion protein (including the leader

sequence) is as follows (SEQ ID NO: 332):

(Signal peptide)

MKWVTFISLLFLFSSAYS (Human IL-21) wo 2021/247604 WO PCT/US2021/035285

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

TCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGI SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE GATVLGLFRVTPEIPAGLPSPRSE

The following experiment was conducted to evaluate whether the CD137L

portion in 7t15-21s137L was intact to bind to CD137 (4.1BB). On day 1, a 96-well plate

was coated with 100 uL (2.5 ug/mL) of GAH IgG Fc (G-102-C, R&D Systems) in R5

(coating buffer), overnight. On day 2, the plates were washed three times and blocked

with 300 uL of 1% BSA in PBS at 37°C for 2 hrs. 10 ng/ml of 4.1BB/Fc (838-4B, R&D

Systems) was added at 100 ul/well for 2 hrs at room temperature. Following three

washes, 7t15-21s137L (long version) or 7t15-21s137Ls (short version) was added

starting at 10 nM, or recombinant human 4. 1BBL starting at 180ng/mL, with 1/3 dilution,

followed by incubation at 4°C overnight. On day 3, the plates were washed three times,

and 500 ng/mL of biotinylate-goat anti-human 4. 1BBL (BAF2295, R&D Systems) was

applied at 100 uL per well, followed by incubation at RT for 2 hrs. The plates were

washed three times, and incubated with 0.25 ug/mL of HRP-SA (Jackson

ImmuneResearch) at 100 uL per well for 30 min. The plates were then washed three

times, and incubated with 100 uL of ABTS for 2 mins at RT. The results were read at

405 nm. As shown in Figure 188, both 7t15-21s137L (long version) and 7t15-21s137L

(short version) could interact with 4.1BB/Fc (dark diamond and gray square) compared to

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the recombinant human 4. 1BB ligand (rhCD137L, light gray star). 7t15-21s137L (long

version) (dark diamond) interacted better with 4. 1BB/Fc as compared to 7t15-21s137L

(short version) (gray square).

The following experiments were conducted to evaluate whether the components

IL7, IL21, IL15, and 4. IBBL in 7t15-21s137L (long version) were intact to be detected

by the individual antibody using ELISA. A 96-well plate was coated with 100 uL (4

ug/mL) of anti-TF (human IgG1) in R5 (coating buffer) and incubated at RT for 2 hrs.

The plates were washed three times, and blocked with 100 uL of 1% BSA in PBS.

Purified 7t15-21s137L (long version) was added starting at 10 nM, and at 1/3 dilution,

followed by incubation at RT for 60 min. The plates were washed three times, and 500

ng/mL of biotinylate-anti-IL7 (506602, R&D Systems), 500 ng/mL of biotinylate-

IL21 (13-7218-81, R&D Systems), 50 ng/mL of biotinylate-anti-IL15 (BAM247, R&D

Systems), or 500 ng/ml of biotinylate-goat anti-human 4.1BBL (BAF2295, R&D

Systems) was added per well and incubated at room temperature for 60 min. The plates

were washed three times and incubated with 0.25 ug/mL of HRP-SA (Jackson

ImmunoResearch) at 100 uL per well for 30 min at RT. The plates were washed four

times, and incubated with 100 uL of ABTS for 2 mins at room temperature. The

absorbance results were read at 405 nm. As shown in Figure 189A-189D, the

components including IL7, IL21, IL15, and 4. 1BBL in 7t15-21s137L (long version) were

detected by the individual antibodies.

The following experiment was conducted to evaluate the activity of IL15 in 7t15-

21s137L (long version) and 7t15-21s137L (short version). The ability of 7t15-21s137L

(long version) and 7t15-21s137L (short version) to promote proliferation of IL2RaBy-

expressing CTLL2 cells was compared with that of recombinant IL15. IL15 dependent

CTLL2 cells were washed five times with IMDM-10% FBS and seeded to the wells at 2

X 104 cells/well. Serially diluted 7t15-21s137L (long version), 7t15-21s137L (short

version), or IL15 were added to the cells. Cells were incubated in a CO2 incubator at 37

°C for 3 days. Cell proliferation was detected by adding 20 uL of PrestoBlue (A13261,

ThermoFisher) to each well on day 3 and incubated for an additional 4 hours in a CO2

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incubator at 37 °C. Raw absorbance at 570-610 nm was read in a micro-titer plate reader.

As shown in Figure 190, 7t15-21s137L (long version), 7t15-21s137L (short version), and

IL15 all promoted CTLL2 cell proliferation. The EC50 of 7t15-21s137L (long version),

7t15-21s137L (short version), and IL15 is 51.19 pM, 55.75 pM, and 4.947 pM,

respectively.

Example 73: Induction of Treg cells by 2t2

The peripheral blood mononuclear cells (PBMC) of a heathy donor (Donor 163)

were isolated from 5 mL of whole blood buffy coats by Ficoll Paque Plus (GE17144003).

The PBMC were then lysed with ACK to remove red blood cells. Cells were washed

with IMDM-10% FBS and counted. 1.8 x106 cells (100 uL/tube) were seeded to the flow

tubes and incubated with 50 uL of descending 2t2 or IL2 (15000, 1500, 150, 15, 1.5,

0.15, or 0 pM) and 50 uL of pre-staining antibodies (anti-CD8-BV605 and anti-CD127-

AF647). Cells were incubated for 30 min at 37 °C in water bath. 200 uL of pre-warmed

BD Phosflow Fix Buffer I (Cat# 557870, Becton Dickinson Biosciences) was added for

10 min at 37° C in water bath to stop the stimulation. Cells (4.5 x105 cells/100 uL) were

transferred to a V-shape 96-well plate and were spun down followed by permeabilization

with 100 uL of -20 °C pre-cooled BD Phosflow Perm Buffer III (Cat# BD Biosciences)

for 30 min on ice. The cells were then extensively washed x2 with 200 uL of FACS

buffer and stained with a panel of fluorescent antibodies (anti-CD25-PE, CD4-PerCP-

Cy5.5, CD56-BV421, CD45RA-PE-Cy7 and pSTAT5a-AF488) to distinguish between

different lymphocyte subpopulations and evaluate the pSTAT5a status. Cells were spun

down and resuspended in 200 uL of FACS buffer for FACSCelesta analysis. As sown in

Figure 191A, 6 pM of 2t2 was sufficient to induce the phosphorylation of Stat5a in

CD4*CD25hi Treg cells while 43.11 pM of IL-2 was required to induce phosphorylation of

Stat5a in the same population of lymphocytes. In contrast, 2t2 was less active (Figure

191B) or equally active (Figure 191C) as compared to IL2 in inducing phosphorylation of

Stat5a in CD4*CD25*Tcon and CD8 Tcon cells. These results suggest that 2t2 is superior

as compared to IL2 in activating Treg in human PBMC, and that 2t2 demonstrates

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increased Treg selectivity compared to IL-2 in human blood lymphocyte pStat5a

responses

Example 74. Improvement in Hair Growth using a Single-Chain Chimeric

Polypeptide

The dorsal hair of 7-week-old C57BL6/J mice was shaved and depilated using

commercial depilatory cream. The mice were injected on the same day subcutaneously

with a single dose of 2t2 or low dose commercially available recombinant IL-2, followed

by daily dosing for four additional days. Untreated mice served as controls. On day 10,

the mice were sacrificed and skin sections of the shaved areas were prepared.

Representative H&E staining of skin sections from C57BL6J mice on day 10 following

depilation are shown in Figures 192A - 192E. Figure 192A shows control mice - only

depilation done after hair was shaved, Figure 192B shows mice where depilation was

followed by low dose IL-2 (1 mg/kg) administration, and Figures 192C-192E shows mice

where depilation was followed by 2t2 administered at 0.3 mg/kg (Figure 192C), 1 mg/kg

(Figure 192D), and 3 mg/kg (Figure 192E). Black arrows indicate anagen-phase hair

follicles that will later extend into dermis and facilitate hair growth. Figure 194 shows

the total number of anagen phase hair follicles counted per 10 fields for each treatment

group. In summary, the data show that the 2t2 molecule resulted in increased numbers of

anagen-phase hair follicles compared to depilation alone. This effect was also dose-

dependent.

Example 75: Differentiation of the Immune Cell into a Memory-Like Immune Cell

Fresh human leukocytes were obtained from the blood bank and CD56+ NK cells

were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The

purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-

BV510, CD25-PE, CD69-APCFire750 (BioLegend). The cells were counted and

resuspended at a density of 2 x 106 cells/mL in RPMI 1640 medium (Gibco)

supplemented with 2 mM L-glutamine (Thermo Life Technologies), antibiotics

(penicillin, 10,000 units/mL; streptomycin, 10,000 ug/mL; Thermo Life Technologies), and 10% FBS (Hyclone). The cells (1 mL) were transferred into a 24-well flat bottom plate, and subjected to either: no treatment, or expanded with 7t15-21s + anti-tissue factor

(TF)-antibody (IgG1) (50 nM) for 14 days with medium. The cells were replenished with

fresh 7t15-21s + anti-TF-antibody (IgG1) (50 nM) to keep the cell density at

approximately 1 X 106 cells/mL.

Unexpanded NK cells to treatment groups were used as positive controls for full

DNA methylation levels (Data not shown). NK cells were pelleted (1 X 106), and

genomic DNA (nDNA) isolated using the QIAamp UCP DNA Micro Kit (Qiagen). 500

ng of purified nDNA was subjected to sodium bisulfite treatment using the EZ DNA

Methylation-Direct kit (Zymo Research) according to the manufacturer's protocol.

Bisulfite treatment introduces methylation-dependent changes in the DNA with

demethylated cytosines being converted into uracil, whereas methylated cytosines remain

unchanged. The bisulfite-treated nDNA (10-50 ng) was used as template to PCR amplify

a 228 bp region of the IFNy promoter containing two CpG sites (CpG -186 and CpG -54,

position relative to the transcription start site, TSS), known to be heavily regulated by

DNA methylation in T cells, using the Pyromark PCR kit (Qiagen) with the forward

primer IFNG127F (5'-ATGGTATAGGTGGGTATAATGG-3') and the biotinylated

reverse primer IFNG355R-bio (biotin-5'-CAATATACTACACCTCCTCTAACTAC-3') (GENEWIZ). The PCR conditions were 15 minutes at 95°C, 48 cycles of 30 seconds at

95 °C, 30 seconds at 56°C, 60 seconds at 72°C followed by 10 minutes at 72°C. The

integrity and quality of the PCR amplified products were visualized on a 1.2% TAE

agarose gel. The DNA methylation status of these two CpG sites was determined by

pyrosequencing, which is the gold standard technique to quantitatively measure DNA

methylation at single CpG-site. Pyrosequencing reactions were performed at Johns

Hopkins University Genetic Resources Core Facility using the DNA sequencing primers

C186-IFNG135F (5'-GGTGGGTATAATGGG-3') (SEQ ID NO: 333) and C54-

IFNG261F (5'-ATTATTTTATTTTAAAAAATTTGTG-3') (SEQ ID NO: 334), specific to the CpG sites -186 and -54, respectively. Commercially available non-methylated and

methylated DNA (Zymo Research) were used as controls for DNA methylation. The

methylation percentages of the two CpG sites (-186 and -54) were pooled for each

PCT/US2021/035285

treatment. The percent difference in DNA methylation was calculated relative to the

levels of DNA methylation at the two CpG sites observed in unexposed NK cells.

Analysis of the DNA methylation status of these two IFNy CpG sites revealed

higher levels of DNA demethylation in NK cells supported by 7t15-21s + anti-TF-

antibody compared to unexposed NK cells (Figure 194). These 7t15-21s + anti-TF-

antibody supported NK cells exhibited 47.70% + 11.76 difference in DNA methylation

(i.e., demethylation) compared to unexposed NK cells. The DNA methylation levels of

these two IFNy CpG sites correlated with increased expression of IFNy following

treatment with 7t15-21s + anti-TF-antibody. These data suggest that long-term exposure

of NK cells (14 days expansion in culture) with a combination regimen of 7t15-21s +

anti-TF-antibody is able to induce DNA demethylation of the two hypomethylated IFNy

CpG sites (-186 and -54) and that 7t15-21s + anti-TF-antibody (IgG1) can epigenetically

reprogram gene expression of IFNy via DNA demethylation of CpG sites leading to

interconversion of NK cells into innate immune memory NK cells.

Example 76: Chemotherapy Induces p21ciP1p21 Senescence-associated Gene

Expression in C57BL/6 Mice Chemotherapy induces p21clPlp21 senescence-associated gene expression in

C57BL/6 Mice. Figure 203A is a schematic showing the treatment regimen. C57BL/6

mice were treated with three doses of chemotherapy docetaxel (DTX) (10 mg/kg) at day

1, day 4 and day 7. At day 9 the mice were sacrificed, and lung and liver tissues were

harvested to evaluate the senescence markers. Figures 203B and 203C show expression

of p21clPlp21 in lung (B) and liver (C) tissues respectively. Lung and liver tissues were

homogenized by using mortar and pestle in liquid nitrogen. Homogenized tissues were

transferred in fresh Eppendorf tubes containing 1 mL of Trizol (Thermo Fischer). Total

RNA was extracted using RNeasy Mini Kit (Qiagen #74106) according to the

manufacturer's instructions. 1 ug of total RNA was used for cDNA synthesis using the

QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with

CFX96 Detection System (Bio-Rad) using FAM labeled predesigned primer p21clPlp21

were purchased from Thermo Scientific. Reactions were run in triplicate for all the genes

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examined. The housekeeping gene 18S ribosomal RNA was used as an internal control to

normalize the variability in expression levels. The expression of each target mRNA

relative to 18S rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Cttarget Ct18s.

As shown in Figures 203A-203C, the senescence marker p21clPlp21 was induced in the

lung and liver tissues of mice treated with docetaxel.

Example 77: Immuno-phenotype and Cell Proliferation following Treatment with

IL-15-based Agents (Day 3 post treatment)

The mouse blood was prepared in order to evaluate the different subsets of

immune cells after treatment with TGFRt15-TGFRs. C57BL/6, 6-week-old mice were

purchased from The Jackson Laboratory. Mice were housed in a temperature and light

controlled environment. Mice were divided into groups as follows: Saline control group

(n =6), docetaxel group (n =6), docetaxel with TGFRt15-TGFRs group (n =6) and

docetaxel with IL-15SA group (n =6). The IL-15 superagonist (IL-15SA) was

constructed and administered as previously described (Zhu et al., J. Immunol.

183(6):3598-3607, 2009). Senescence was induced in mice with three doses of docetaxel

(10 mg/kg) at day 1, 4 and 7. On day 8, mice were treated subcutaneously with either

PBS or with TGFRt15-TGFRs (3 mg/kg) or with IL-15SA (0.2 mg/kg). The mouse blood

was collected from submandibular vein on Day 3 post treatment in EDTA contained

tubes. The whole blood was centrifuged to collect plasma @ 3000 RPM for 10 minutes in

a micro centrifuge. Plasma was stored at -80°C and whole blood was processed for

immune cells phenotyping by flow cytometry. Whole bloods were lysed in ACK buffer

for 5 minutes at room temperature. Cell were washed in FACS buffer (1X PBS (Hyclone)

with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)). To assess the

different types of immune cells in blood, cells were stained for cell-surface CD4, CD45,

CD8 and NK1.1 (BioLegend) for 30 minutes at RT. After surface staining, cells were

washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (1X PBS

(Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)). Cells

were treated with permeabilization buffer (Invitrogen) for 20 min at 4°C followed by

wash with Perm buffer (Invitrogen). Cells were then stained for intracellular markers wo 2021/247604 WO PCT/US2021/035285 PCT/US2021/035285

(Ki67) and FoxP3 for 30 min at room temperature. After two washes, cells were

resuspended in fixation buffer and analyzed by Flow Cytometry (Celesta-BD

Bioscience). These data show that IL-15-based agents TGFRt15-TGFRs and IL-15SA

can stimulate and promote the expansion and proliferation of NK and CD8+ T cells after

docetaxel treatment (Figure 204).

Example 78: TGFRt15-TGFRs Treatment Reduces Senescence-associated Gene

Expression in C57BL/6 Mice

Chemotherapy induced senescence-associated gene expression was significantly

reduced with TGFRt15-TGFRs in the lung and liver of C57BL/6 mice. C57BL/6 mice

were treated with three doses of chemotherapy docetaxel (10 mg/kg) at day 1, day 4 and

day 7. On day 8, docetaxel treated mice were divided into three groups. The first group

received no treatment, second group received TGFRt15-TGFRs and third group received

IL-15SA. Saline treated mice were used as controls. The TGFRt15-TGFRs was

administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.2 mg/kg. On

Day 3 post-study drug treatment, the mice were sacrificed and lung and liver were collected.

Figures 205A-205C show expression of p21clPlp21 and CD26 in lung (A and B) and

p21clPlp21 in liver (C) tissues respectively. Lung and liver tissues were homogenized by

using mortar and pestle in liquid nitrogen. Homogenized tissues were transferred in fresh

Eppendorf tubes containing 1mL of Trizol (Thermo Fischer). Total RNA was extracted

using RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's instructions. 1

ug of total RNA was used for cDNA synthesis using the Quanti Tect Reverse

Transcription Kit (Qiagen). Real-time PCR was carried out with CFX96 Detection

System (Bio-Rad) using FAM labeled predesigned primers p21 CIP 1p21 and CD26 were

purchased from Thermo Scientific. Reactions were run in triplicate for all the genes

examined. The housekeeping gene 18S ribosomal RNA was used as an internal control to

normalize the variability in expression levels. The expression of each target mRNA

relative to 18S rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Cttarget Ctiss.

As shown in Figures 205A-205C, the therapy-induced senescence marker

p21clP1p21 was significantly reduced in the lung and liver tissues of mice treated with

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TGFRt15-TGFRs. The therapy-induced senescence marker CD26 was also significantly

reduced in the lung tissues of mice treated with TGFRt15-TGFRs.

Example 79: Immuno-Phenotype Following Treatment with IL-15-based Agents

The mouse blood was prepared in order to evaluate the different subsets of

immune cells after treatment with IL-15-based agents: TGFRt15-TGFRs, an IL-15

superagonist (IL-15SA) and an IL-15 fusion with a D8N mutant knocking out the IL-15

activity (TGFRt15*-TGFRs). C57BL/6, 6-week-old mice were purchased from The

Jackson Laboratory. Mice were housed in a temperature and light controlled

environment. Mice were divided into groups (n =6/group) and treated with the following:

1) PBS (saline) control, 2) docetaxel, 3) docetaxel with TGFRt15-TGFRs, 4) docetaxel

with IL-15SA, 5) docetaxel with an IL-15 mutant (TGFRt15*-TGFRs) and 6) docetaxel

with an IL-15 superagonist (IL-15SA) plus TGFRt15*-TGFRs. Senescence was induced

in mice with three dose of docetaxel (10 mg/kg) at day 1, 4 and 7. On day 8, the mice

were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA

or in combinations as discussed above. TGFRt15-TGFRs and TGFRt15*-TGFRs were

administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. The

mouse blood was collected from the submandibular vein on day 3 post-study drug

treatment into EDTA tubes. The whole blood was centrifuged to collect plasma at 3000

RPM for 10 minutes in a micro centrifuge. Plasma was stored at -80°C and whole blood

was processed for immune cell phenotyping by flow cytometry. Whole blood was lysed

in ACK buffer for 5 minutes at 37°C. Cell were washed in FACS buffer (1X PBS

(Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)). To

assess the different types of immune cells in the blood, cells were stained for cell-surface

CD4, CD45, CD19 CD8 and NK1.1 (BioLegend) for 30 minutes at room temperature

(RT). After surface staining, cells were washed (1500 RPM for 5 minutes at room

temperature) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and

0.001% Sodium Azide (Sigma)). Cells were treated with permeabilization buffer

(Invitrogen) for 20 min at 4°C followed by wash with Perm buffer (Invitrogen). Cells

were then stained for intracellular markers (Ki67) for 30 min at RT. After two washes,

WO wo 2021/247604 PCT/US2021/035285

cells were resuspended in fixation buffer and analyzed by Flow Cytometry (Celesta-BD

Bioscience) (Figures 206 and 207).

These data show that IL-15-based agents TGFRt15-TGFRs and IL-15SA can

stimulate and promote the expansion and proliferation of NK and CD8+ T cells after

docetaxel treatment. Increased NK and CD8+ T cell expansion and proliferation was not

seen with fusion proteins lacking IL-15 activity (i.e., TGFRt15*-TGFRs).

Example 80: Evaluation of Senescence Markers p21clP1p21 and CD26 in Lung and

Liver Tissues

Markers for cellular senescence were evaluated in tissues of normal mice

following chemotherapy and administration of study treatments. C57BL/6, 6-week-old

mice were purchased from The Jackson Laboratory. Mice were housed in a temperature

and light controlled environment. Mice were divided into six groups and treated with the

following: 1) PBS (saline) control (n =5), 2) docetaxel (n =8), 3) docetaxel with

TGFRt15-TGFRs (n =8), 4) docetaxel with IL L15SA (n =8), 5) docetaxel with an IL-15

mutant (TGFRt15*-TGFRs) (n =8) and 6) docetaxel with an IL-15 superagonist (IL-

15SA) plus TGFRt15*-TGFRs (n =6). Senescence was induced in mice with three doses

of docetaxel (10 mg/kg) at day 1, 4 and 7. On day 8, the mice were treated

subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA or in

combinations as discussed below. TGFRt15-TGFRs and TGFRt15*-TGFRs were

administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. The

mouse tissues were prepared in order to evaluate the different senescence markers. Mice

were euthanized on day 7 post-study drug treatment and the liver and lung tissues were

harvested and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were

homogenized by using mortar and pestle in liquid nitrogen. Homogenized tissues were

transferred in fresh Eppendorf tubes containing 1 mL of Trizol (Thermo Fischer). Total

RNA was extracted using RNeasy Mini Kit (Qiagen #74106) according to the

manufacturer's instructions and 1 ug of total RNA was used for cDNA synthesis using

the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with

CFX96 Detection System (Bio-Rad) using FAM labeled predesigned primers purchased

WO wo 2021/247604 PCT/US2021/035285

from Thermo Scientific. Reactions were run in triplicate for all the genes examined. The

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in expression levels. The expression of each target mRNA relative

to 18S rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Cttarget Ctiss.

As shown in Figures 208A-208C, the senescence markers p21 and CD26 were induced in

the lung [(A) and (B), respectively] and p21clPlp21 in liver (C) tissues of mice treated

with docetaxel. The senescence markers p21clPlp21 and CD26 in the lungs and

p21clP1p21 in the liver were reduced of the mice treated with TGFRt15-TGFRs, IL-15SA

and combination of IL-15SA and TGFRt15*-TGFRs mutant. However, the TGFRt15*-

TGFRs mutant treated mice lung failed to eliminate the senescence markers in these

tissues. These results show that IL-15 activity is important for clearance of TIS

senescence cells.

Example 81: Immuno-Phenotype Following Treatment with TGFRt15-TGFRs

The mouse blood was prepared in order to evaluate the different subsets of

immune cells after treatment with TGFRt15-TGFRs. C57BL/6, 76-week-old aged mice

were purchased from The Jackson Laboratory. Mice were housed in a temperature and

light controlled environment. Mice were divided into two groups as follows: PBS control

group (n=6) and TGFRt15-TGFRs group (n =6). Mice were treated subcutaneously with

either PBS or with TGFRt15-TGFRs at a dosage of 3 mg/kg on Day 0. On Day 4

following the first dose of study treatment, the mouse blood was collected from the

submandibular vein in EDTA contained tubes. The whole blood was centrifuged to

collect plasma at 3000 RPM for 10 minutes in a micro centrifuge. Plasma was stored at -

80°C and the blood was processed for immune cell phenotyping by flow cytometry.

Whole blood was lysed in ACK buffer for 5 minutes at room temperature. Cells were

washed in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and

0.001% Sodium Azide (Sigma)). To assess the different types of immune cells in blood,

cells were stained for cell-surface CD4, CD45, CD19 CD8 and NK1.1 (BioLegend) for

30 minutes at room temperature (RT). After surface staining, cells were washed (1500

RPM for 5 minutes at RT) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD

WO wo 2021/247604 PCT/US2021/035285

Millipore) and 0.001% Sodium Azide (Sigma)). Cells were treated with permeabilization

buffer (Invitrogen) for 20 min at 4°C followed by wash with Perm buffer (Invitrogen).

Cells were then stained for intracellular markers (Ki67) for 30 min at RT. After two

washes, cells were resuspended in fixation buffer and analyzed by flow cytometry

(Celesta-BD Bioscience).

As shown in Figure 195, the percentages of CD8+ T cells and proliferation of

CD8+ T cells, which was measured by Ki67, significantly increased, 4 days after the first

dose of TGFR115-TGFRs. We also observed an increase in NK cells and proliferation of

NK cells as shown in Figure 196. We observed significant decreases in CD19+ cells after

the first dose of TGFRt15-TGFRs. These results demonstrate that a single dose of

TGFRt15-TGFRs administered subcutaneously can stimulate immune cells, such as

CD8+ T cells and NK cells to proliferate in the blood of aged mice.

Example 82: TGFRt15-TGFRs Reduces Senescence-Associated B-Gal from Liver

and Lung Tissues

The mouse liver and lungs were prepared in order to evaluate the senescence-

associated B-gal in tissues after treatment with TGFRt15-TGFRs. C57BL/6, 76-week-old

aged mice were purchased from The Jackson Laboratory. Mice were housed in a

temperature and light controlled environment. Mice were divided into two groups as

follows: PBS control group (n =6) and TGFRt15-TGFRs group (n =6). Mice were treated

subcutaneously with either PBS or with TGFRt15-TGFRs at a dosage of 3 mg/kg on Day

0 and Day 10. On Day 7 following the second dose of study treatment, mice were

euthanized and liver and lungs were harvested, homogenized in PBS containing 2% PBS,

and filtered in 70-micron filter to obtain a single cell suspension. Cells were spun down

then resuspended in 5 mL RPMI containing 0.5 mg/mL collagenase IV and 0.02 mg/mL

DNAse in 14 mL round bottom tubes. Then, the cells were shaken on orbital shaker for 1

hr at 37°C. The cells were washed twice with RPMI. Cells were resuspended at 2 X

106/mL in a 24 well flat bottom plate in 2 mL of complete media (RPMI 1640 (Gibco)

supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo

Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone))

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and cultured for 48 hrs at 37°, 5% CO2. Cells were harvested, washed once in warm

complete media at 1000 rpm for 10 minutes at room temperature. The cell pellet was

resuspended in 500 uL of fresh media containing 1.5 uL of Senescence Dye per tube.

Then, the cells were further incubated for 1-2 hr at 37°C, 5% CO2 and washed 2X with

500 uL Wash buffer. Cell pellet was resuspended cells in 500 uL of wash buffer and was

analyzed immediately by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 197, the percentages of senescence-associated B-gal+ cells

decreased 7 days following the second dose of TGFRt15-TGFRs. These results

demonstrate that TGFRt15-TGFRs can reduce the senescence-associated B-gal in tissues

of aged mice.

Example 83: Senescence Markers CD26, IL-1a, p16INK4 and p21 CIP1 in Kidney,

Skin, Liver and Lung Tissues

The mouse kidney, skin, liver and lungs were harvested in order to evaluate the

senescence markers CD26, IL-1a, p16 and p21 by quantitative PCR in tissues after

treatment with TGFRt15-TGFRs or the PBS control group. C57BL/6, 76-week-old aged

mice were purchased from The Jackson Laboratory. Mice were housed in a temperature

and light controlled environment for one week before performing any study. Mice were

divided into two groups as follows: PBS control group (n =6) and TGFRt15-TGFRs

group (n =6). Mice were treated subcutaneously either with PBS or with TGFRt15-

TGFRs at a dosage of 3 mg/kg on Day 0 and Day 10. On Day 7 following the second

dose of study treatment, mice were euthanized and the kidney, skin, liver and lung were

harvested and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were

homogenized by using mortar and pestle in liquid nitrogen. Homogenized tissues were

transferred in fresh Eppendorf tubes containing 1 mL of Trizol (Thermo Fischer). Total

RNA was extracted using RNeasy Mini Kit (Qiagen #74106) according to the

manufacturer's instructions and 1 ug of total RNA was used for cDNA synthesis using

the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with

CFX96 Detection System (Bio-Rad) using FAM labeled predesigned primers purchased

from Thermo Scientific. Reactions were run in triplicate for all the genes examined. The

WO wo 2021/247604 PCT/US2021/035285 PCT/US2021/035285

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in expression levels. The expression of each target mRNA relative

to 18S rRNA was calculated based on Ct as 2-^(ACt)_ in which ACt = Cttarget Ctiss.

As shown in Figures 198-201, there was no difference in senescence markers

CD26 and IL-1a, however p21 CIP1 showed decreased expression in the liver (Figure 198),

lung (Figure 201) and skin (Figure 200) of TGFRt15-TGFRs-treated-mice. In the kidney

(Figure 199), both p21CIPI and IL ,la markers were significantly decreased in the aged

mice 7 days after the second dose of TGFRt15-TGFRs.

Example 84: B-Gal Staining on Kidney Tissues by Histology

The mouse kidney was prepared in order to evaluate senescence marker B-gal in

kidney tissues after treatment with TGFRt15-TGFRs. C57BL/6, 76-week-old aged mice

were purchased from The Jackson Laboratory. Mice were housed in a temperature and

light controlled environment. Mice were divided into two groups as follows: PBS control

group (n=6) and TGFRt15-TGFRs group (n =6). Mice were treated subcutaneously with

either PBS or with TGFRt15-TGFRs at a dosage of 3 mg/kg on Day 0 and Day 10. On

Day 7 following the second dose of study treatment, mice were euthanized and the

kidneys were harvested, and half of the kidney tissue was embedded in tissue-tek

cyromolds contain OCT compound. Tissue-tek cyromolds containing tissue were

immediately frozen down in the vapor phase of liquid nitrogen. Samples were further

processed to cut 4-8 um thick cryostat sections (Lecia Cm 1800 Cryostat) and mounted

on superfrost plus slides. Slides with sections were processed for senescence b-

galactosidase staining kit (Cell Signaling) as per manufacturer's protocol. Tissue

sections were observed under microscope.

As shown in Figure 202, decreased numbers of senescence-associated B-gal cells

were observed in TGFRt15-TGFRs treated mice compared to control mice (n=3). These

results demonstrate that TGFRt15-TGFRs treatment is able to reduce senescence-

associated B-gal in tissues of aged mice.

Example 85: TGFRt15*-TGFRs fusion protein generation

A fusion protein complex was generated comprising of TGFR/IL15RaSu and

TGFR/TF/IL-15D8N fusion proteins (Figures 209 and 210). The human TGF-B receptor

(TGFR), IL-15 alpha receptor sushi domain (IL15RaSu), tissue factor (TF) and IL-15

with D8N mutant (IL15D8N) sequences were obtained from the GenBank website and

DNA fragments for these sequences were synthesized by Genewiz. Specifically, a

construct was made linking the TGFR sequence to the N-terminus coding region of

IL15RaSu and the TGFR sequence to the N-terminus of tissue factor 219 followed by the

N-terminus coding region of IL-15D8N.

The nucleic acid sequence of the TGFR/IL15RaSu_construct (including signal

peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Single chain Human TGF-beta Receptor II homodimer)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCA CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA AAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGA GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCC CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCG/ CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA

AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCG AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA wo WO 2021/247604 PCT/US2021/035285

GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Sushi domain of IL15 receptor alpha chain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence of the TGFR/TF/IL15D8N construct (including signal

peptide sequence) is as follows:

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC (Single chain Human TGF-beta Receptor II homodimer)

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACT GACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGA GATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCA CTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGA GACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACC GACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACC ATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAA ATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAA AAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCA ATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAG GGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCAC TGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGC GAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGAC AACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCO GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA/ GGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAA GACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA ACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTT

TAGCGAGGAATACAATACCAGCAACCCCGA0 731 wo WO 2021/247604 PCT/US2021/035285

(Human Tissue Factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCA ACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCT ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTT ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTA CACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTG/ CACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAA GCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGO ACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTA CTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA CCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA ACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAAC

ACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACT TTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACT AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTC AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCA AGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTA GAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA GAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA (Human IL-15D8N)

ACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATT ATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAG TTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCAC TTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCAC TTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCT TTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCT AGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAA GAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTG GAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTC TACATATTGTCCAAATGTTCATCAACACTTCT ACATATTGTCCAAATGTTCATCAACACTTCT

The amino acid sequence of TGFR/IL15RaSu fusion protein (including signal

peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS (Single chain Human TGF-beta Receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS) TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK wo WO 2021/247604 PCT/US2021/035285

PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVOKSVI NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD CNDNIFSEEYNTSNPD (Human IL-15 receptor a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA' ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of TGFR/TF/IL15D8N fusion protein (including signal

peptide sequence) is as follows:

(Signal peptide)

MGVKVLFALICIAVAEA (Single chain Human TGF-beta Receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKE PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVI PGETFFMCSCSSDECNDNIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD

(Tissue factor)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK YTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTE FYTTDTECDLTDEIVKDVKOTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (IL-15D8N)

NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS MFINTS

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The TGFR/IL15RgSu and TGFR/TF/IL-15D8N constructs were cloned into a

modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al). The expression vectors were transfected

into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for

formation and secretion of the soluble TGFR/IL15RaSu - TGFR/TF/IL-15D8N protein

complex (referred to as TGFRt15*-TGFRs), which can be purified by anti-TF antibody

affinity.

Example 86: Binding Activity of TGFRt15-TGFRs and TGFRt15*-TGFRs to TGF-

B1 and LAP Binding activity of TGFRt15-TGFRs to TGF-B1 and LAP was determined by

ELISA. TGFRt15-TGFRs (5 mg/mL) was used to capture the titrated TGF-B1 (labeled

as TGFß1, BioLegend) and latent associated peptide of TGF-B1 (LAP, R&D Systems).

TGF-B1 was detected by biotinylated anti-TGF-31 (0.2 mg/mL, R&D Systems) and LAP

by biotinylated anti-LAP (0.2 mg/mL, R&D Systems) followed by peroxidase conjugated

streptavidin (Jackson ImmunoResearch Lab). 2,2'-azino-bis (3-ethylbenzothiazoline-6-

sulphonic acid) (ABTS, Surmodics IVD) was used as a substrate and measured by a plate

reader. As shown in Figure 211A, the results demonstrate that TGFRt15-TGFRs binds to

TGF-31 and LAP similarly, and more strongly than the Fc fusion.

Binding activity of TGF-B1 receptor/Fc fusion to TGF-B1 and LAP was

determined by ELISA. A commercial TGF-B1 receptor II - Fc fusion (TGFRII/Fc) was

used to compare the binding activity of TGFRt15-TGFRs to TGF-B1 and LAP.

TGFRII/Fc (5 mg/mL, R&D Systems) was used to capture the titrated TGF-B1 and LAP.

Other procedures were the same as described above. As shown in Figure 211B, the

results demonstrate that TGFRII/Fc binds to TGF-B1 and LAP similarly and its binding is

comparable with TGFRt15-TGFRs, and stronger than the Fc fusion.

Binding Activity of TGFRt15-TGFRs and TGFR11**TGFRs to TGF-B1 and LAP

TGFRt15-TGFRs and TGFRt15*-TGFRs (10 mg/mL) were used to capture the

titrated TGF-31 LAP. Other procedures were the same as described above. As shown in

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Figure 211C and D, the results demonstrate that TGFRt15*-TGFRs binds to TGF-31 and

LAP similarly and its binding is comparable with TGFRt15-TGFRs, and stronger than

the Fc fusion.

Binding of TGFRt15-TGFRs and TGFR11**TGFRs to CTLL-2 Cells

IL-2-dependent CTLL-2 cells were stained with TGFRt15-TGFRs (50 0 MM),

TGFRt15*-TGFRs (50 nM), 7t15-21s (50 nM, IL-7-TF-IL15 and IL-21-IL-15RaSu) (as a

control fusion molecule, which does not contains TGF-B1 receptor II), and PBS (as a

negative control) for 60 minutes and probed by biotinylated second staining antibodies

(Anti-TF: anti-human tissue factor, HCW Biologics and Anti-TGFR: anti-TGF-ß receptor

II: R&D Systems) and then followed by R-phycoerythrin-streptavidin (Jackson

ImmunoResearch Lab). The mean fluorescent intensity (MFI) of staining was measured

by flow cytometry. As shown in Figure 211E, the results show that TGFRt15-TGFRs

bound to CTLL-2 cells significantly better than other molecules, TGFRt15*-TGFRs less

than TGFRt15-TGFRs because of the IL-15 mutant. However, 7t15-21s binding to

CTLL-2 cells could be detected with anti-TF but not anti-TGFR.

Example 87: Biological Activities of TGFRt15-TGFRs and TGFRt15*-TGFRs with

Cell-Based Assays

TGF- B1 Blocking Activities of TGFR115-TGFRs and TGFR115*-TGFRs.

HEK-Blue TGF-B cells (InvivoGen) were incubated in IMDM-10 with titrated

TGFRt15-TGFRs, TGFRt15*-TGFRs and TGFRII/Fc as a control in the presence of

TGF-B1 (0.1 nM, BioLegend). TGFRII/Fc is a commercial TGF-B1 receptor II - Fc

fusion (R&D Systems). After 24 hours of incubation, the culture supernatants were

mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hrs. The OD620 values

were measured by a plate reader. As shown in Figure 212A, TGFRt15-TGFRs and

TGFRt15*-TGFRs had the same TGF-B1 blocking activity. In contrast, TGFRII/Fc

(IC50=470.2 pM) had about 10 fold lower TGF-B1 blocking activity than TGFRt15-

TGFRs (IC50=43.2 pM) or TGFRt15*-TGFRs (45.2 pM). The blocking activity was

calculated with GraphPad Prism 7.04.

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IL-15 Activity of TGFR+15-TGFRs and TGFR11**TGFRs

IL-15 dependent 32DB cells were cultured in IMDM-10 with titrated TGFRt15-

TGFRs, TGFRt15*-TGFRs and IL15 as a control. WST-1 (Fisher Scientific) was added

2 days later and the OD450 values were measured by a plate reader. As shown in Figure

212B, TGFRt15-TGFRs (EC50=1641 pM) had about 20 fold lower IL-15 biological

activity than IL-15 itself (IC50=81.8 pM). As expected, TGFRt15*-TGFRs had no

detectable IL-15 activity. The IL-15 activity was calculated with GraphPad Prism 7.04.

Reversal of TGF-6 Growth Suppression of CTLL-2 by TGFR11**TGFRs

TGF- includes three isoforms (TGF-B1, TGF-B2 and TGF-B3). which have

similar biological functions. CTLL-2 cells were used to compare biological blocking

activity of TGFRt15*-TGFRs in this study. TGFRt15*-TGFRs is structurally very

similar to TGFRt15-TGFRs, which cannot be used to do SO due to the IL-15 activity of

TGFRt15-TGFRs. CTLL-2 cells were cultured in RPMI-10 with titrated mouse IL-4

(Biolegend), TGF-B (5 ng/ml, TGF-31 (Biolegend), TGF-B2, 33 (R&D Systems)) and

TGFRt15*-TGFRs (21 nM; TGFRt15*-TGFRs:TGF-B molar ratio=100:1) for 5 days.

Cell proliferation (OD570-600 value) was determined by a plate reader after adding

PrestoBlue (Fisher Scientific) at the last day culture. Figure 212C shows that all three

TGF-B similarly inhibited IL-4 induced CTLL-2 growth in the absence of TGFRt15*.

TGFRs. Figure 212D shows that TGFRt15*-TGFRs (21 nM; TGF-B:TGFRt15*-TGFRs

molar ratio=1:100) significantly reversed the inhibition of TGF-B1 and TGF-B3 of IL-4-

induced CTLL-2 cell growth, In contrast, TGFRt15*-TGFRs had minimum reversal

TGF-B2 inhibitory activity.

Example 88: Stability of TGFRt15-TGFRs

Stability of TGFRt15-TGFRs by ELISA. TGFRt15-TGFRs was preincubated in

RPMI medium with 50% human serum at 4°C, room temperature (RT) or 37 °C for 10

days. IL-15 domain and TGFßRII domain of TGFRt15-TGFRs were evaluated by

ELISA. Anti-TF antibody (HCW Biologics) was used to capture TGFRt15-TGFRs

molecules and biotinylated anti-IL-15 (R&D Systems) was used to detect IL-15 domain

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and biotinylated anti-TGF3RII (R&D Systems) was used to detect TGFßRII domain.

Biotinylated detection antibodies were probed by peroxidase-streptavidin (Jackson

ImmunoResearch Lab). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS,

Surmodics IVD) was used as a substrate and OD405 value was measured by a plate

reader. As shown in Figure 213A and B, the results show that there were no significant

changes in the domains of TGFRt15-TGFRs following 10 day incubation 4°C, RT, or

37°C. These findings demonstrate that IL-15 domain and TGFßRII domain of TGFRt15-

TGFRs remain intact when incubated with human serum under the evaluated conditions.

Stability of TGFR15-TGFRs Biological Activities with Cell-based Assays

TGFRt15-TGFRs was preincubated in RPMI-10 with 50% human serum at 4 °C,

room temperature (RT) or 37°C for 10 days. TGF-B1 neutralizing activity of TGFRt15-

TGFRs was accessed with HEK-Blue TGF-B cells (TGF-B1 activity report cell line,

InvivoGen). HEK-Blue TGF-B cells were incubated in IMDM-10 with titrated

TGFRt15-TGFRs in the presence of TGF-B1 (0.1 nM). After 24 hours of incubation, the

culture supernatants were mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3

hrs. The OD620 values were measured by a plate reader. As shown in Figure 213C, the

results show that there were no changes in the TGF-31 neutralizing activity of TGFRt15-

TGFRs following incubation in human serum for 10 days at 4 °C, RT, or 37 °C. IL-15

activity of TGFRt15-TGFRs was evaluated with IL-15 dependent 32DB cells. 32DB cells

were cultured in IMDM-10 with titrated TGFRt15-TGFRs. WST-1 (InvitroGen) was

added 2 days later and the OD450 values were measured by a plate reader. As shown in

Figure 213D, the results show that there were no changes in the IL-15 activity of

TGFRt15-TGFRs following incubation in human serum for 10 days at 4 °C, RT, or 37

°C.

Example 89: Reversal of TGF-B1 Immunosuppression for Human NK Cells and

PBMC by TGFRt15-TGFRs and TGFRt15*-TGFRs Human NK cells were purified from blood buffy coats (4 donors, One Blood)

with RosetteSepTM Human NK Cell Enrichment Cocktail (StemCell) according to wo 2021/247604 WO PCT/US2021/035285

StemCell instruction and PBMCs were isolated from blood buffy coats (6 donors) with

Ficoll-Paque (Sigma-Aldrich) density centrifugation. NK cells and PBMCs were

cultured in RPMI-10 with IL-15 (10 ng/mL, PeproTech) and/or TGF-B1 (10 ng/mL,

Biolegend), TGFRt15-TGFRs (42 nM or 4.2 nM) or TGFRt15*-TGFRs (42 nM or 4.2

nM) for 3 days. The cultures were harvested and used for the following assays: cell

mediated cytotoxicity assay (Figures 214A and B) and flow cytometry analyses for

intracellular granzyme B (Figures 214C and D) and Interferon gamma (IFNy, Figures

214E and F).

Cultured NK cells and PBMCs were used as effector cells and K562 tumor cells

(ATCC) as target cells in cell mediated cytotoxicity assay. The mixtures of the effector

cells and K562 tumor cells were incubated in RPMI-10 at 37°C for 4 hours at E:T

ratio=4:1 for NK cells (Figure 214A) or 20:1 for PBMCs (Figure 214B). The levels of

dead K562 cells were determined by flow cytometry. As shown in Figures 214A and B,

the results showed that there were significantly less dead K562 target cells in the

presence of TGF- B1 than were observed medium control cultures, indicating that TGF-

B1 inhibits immune cell cytotoxicity. However, there were significantly more dead K562

target cells in the presence of TGF-B1 and TGFRt15-TGFRs or TGFRt15*-TGFRs than

was observed cultures incubated with TGF-B1 alone conditions. These findings

demonstrate TGFRt15-TGFRs and TGFRt15*-TGFRs significantly reduced TGF-B1

immunosuppression and enhanced the cytotoxicity of human NK cells and PBMCs

against K562 target cells in a concentration dependent manner. Additionally, the IL-15

activity of TGFRt15-TGFRs further enhances cytotoxicity of human NK cells and

PBMCs when compared to the activity of TGFRt15*-TGFRs.

Cultured NK cells and PBMCs were stained with fluorochrome labeled anti-CD56

and anti-CD16 human NK cell surface markers and then with fluorochrome-labeled

granzyme B and IFNy intracellular molecules (BioLegend). The granzyme B and IFNy

expression (MFI: mean fluorescence intensity) in the purified NK cells and gated NK

cells (CD56+ and/or CD16*) of PBMC cultures were analyzed by flow cytometry. As

shown in Figures 214C and D, there was significantly less granzyme B (Figures 214C

and 214D) and IFNy (Figures 214E and 214F) expression in NK cells cultured in the

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presence of TGF-B1 than was observed in cells cultured in medium alone, indicating that

TGF-B1 inhibits immune cell activation. However, there was significantly higher

granzyme B and IFNy expression NK cells cultures in the presence of TGF-B1 and

TGFRt15-TGFRs or TGFRt15*-TGFRs than was observed in cells cultured in TGF-B1

alone. The TGFRt15*-TGFRs had a minimum effect on granzyme B and IFNy

expression at 4.2 nM concentration. These findings demonstrate TGFRt15-TGFRs and

TGFRt15*-TGFRs significantly enhanced the granzyme B and IFNy expression of

human NK cells in a concentration-dependent manner through the activities of the IL-15

and TGFßRII domains.

Example 90: Half-life of TGFRt15-TGFRs in C57BL/6 Mice

The pharmacokinetics (half-life, t1/2) of TGFRt15-TGFRs was evaluated in

female C57BL/6 mice. The mice were treated subcutaneously with TGFRt15-TGFRs at

a dosage of 3 mg/kg. The mouse blood was collected from tail vein at various time

points and the serum was prepared. The TGFRt15-TGFRs concentrations in mouse

serum was determined with ELISA. Anti-TF antibody (anti-human tissue factor antibody

generated in HCW Biologics) was used to capture TGFRt15-TGFRs molecules and

biotinylated anti-TGF3RII (R&D Systems) was used to detect TGFßRII domain.

Biotinylated detection antibodies were probed by peroxidase-streptavidin (Jackson

ImmunoResearch Lab). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS,

Surmodics IVD) was used as a substrate and the OD405 values were measured by a plate

reader. As shown in Figure 215, the half-life of TGFRt15-TGFRs was 18.22 hours in

C57BL/6 mice calculated with GraphPad Prism 7.04.

Example 91: Toxicity of TGFRt15-TGFRs in C57BL/6 Mice

A single dose of TGFRt15-TGFRs (50-400 mg/kg) was subcutaneously injected

into C57BL/6 female mice (7 weeks old, n=4). Mouse bodyweight was measured as

shown in Figure 216 and clinical signs (mortality, morbidity, ruffled fur, hunched

posture, lethargy, etc.) were assessed during experimental period. The mice that received

200 mg/kg or 400 mg/kg of TGFRt15-TGFRs showed less activity 6-8 days post- treatment and without other significant clinical signs. TGFRt15-TGFRs at 200 mg/kg or

400 mg/kg caused loss in mouse body weight compared with PBS group especially on

day 7 after treatment (p<0.05). The affected mice gradually recovered after 10 days

without mortality or morbidity. As shown in Figure 216, these findings indicate that

C57BL/6 mice can tolerate single dose TGFRt15-TGFRs at up to 100 mg/kg.

Example 92: Antitumor Activity of TGFRt15-TGFRs in a C57BL/6 Murine

Melanoma Model Mouse B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice

(The Jackson Laboratory) to establish the mouse melanoma model. Four days after

tumor cell injection, the mice were divided into different groups to receive the following

immunotherapies: Group 1: PBS vehicle control; Group 2: antitumor antibody TA99 (10

mg/kg) alone control; Group 3: TA99 combined with IL-15SA (0.05 mg/kg); Group 4:

TA99 combined with TGFRt15-TGFRs (4.93 mg/kg, equivalent IL-15 activity of 0.05

mg/kg IL-15SA); and Group 5: TA99 combined with TGFRt15*-TGFRs (4.93 mg/kg.

IL-15D8N mutant without IL-15 activity). The tumor volume was measured and

calculated using the formula: length X width X width/2 formula. As shown in Figure 217,

the results indicated that the mice receiving antitumor antibody TA99 combined with

TGFRt15-TGFRs or IL15SA had significantly smaller tumors at day 11 after tumor

inoculation, when compared to the PBS, TA99 antibody alone, and TA99 with

TGFRt15*-TGFRs groups (p<0.05). There was no significant difference among groups

1, 2, and 5 and between groups 3 and 4. These findings demonstrated that IL-15 activity

of TGFRt15-TGFRs was important for antitumor activity of TGFRt15-TGFRs.

Example 93: Model of Lung Fibrosis - Treatment with TGFRt15-TGFRs

Inflammatory and fibrotic lung diseases (including idiopathic pulmonary fibrosis,

chronic obstructive pulmonary disease and cystic fibrosis) are major causes of death with

limited treatment options. Additionally, various therapies result in lung injury side

effects leading to pulmonary fibrosis. For example, lung toxicity develops in ~ ~10% of

cancer patients receiving bleomycin chemotherapy. These effects have led to the use of

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bleomycin treatment in rodents to model pulmonary fibrosis for the study of mechanisms

involved in fibrogenesis and for evaluation of potential therapies. To assess the activity

of TGFRt15-TGFRs in this model, nine-week old C57B16/j male mice were given 50 uL

of bleomycin (2.5 mg/kg, single dose) through the oropharyngeal route. Mice were given

TGFRt15-TGFRs subcutaneously (3 mg/kg) on day 17 following bleomycin treatment.

Mice were sacrificed on day 28 post-bleomycin. Lungs were isolated and left lung was

homogenized and 100 uL of homogenate was assayed for hydroxyproline content as a

measure of collagen deposition using commercially available kit according to

manufacturer's instructions. The data was expressed as ug of hydroxyproline content per

gram of lung. As shown in Figure 218, the results indicate that TGFRt15-TGFRs therapy

significantly reduced collagen deposition (i.e., fibrosis) in the lungs of bleomycin-treated

mice.

Example 94: In Vivo Characterization of the Activities of TGFRt15-TGFRs and

TGFRt15*-TGFRs TGFRt15*-TGFRs It has been shown that protection from obesity and diabetes in leptin deficient

ob/ob mice can be achieved by blockade of TGF-B/Smad3 signaling. To assess if

TGFRt15-TGFRs or TGFRt15*-TGFRs can protect mice from obesity and diabetes by

blockade of TGF-B/Smad3 signaling, the leptin receptor deficient db/db mouse strain

(BKS.Cg Dock7m Leprdb/J) was used for the study. Six-week-old db/db mice were

divided to three groups (N=8 per group). Mice were injected subcutaneously with

TGFRt15-TGFRs, TGFRt15*-TGFRs, or PBS at 3 mg/kg. Blood was collected at day 4

post-injection through the submandibular vein after the mice had been fasting for 20

hours. The fasting blood glucose was measured with OneTouch UltraMini meter

immediately after blood was drawn. As shown in Figure 219, both TGFRt15-TGFRs and

TGFRt15*-TGFRs can reduce the fasting plasma glucose levels significantly.

The plasma TGFß1-3 levels were assessed to identify the cause of treatment-

related reduction of fasting plasma glucose in db/db mice. Four days after treatment,

plasma was isolated and 30 uL of plasma was sent to EVE Technologies (Calgary, AB

Canada) to assess TGFß1-3 levels by the TGF-3 3-Plex (TGFB1-3) assay. As shown in

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Figures 220A-C, both TGFRt15-TGFRs and TGFRt15*-TGFRs completely depleted

plasma TGFß1 (Figure 220A), partially reduced TGFB2 (Figure 220B), and had no effect

on TGFB3 (Figure 220C).

The lymphocyte subsets were assessed to identify the cause of treatment-related

reduction of fasting plasma glucose in db/db mice. Four days after treatment, whole

blood cells (50 ul) were treated with ACK (Ammonium-Chloride-Potassium) lysing

buffer to lyse red blood cells. The lymphocytes were then stained with PE-Cy7-anti-

CD3, BV605-anti-CD45, PerCP-Cy5.5-anti-CD8a, BV510-anti-CD4, and APC-anti-

NKp46 (all antibodies from BioLegend) to assess the populations of T cells and NK cells.

The cells were further permeabilized and fixed with eBioscience Foxp3/Transcription

factor staining buffer set (Cat# 00-5523-00, ThermoFisher) and stained with AF700-anti-

Ki67 and FITC-anti-Granzyme B in eBioscience Permeabilization buffer (Cat# 00-8333-

56, ThermoFisher) to assess the proliferation and activation of T cells and NK cells.

Another set of lymphocytes were stained with PE-Cy7-anti-CD3, BV605-anti-CD45,

BV510-anti-CD4 and apc-Cy7-anti-CD25 first, and then permeabilized and fixed with

eBioscience Foxp3/Transcription factor staining buffer set (Cat# 00-5523-00,

ThermoFisher) and stained with PE-anti-Foxp3 in eBioscience Permeabilization buffer

(Cat# 00-8333-56, ThermoFisher) to assess the population of Treg cells.

TGFRt15-TGFRs increased the population of NK cells (Figure 221A) and CD8+

T cells (Figure 221D), stimulated the proliferation of NK cells (Figure 221B) and CD8+ T

cells (Figure 221E), and activated NK cells (Figure 221C). TGFRt15*-TGFRs had no

effect on either cell population (Figure 221A-E). Both TGFRt15-TGFRs and TGFRt15*-

TGFRs had no effect on CD4+ T cells, CD19+ B cells, and CD4*CD25*Foxp3* Treg

cells.

In conclusion, in db/db mice, both TGFRt15-TGFRs and TGFRt15*-TGFRs

reduced fasting plasma glucose levels and both TGFRt15-TGFRs and TGFRt15*-TGFRs

completely depleted plasma TGFB1. However, only TGFRt15-TGFRs activated NK

cells and enhanced CD8+ T cells and NK cells proliferation. Based on these results, the

depletion of TGFB1 likely was involved in the reduction of fasting plasma glucose,

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showing that blockade of TGF-B/Smad3 signaling played a role in prevention of obesity

and diabetes in ob/ob mice.

Example 95: In Vitro Characterization of the Activities of TGFRt15-TGFRs and

TGFRt15*-TGFRs TGFRII was demonstrated to interact with TGF31-3. There is no report in the

literature demonstrating interactions between TGFRII and latent TGFB. To assess

whether TGFRt15-TGFRs, TGFRt15*-TGFRs, and TGFRII-Fc interacts with latent

TGFß we applied 2.5 nM of human latent TGFB1-his tag (Cat# TG1-H524x, Acro

Biosystems) or a control protein CD39-his tag (Lot# 58-49/51, HCW Biologics) in 50

mM carbonate buffer pH 9.4 (100ul/well) to coat an ELISA plate (Cat# 80040LE 0910,

ThermoFisher) overnight at 4 °C. Next day, the plate was washed with ELISA washing

buffer (phosphate-buffered saline with 0.05% Tween 20) three times, the plate was

blocked with the blocking buffer (1% BSA-PBS) for 1 hour, and then descending

concentrations of TGFRt15-TGFRs, TGFRt15*-TGFRs, or TGFRII-Fc from 200 nM to

0.09 nM in blocking buffer were added to the plate and the plate was incubated for 1 hour

at 25 °C. The plate was washed three times with ELISA washing buffer. A detection

antibody, biotinylated anti-TGFRII antibody (Cat# BAF241, R&D Systems), at 0.1

ug/mL was added to the plate and incubated at 25 °C for 1 hour. The plate was washed

and horseradish peroxidase-streptavidin (code#016-030-084, Jackson ImmunoResearch)

at 0.25 ug/mL was added to the plate and incubated at 25 5°C for 30 minutes. The plate

was washed and a substrate of HRP, ABTS (Cat# ABTS-1000-01, Surmodics) was added

to the plate and incubated for 20 minutes at 25 °C. The plate was read with a microplate

reader (Multiscan Sky, Thermo Scientific) at OD405 nm. As shown in Figure 222A,

both TGFRt15-TGFRs and TGFRt15*-TGFRs interacted with latent TGFß1 similarly.

However, TGFRII-Fc interacted with latent TGFB1 with lower affinity than was seen

with TGFRt15*-TGFRs (Figure 222B). The results demonstrated TGFRt15-TGFRs,

TGFRt15* TGFRs, and TGFRII-Fc can interact with latent TGFß1, with TGFRt15-

TGFRs, TGFRt15*-TGFRs surprisingly showing higher affinity interaction than

TGFRII-Fc.

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Example 96: Prothrombin Time Test Prothrombin time (PT) test is designed to measure the time it takes for plasma to

clot after mixing with tissue factor and an optimal concentration of calcium. Tissue

factor mixture with phospholipids (called Thrombinplastin) acts as an enzyme to convert

prothrombin to thrombin, which in turn causes blood clotting by converting fibrinogen to

fibrin. Innovin is a lipidated recombinant human TF243 and is used as the standard in

our experiment. In the PT assay, shorter PT time (clotting time) indicates a higher TF-

dependent clotting activity while longer PT (clotting time) means lower TF-dependent

clotting activity.

Briefly, 0.1 mL of normal human plasma (Ci-Trol Coagulation Control, Level I)

was prewarmed at 37 °C for 3 minutes. Plasma clotting reactions were initiated by

adding 0.2 mL of various dilutions of Innovin or testing sample (TGFRt15-TGFRs)

diluted in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaCl2, 0.1% BSA) to the

plasma. Clotting time was monitored and reported by STart PT analyzer (Diagnostica

Stago, Parsippany, NJ).

As seen in Figure 223, different amounts of Innovin (Innovin reconstituted with

purified water equivalent to 10 nM of lipidated recombinant human TF243 is considered

to be 100% Innovin) added to the PT assay indeed demonstrated an inverse relationship

between the amount of TF243 added in the PT assay and the PT time. For example, 1%

Innovin had a PT time of about 25.0 seconds, while 100% Innovin had a PT time of 8.5

seconds.

Figure 224 shows the result of the PT test on TGFRt15-TGFRs. In contrast to

Innovin, TGFRt15-TGFRs exhibited prolonged PT times which were almost the same as

buffer, indicating extremely low or no clotting activity.

The clotting effect of TGFRt15-TGFRs in the presence of CTLL cells was also

evaluated. The binding experiment conducted confirmed that TGFRt15-TGFRs can bind

to CTLL cells. The TGFRt15-TGFRs clotting test in the presence of CTLL cells will

reflect more closely with the potent clotting activity in vivo. TGFRt15-TGFRs was

preincubated with CTLL cells for 20-30 min at 37 °C in PT assay buffer. Then we

proceeded with the PT assay as described above. Figure 224 shows that mixture of

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TGFRt15-TGFRs with CTLL cells had a bit shorter clotting time (154.6 sec) than

TGFRt15-TGFRs alone (167.6 sec) or CTLL cells alone (161.9 sec). However, the

clotting time of 154.6 seconds is still significantly longer than the Innovin clotting time

of 8.5 seconds.

In summary, TGFRt15-TGFRs has extremely low or no TF-dependent clotting

activity (i.e., in the physiological ranges of coagulation factors in human plasma), even in

the presence of cells capable of binding TGFRt15-TGFRs.

Example 97: Gene Expression of Senescence Markers in Tissues of Young Mice, and

of Aged Mice Following Treatment with TGFRt15-TGFRs or PBS and Short-Term

(10 days) or Long-Term (60 days) Follow-Up

C57BL/6, 72-week-old mice were purchased from the Jackson Laboratory. Mice

were housed in a temperature and light controlled environment. Mice were divided into

two groups and treated subcutaneously with either PBS (PBS control group) or TGFRt15-

TGFRs at a dosage of 3 mg/kg (TGFRt15-TGFRs group). Either at day 10 or day 60

post-treatment, mice were euthanized, and kidneys were harvested in order to evaluate

the expression levels of senescence markers PAI1, IL-1a, IL6, and TNFa by quantitative-

PCR. Harvested kidneys were stored in liquid nitrogen in 1.7 mL Eppendorf tubes.

Samples were homogenized by using homogenizer in 1 mL of Trizol (Thermo Fischer).

Homogenized tissues were transferred in fresh Eppendorf tubes. Total RNA was

extracted using RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's

instructions. One ug of total RNA was used for cDNA synthesis using the Quanti

Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with CFX96

Detection System (Bio-Rad) using FAM labeled predesigned primers purchased from

Thermo Scientific. Reactions were run in triplicate for all the genes examined. The

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in expression levels. The expression of each target mRNA relative to 18S

rRNA was calculated based on Ct as 2-A(ACt) , in which ACt=Ct target- Ct18S. Untreated

6-week-old mice (Young) were used as a control to compare the gene expression level to

aged mice.

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As shown in Figure 225, the results show that gene expression of PAI-1, IL-1a,

IL6, and IL-1B in kidney increased with the age of the mice as expected with the age-

dependent increase in cellular senescence. Treatment of 72-month old mice with a single

dose of TGFRt15-TGFRs resulted in a significant and long-lasting effect in reducing

gene expression of senescence markers in kidneys, suggesting a treatment associated

decrease in naturally-occurring senescent cells in the kidneys of aged mice.

As shown in Figure 226, the results showed that treatment of 72-month old mice

with a single dose of TGFRt15-TGFRs mediated in a significant and long-lasting effect

in reducing IL-1a and IL6 gene expression in liver, suggesting a treatment associated

decrease in naturally-occurring senescent cells in the liver of aged mice.

C57BL/6, 72-week-old mice were purchased from the Jackson Laboratory. Mice

were housed in a temperature and light controlled environment. Mice were divided into

two groups and treated subcutaneously with either PBS (PBS control group) or TGFRt15-

TGFRs at a dosage of 3 mg/kg (TGFRt15-TGFRs group). Either at day 10 or day 60

post-treatment, mice were euthanized, and kidneys were harvested in order to evaluate

the proteins levels of the senescence marker PAI-1 by a tissue ELISA. Harvested

kidneys were stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were

homogenized by using homogenizer in 0.3 mL of extraction buffer (Abcam).

Homogenized tissues were transferred in fresh Eppendorf tubes. Protein level in

homogenized tissue was quantified using BCA Protein Assay Kit (Pierce). Mouse PAI-1

ELISA (R&D System) was performed with 200 mg of tissue homogenate. Based on a

standard curve, the concentration of PAI-1 was calculated as picograms per milligram of

tissue.

As shown in Figure 227, the protein levels of senescence markers PAI-1

decreased in the kidneys of TGFRt15-TGFRs treated aged mice compared to PBS group

at 60 days post-treatment. These results are consistent with the effects of TGFRt15-

TGFRs treatment on the PAI-1 gene expression in the kidneys of aged mice. Together,

these results indicate that a single treatment of TGFRt15-TGFRs resulted in a significant

and long-lasting effect in reducing naturally-occurring senescent cells (as measured by

reduced gene and protein expression of senescence markers) in the tissues of aged mice.

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Example 98: Comparison of TGFRt15-TGFRs and TGFRt15*-TGFRs (IL-15 mutant) Treatment in Reducing Gene Expression of Senescence Markers in Tissues

of Aged Mice

C57BL/6, 72-week-old mice were purchased from the Jackson Laboratory. Mice

were housed in a temperature and light controlled environment. Mice were divided into

five groups as follows: saline control group (n =8); TGFRt15-TGFRs group (n =8);

IL15SA group (n =8); TGFRt15*-TGFRs group (n =8); and IL15SA + TGFRt15*.

TGFRs group (n =8). Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3

mg/kg), TGFRt15*-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg), or TGFRt15*-TGFRs (3

mg/kg) plus IL15SA (0.5 mg/kg). Mouse blood was prepared in order to evaluate

changes in the different subsets of immune cells after treatment with TGFRt15-TGFRs

and other agents. The mouse blood was collected from submandibular vein on Day 17

post-treatment in tubes containing EDTA. The whole blood was centrifuged to collect

plasma at 3000 RPM for 10 minutes in a micro centrifuge. Plasma was stored at -80 °C

and whole blood was processed for immune cell phenotyping by flow cytometry. Whole

blood RBCs were lysed in ACK buffer for 5 minutes at room temperature. Remaining

cells were washed in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore)

and 0.001% Sodium Azide (Sigma)). To assess the different types of immune cells in

blood, cells were stained with antibodies specific to cell-surface CD3, CD45, CD8, and

NK1.1 (BioLegend) for 30 minutes at room temperature (RT). After surface staining,

cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (1X

PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)).

After two washes, cells were resuspended in fixation buffer and analyzed by flow

cytometry (Celesta-BD Bioscience).

As shown in Figure 228, the results indicate that treatment of aged mice with

TGFRt15-TGFRs. IL15SA (positive control) or TGFRt15*-TGFRs + IL15SA mediated

an increase in the percentages of CD3*CD8*, CD3*NK1.1*, and CD3*CD45+ immune

cells in the blood, whereas treatment with TGFRt15*-TGFRs had little or no effect on the

percentage of these cell populations. These results suggest that IL-15 activity of

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TGFRt15-TGFRs plays a role in increasing CD8+ T cells and NK cells in the blood of

aged mice.

As shown in Figure 229, the results indicate that treatment of aged mice with

TGFRt15-TGFRs. IL15SA (positive control) or TGFRt15*-TGFRs + IL15SA mediated

an increase in the percentages of CD3*CD8 CD3*NK1.1*, and CD3+CD45 immune

cells in the spleen, whereas treatment with TGFRt15*-TGFRs had little or no effect on

the percentage of these cell populations. These results suggest that IL-15 activity of

TGFRt15-TGFRs plays a role in increasing CD8+ T cells and NK cells in the spleen of

aged mice.

C57BL/6, 72-week-old mice were purchased from the Jackson Laboratory. Mice

were housed in a temperature and light controlled environment. Mice were divided into

five groups as follows: saline control group (n =8); TGFRt15-TGFRs group (n =8);

IL15SA group (n =8); TGFRt15*-TGFRs group (n =8); and IL15SA with TGFRt15*-

TGFRs group (n =8). Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3

mg/kg), TGFRt15*-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg), or TGFRt15*-TGFRs (3

mg/kg) plus IL15SA (0.5 mg/kg). The mouse kidney, liver, and lungs were harvested in

order to evaluate the gene expression of senescence markers p21, PAI1, IL-1a, and IL6

by quantitative-PCR in tissues after treatment with TGFRt15-TGFRs, TGFRt15*-TGFRs,

or control groups. Mice were euthanized day 17 post-treatment and kidney, liver, and

lung were harvested and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples

were homogenized by using homogenizer in 1 mL of Trizol (Thermo Fischer).

Homogenized tissues were transferred in fresh Eppendorf tubes. Total RNA was

extracted using RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's

instructions. One ug of total RNA was used for cDNA synthesis using the QuantiTect

Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with CFX96

Detection System (Bio-Rad) using FAM labeled predesigned primers purchased from

Thermo Scientific. Reactions were run in triplicate for all the genes examined. The

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in expression levels. The expression of each target mRNA relative to 18S

rRNA was calculated based on Ct as 2-A(ACt) in which ACt= Ct target- Ct18S.

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As shown in Figure 230A-D, treatment of 72-month old mice with a single dose

of TGFRt15-TGFRs or TGFRt15*-TGFRs mediated in a significant decrease in p21,

PAI1, IL-1a, and IL6 gene expression in kidney and liver, suggesting a treatment

associated decrease in naturally-occurring senescent cells in the kidney and liver of aged

mice. The results of this study suggest that both the IL-15 and TGF-B trap activities of

TGFRt15-TGFRs are capable of reducing naturally-occurring senescent cells in the

tissues of aged mice.

Example 99: Immuno-Phenotype Following Treatment with IL-15-based Agents

The mouse blood was prepared in order to evaluate changes in the different

subsets of immune cells after treatment with IL-15-based agents: TGFRt15-TGFRs, an

IL-15 superagonist (IL-15SA), and an IL-15 fusion with a D8N mutant knocking out the

IL-15 activity (TGFRt15*-TGFRs). C57BL/6, 6-week-old mice were purchased from

Jackson Laboratory. Mice were housed in a temperature and light controlled

environment. Mice were divided into groups (n =6/group) and treated with the following:

1) PBS (saline) control, 2) docetaxel, 3) docetaxel with TGFRt15-TGFRs, 4) docetaxel

with IL15SA, 5) docetaxel with an IL-15 mutant (TGFRt15*-TGFRs), and 6) docetaxel

with an IL-15 superagonist (IL-15SA) plus TGFRt15*-TGFRs. Senescence was induced

in mice with three doses of docetaxel (10 mg/kg) at day 1, 4, and 7. On day 8, the mice

were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs IL-15SA

or in combinations as discussed above. TGFRt15-TGFRs and TGFRt15*-TGFR: were

administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. The

mouse blood was collected from the submandibular vein on day 3 post-study drug

treatment into EDTA tubes. The whole blood was centrifuged to collect plasma at 3000

RPM for 10 minutes in a microcentrifuge. Plasma was stored at -80 °C and whole blood

was processed for immune cell phenotyping by flow cytometry. RBCs were lysed in

ACK buffer for 5 minutes at 37 °C. The remaining cells were washed in FACS buffer

(1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide

(Sigma)). To assess the different types of immune cells in the blood, cells were stained

with antibodies for cell-surface CD4, CD45, CD19, CD8, and NK1.1 (BioLegend) for 30

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minutes at room temperature (RT). After surface staining, cells were washed (1500 RPM

for 5 minutes at room temperature) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA

(EMD Millipore) and 0.001% Sodium Azide (Sigma)). The cells were treated with

permeabilization buffer (Invitrogen) for 20 minutes at 40 °C followed by wash with

permeabilization buffer (Invitrogen). The cells were then stained for an intracellular

marker for proliferation (Ki67) for 30 minutes at RT. After two washes, the cells were

resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figures 231A and 231B, the results indicate that treatment of mice

with TGFRt15-TGFRs, IL15SA (positive control), or TGFRt15*-TGFRs + IL15SA

mediated an increase in the percentages and proliferation (as measured by Ki67) of CD8+

T cells and NK1.1+ cells in the blood, whereas treatment with TGFRt15*-TGFRs had

little or no effect on the percentage of these cell populations. These results suggest that

IL-15 activity of TGFRt15-TGFRs plays a role in increasing CD8+ T cells and NK cells

in the blood of mice following chemotherapy.

Example 100: Evaluation of Gene Expression of Senescence Markers p21 and CD26

in Lung and Liver Tissues of Mice Following Chemotherapy and Treatment with

IL-15-based Agents

Gene expression of markers for cell senescence were evaluated in tissues of

normal mice following chemotherapy and administration of study treatments. C57BL/6,

6-week-old mice were purchased from Jackson Laboratory. Mice were housed in a

temperature and light controlled environment. Mice were divided into six groups and

treated with the following: 1) PBS (saline) control (n =5), 2) docetaxel (n =8), 3)

docetaxel with TGFRt15-TGFRs (n =8), 4) docetaxel with IL 15SA (n =8), 5) docetaxel

with an IL-15 mutant (TGFRt15*-TGFRs) (n =8), and 6) docetaxel with an IL-15

superagonist (IL-15SA) plus TGFRt15*-TGFRs (n =6). Senescence was induced in mice

with three doses of docetaxel (10 mg/kg) at day 1, 4, and 7. On day 8, the mice were

treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA, or in

combinations as discussed below. TGFRt15-TGFRs and TGFRt15*-TGFRs were

administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.5 mg/kg. The

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mouse tissues were prepared in order to evaluate the different gene expression of

senescence markers. Mice were euthanized on day 7 post-study drug treatment and the

liver and lung tissues were harvested and stored in liquid nitrogen in 1.7 mL Eppendorf

tubes. Samples were homogenized by using mortar and pestle in liquid nitrogen.

Homogenized tissues were transferred in fresh Eppendorf tubes containing 1 mL of

Trizol (Thermo Fischer). Total RNA was extracted using RNeasy Mini Kit (Qiagen

#74106) according to the manufacturer's instructions and 1 ug of total RNA was used for

cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time

PCR was carried out with CFX96 Detection System (Bio-Rad) using FAM labeled

predesigned primers purchased from Thermo Scientific. Reactions were run in triplicate

for all the genes examined. The housekeeping gene 18S ribosomal RNA was used as an

internal control to normalize the variability in expression levels. The expression of each

target mRNA relative to 18S rRNA was calculated based on Ct as 2-A(ACi) in which ACt =

Ct target- Ct18S.

As shown in the Figure 232, gene expression of the senescence markers p21 and

CD26 was induced in the lung (Figure 232A) and (Figure 232B), and p21 in liver (Figure

232C) tissues of mice treated with docetaxel, as compared to gene expression in tissue of

saline-treated mice. Gene expression of senescence markers p21 and CD26 in the lungs

and p21 in the liver were reduced of the chemotherapy-treated mice following subsequent

treatment with TGFRt15-TGFRs, IL-15SA, and combination of IL-15SA and TGFRt15*-

TGFRs mutant, as compared to the chemotherapy-treated controls. However, the

TGFRt15*-TGFRs mutant treatment failed to effect the chemotherapy-induced

senescence marker gene expression in these tissues. These results show that IL-15

activity is important for clearance of TIS senescence cells in normal tissues of mice.

Example 101: TGFRt15-TGFRs Treatment Enhances the Immune Cell

Proliferation, Expansion, and Activation in the Peripheral Blood of B16F10 Tumor

Bearing Mice C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

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(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Blood was drawn from submandibular

vein on days 3, 5, and 10 after immunotherapy treatment (day 8). The RBCs were lysed

in ACK lysis buffer and the lymphocytes were washed and stained with antibodies

specific to cell-surface expression of NK, CD8, CD25, and Granzyme B (GzB)

(BioLegend) for 30 minutes at room temperature (RT). After surface staining, the cells

were washed (1500 RPM for 5 minutes at RT) in FACS buffer (1X PBS (Hyclone) with

0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)). After two washes, the

cells were resuspended in fixation buffer. After fixation, the cells were washed and

treated with permeabilization buffer (Invitrogen) for 20 minutes at 4 °C followed by wash

with permeabilization buffer (Invitrogen). The cells were then stained for an intracellular

marker for proliferation (Ki67) for 30 minutes at RT. After two washes, the cells were

resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figures 233A and B, peripheral blood analysis showed that

proliferative Ki67-positive NK and CD8+ cells were predominantly present at day 3 post-

TGFRt15-TGFRs+TA99 therapy, when compared to the saline or chemotherapy

treatment groups. The expansion of NK and CD8+ cells was found on days 3 and 5 post-

immunotherapy. While the NK cells were still expanding, the CD8+ cells was not found

to be expanding in the blood at day 10 post-immunotherapy. These cells also expressed

the activation markers CD25 and granzyme B post-TGFRt15-TGFRs+TA99 therapy,

when compared to immune cells of the saline or chemotherapy treatment groups. These

effects are consistent with the immunostimulatory activities of TGFRt15-TGFRs.

Example 102: TGFRt15-TGFRs treatment decreases levels of TGFB in the plasma

of B16F10 tumor bearing mice C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

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with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Blood was collected from the

submandibular on days 1, 3, 5, and 10 after immunotherapy treatment in tubes containing

EDTA and immediately placed on ice. The blood was centrifuged for 15 minutes at

3,000 rpm at room temperature to separate plasma. Plasma samples were aliquoted and

stored at -80 °C. The plasma TGFB levels were analyzed by using cytokine array, TGFß

3-plex (TGFß 1-3) from Eve Technologies, Calgary, AL, Canada.

As shown in Figure 234, the results show that administration of TGFRt15-

TGFRs+TA99 led to a reduction in the plasma levels of TGF-31, TGF-B2, and TGF-B3 in

tumor-bearing mice for 3 to 5 days post-treatment, when compared to the saline or

chemotherapy treatment groups. This effect is consistent with the TGF-B agonistic

activity of TGFRt15-TGFRs.

Example 103: TGFRt15-TGFRs Treatment Reduces Levels of Proinflammatory

Cytokines in the Plasma of B16F10 Tumor Bearing Mice

C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Blood was drawn from submandibular

vein on days 1, 3, 5, and 10 after immunotherapy treatment (day 8) in tubes containing

with EDTA and immediately placed on ice. The blood was centrifuged for 15 minutes at

3,000 rpm at room temperature to separate plasma. Plasma samples were aliquoted and

stored at -80 °C. Aliquots were diluted 2-fold in PBS and analyzed using a Mouse

Cytokine Array Proinflammatory Focused 10-plex (MDF10) assay.

As shown in Figure 235, the results show that administration of TGFRt15-

TGFRs+TA99 reduced in plasma levels of IL2, IL-1B, IL6, MCP-1, and GM-CSF in

tumor-bearing mice on day 10 post-treatment, when compared to the chemotherapy treatment group. This effect is consistent with the immunostimulatory activities of

TGFRt15-TGFRs.

Example 104: TGFRt15-TGFRs Treatment Enhances NK and CD8+ expansion in

the Spleen of B16F10 Tumor Bearing Mice

C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 0 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Mice were sacrificed and the spleens were

harvested at days 3, 5, and 10 post-immunotherapy (day 8). The spleens were crushed

with flat back end of the sterile piston/plunger of 3 cc syringe to release the splenocytes.

The splenocytes were passed through a 70-uM cell strainer and homogenized into a

single cell suspension. The RBCs were lysed in ACK lysis buffer and the splenocytes

were washed and stained with antibodies for cell-surface expression of NK and CD8

(BioLegend), for 30 minutes at RT. After two washes, the cells were resuspended in

fixation buffer and analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in the Figure 236, the expansion of NK and CD8+ cells were seen in the

spleen at days 3 and 5 post-TGFRt15-TGFRs+TA99 therapy, when compared to the

saline or chemotherapy treatment groups. Levels of NK cells (but not the CD8+ cells)

were still found to be elevated at day 10 post-immunotherapy in the spleen of tumor-

bearing mice, when compared levels in the spleens of the chemotherapy treatment group.

These effects are consistent with the immunostimulatory activities of TGFRt15-TGFRs.

Example 105: TGFRt15-TGFRs Treatment Enhances Glycolytic Activity of

Splenocytes in B16F10 Tumor Bearing Mice

C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

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with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Mice were sacrificed and the spleens were

harvested at days 3, 5, and 10 post-immunotherapy (day 8). The spleens were crushed

with flat back end of the sterile piston/plunger of 3 CC syringe to release the splenocytes.

The splenocytes were passed through a 70-uM cell strainer and homogenized into a

single cell suspension. The RBCs were lysed in ACK lysis buffer and the splenocytes

were washed and counted. To measure the glycolytic activity of the splenocytes, the cells

were washed and resuspended in seahorse media and resuspended in 4 X 106 cells/mL.

The cells were seeded at 50 uL/well in Cell-Tak-coated Seahorse Bioanalyzer XFe96

culture plates in Seahorse XF RPMI medium, pH 7.4 supplemented with 2 mM L-

glutamine for glycolysis stress test. The cells were allowed to attach to the plate for 30

minutes at 37 °C. Additionally, 130 uL of the assay medium was added to each well of

the plate (also the background wells). The plate was incubated in 37 °C, non-CO2

incubator for 1 hr. For glycolysis stress test the calibration plate contained 10x solution

of glucose/oligomycin/2DG prepared in Seahorse assay media and 20 uL of

glucose/oligomycin/2DG were added to each of the ports of the extracellular flux plate

that was calibrated overnight. The glycolysis stress test is based on extracellular

acidification rate (ECAR) and measures three key parameters of glycolytic function

including glycolysis, glycolytic capacity, and glycolytic reserve. Complete ECAR

analysis consisted of four stages: non glycolytic acidification (without drugs), glycolysis

(10 mM glucose), maximal glycolysis induction/glycolytic capacity (2 uM oligomycin),

and glycolysis reserve (100 mM 2-DG). At the end of the experiment the data was

exported as a Graph Pad Prism file. The XF glycolysis stress test report generator

automatically calculated the XF cell glycolysis stress test parameters from the Wave data.

The data was analyzed using the Wave software (Agilent).

As shown in the Figures 237A and B, the splenocytes isolated from tumor-bearing

mice at day 3 and day 5 after TGFRt15-TGFRs+TA99 therapy showed enhanced basal

glycolysis, capacity and reserve rate, when compared to splenocytes of the saline or

chemotherapy treatment groups. However no significant difference in the splenocyte

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glycolytic activity was observed at day 10 post-immunotherapy. These effects are

consistent with the immunostimulatory activities of TGFRt15-TGFRs.

Example 106: TGFRt15-TGFRs Treatment Enhances Mitochondrial Respiration of

Splenocytes in B16F10 Tumor Bearing Mice

C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Mice were sacrificed and the spleens were

harvested at days 3, 5, and 10 post-immunotherapy (day 8). The spleens were crushed

with flat back end of the sterile piston/plunger of 3 cc syringe to release the splenocytes.

The splenocytes were passed through a 70 uM cell strainer and homogenized into a single

cell suspension. The RBCs were lysed in ACK lysis buffer and the splenocytes were

washed and counted. To measure the mitochondrial respiration of the splenocytes, the

cells were washed and resuspended in seahorse media and resuspended in 4 x 106

cells/mL. The cells were seeded at 50 uL/well in Cell-Tak-coated Seahorse Bioanalyzer

XFe96 culture plates in Seahorse XF RPMI medium, pH 7.4 supplemented with 2 mM L-

glutamine for glycolysis stress test. For mitochondrial stress test, the cells were seeded in

Seahorse XF RPMI medium, pH 7.4 supplemented with 10 mM glucose and 2 mM L-

glutamine. The cells were allowed to attach to the plate for 30 minutes at 37 °C.

Additionally, 130 uL of the assay medium was added to each well of the plate (also the

background wells). The plate was incubated in 37 °C, non-CO2 incubator for 1 hr. For

mitochondrial stress test, the Calibration plate contained 10x solution of

oligomycin/FCCP/rotenone prepared in Seahorse assay media and 20 uL of oligomycin,

FCCP, and rotenone was added to each of the ports of the extracellular flux plate that was

calibrated overnight. Oxygen Consumption Rate (OCR) was measured using an XFe96

Extracellular Flux Analyzer. Complete OCR analysis consisted of four stages: basal

respiration (without drugs), ATP-linked respiration/Proton leak (1.5 uM mM

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Oligomycin), maximal respiration (2 FCCP), and spare respiration (0.5 uM

Rotenone). At the end of the experiment, the data was exported as a Graph Pad Prism

file. The XF mitochondrial stress test report generator automatically calculates the XF

mitochondrial stress test parameters from the Wave data that have been exported to

Excel. The data was analyzed by using the Wave software (Agilent).

As shown in the Figures 238A and B, the splenocytes isolated from tumor-bearing

mice at day 3 and day 5 after TGFRt15-TGFRs+TA99 therapy showed enhanced basal

respiration, mitochondria respiration, capacity and ATP production, when compared to

splenocytes of the saline or chemotherapy treatment groups. However no significant

difference in the splenocyte mitochondrial respiration was observed at day 10 post-

immunotherapy. These effects are consistent with the immunostimulatory activities of

TGFRt15-TGFRs. Metabolic pathways like oxidative metabolism and glycolysis are

known to preferentially fuel the cell fate decisions and effector functions of immune

cells. Therefore, TGFRt15-TGFRs mediated increased glycolytic activity and

mitochondrial respiration might be associated with the activation of NK and CD8+

immune cells in the blood, spleen, and tumor of the mice.

Example 107: TGFRt15-TGFRs Treatment Enhances NK and CD8 Immune Cell Infiltration (TILs) into Tumors of B16F10 Tumor Bearing Mice

C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Mice were sacrificed and the tumors were

harvested at days 3, 5, and 10 post-immunotherapy. The tumor tissue was dissociated

into single cell suspension by collagenase digestion to determine the tumor-infiltrating

immune cells. The single cell suspension was layered on Ficoll-Paque media followed by

density gradient centrifugation to separate the lymphocytes and tumor cells. The cells

were centrifuged at 1000 g for 20 minutes at 20 °C with slow acceleration and break

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turned off. After centrifugation the Ficoll-Paque results in a distinct separation between

two layers. The TILs are found on the interface between the media and Ficoll-Paque,

while the pellet consists of the tumor cells. The TILs were carefully removed from the

interface and washed with complete RPMI media. After washing, the RBCs were lysed

in ACK buffer for 5 minutes at room temperature. The cells were washed in FACS

buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide

(Sigma)). To assess the different types of immune cells in tumor, the cells were stained

with antibodies for cell-surface CD8, NK1.1, CD25, and GzB (BioLegend) for 30

minutes at RT. After surface staining, the remaining cells were washed (1500 RPM for 5

minutes at room temperature) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD

Millipore) and 0.001% Sodium Azide (Sigma)). After two washes, the cells were

resuspended in fixation buffer. After fixation cells were washed and treated with

permeabilization buffer (Invitrogen) for 20 minutes at 4 °C followed by wash with

permeabilization buffer (Invitrogen). The cells were then stained for intracellular

markers for proliferation (Ki67) for 30 minutes at RT. After two washes, the cells were

resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figures 239A and B, tumor analysis showed high levels of Ki67-positive

NK and CD8 cells at day 3 post-therapy. Expansion of NK and CD8+ cells (based on %

of lymphocytes in tumors) was found at day 3 and day 5 post-TGFRt15-TGFRs+TA99

therapy, when compared to the chemotherapy treatment group. Tumors CD8+ cells were

elevated even at day 10 post-immunotherapy. Both NK and CD8+ showed the expression

of activation markers CD25 and granzyme B at day 3 post-TGFRt15-TGFRs+TA99

therapy, when compared to immune cells of the chemotherapy treatment group. These

effects are consistent with the immunostimulatory activities of TGFRt15-TGFRs and are

comparable to changes seen in the blood and splenocytes of tumor-bearing mice.

Example 108: Histopathological Analysis of Tumors Following TGFRt15-TGFRs

Treatment C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

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(10 mg/kg) on days 1, 4, and 7 and single dose of TGFRt15-TGFRs (3 mg/kg) combined

with monoclonal antibody targeting a tumor antigen anti-TYRP-1 antibody TA99 (200

ug) on day 8. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10

mg/kg) on days 1, 4, and 7 served as controls. Blood was drawn from submandibular

vein on days 1, 3, 5, and 10 after immunotherapy treatment (day 8). On day 10 post-

immunotherapy, the mice were sacrificed, and tumors were isolated. For the histological

analysis, tumor samples were fixed in 10% formalin solution and were embedded in

paraffin and cut at 5 um. The sections were stained with H & E to assess tissue and

cellular morphology. The slides were scored based on the mitotic and necrotic activity of

the tumor. The percentage necrosis in the tumor was scored as, +1 (0-20%), +2 (20-

40%), and +3 (40-60%). The Mitotic Index of the tumor was scored as +1=Moderate (1-

5 per high power field) and =Extensive (>5 per high power field).

As shown in Figure 240, following TGFRt15-TGFRs+TA99 treatment, tumors

displayed less mitotic and necrotic activity. The mitotic index is correlated to the

dividing cells and presence of necrosis is a measure of more aggressive features and poor

prognosis. Hence TGFRt15-TGFRs is a promising therapy in pre-clinical murine models

for testing of combination tumor immunotherapy.

Example 109: Anti-PD-L1 Antibody in Combination with TGFRt15-TGFRs+TA99

and Chemotherapy in B16F10 Melanoma Mouse Model C57BL/6 mice were subcutaneously injected with 0.5x106 B16F10 cells. After

tumor inoculation (day 0), the mice were given three doses of doxetaxel chemotherapy

(10 mg/kg) on days 1, 4, and 7. Tumor-bearing mice treated with only saline or

doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 served as controls. The

remaining mice were randomized in two groups, one group was treated with anti-mPD-

L1 antibody (2 X 10 mg/kg) and the other group was treated with TGFRt15-TGFRs (3

mg/kg) with TA99 (200 ug) on day 8. After 6 days, the mice which received the

TGFRt15-TGFRs with TA99 were given anti-mPD-L1 antibody (2 X 10 mg/kg) and mice

which received anti-mPD-L1 antibody were treated with TGFRt15-TGFRs (3 mg/kg)

with TA99 (200 ug). The anti-mPD-L1 antibody was given as two doses on days 8 and

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10 or days 14 and 16. Tumor growth was monitored by caliper measurement, and tumor

volume was calculated using the formula V= (LxW2)/2, where L is the largest tumor

diameter and W is the perpendicular tumor diameter. N=6-8 mice/group.

As shown in the Figure 241, TGFRt15-TGFRs+TA99 administration following

by anti-PD-L1 antibody treatment resulted in better antitumor activity in B16F10 tumor-

bearing mice as compared to treatment with anti-PD-L1 antibody and then TGFRt15-

TGFRs+TA99. Therefore, combining TGFRt15-TGFRs with anti-PD-L1 antibody may

be advantageous in treating tumors that are resistance to anti-PD-L1 antibody therapy.

Example 110: Anti-tumor efficacy of TGFRt15-TGFRs in B16F10 Melanoma Mouse

Model is Dependent on NK and CD8+ T Cells

Groups of C57BL/6 mice (N=6-8 mice/group) were treated with three doses of

NK1.1 Ab (500 ug) or CD8+a (500 ug) antibody intraperitoneal every third day to deplete

the NK and CD8 cells. Blood was drawn and analyzed for NK and CD8+ lymphocyte

levels before the B16F10 tumor implantation. Untreated mice served as

immunocompetent controls. C57BL/6 mice were subcutaneously injected with 0.5x106

B16F10 cells. After tumor inoculation (day 0), the mice were given three doses of

docetaxel (10 mg/kg) on days 1, 4, and 7, followed by single dose of TGFRt15-TGFRs (3

mg/kg) + TA99 (200 ug) on day 8. Tumor growth was monitored by caliper

measurement, and tumor volume was calculated using the formula V = (L X W2)/2,

where L is the largest tumor diameter and W is the perpendicular tumor diameter.

As shown in Figure 242, B16F10 tumor bearing mice treated with TGFRt15-

TGFRs in combination with TA99 and chemotherapy showed a significant reduction in

B16F10 tumor volume, when compared to tumors of the saline or chemotherapy

treatment groups. However, when the mice were depleted for NK and CD8+ cell subsets,

there was no effect of immunotherapy on the anti-antitumor activity. This experiment

shows that both the NK and CD8+ immune cells play an important role in TGFRt15-

TGFRs mediated anti-tumor activity.

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Example 111: Comparison of TGFRt15-TGFRs and TGFRt15*-TGFRs Treatment in Reducing Senescence Markers in Liver and Lung Tissues of B16F10 Tumor-

bearing Mice Following Chemotherapy

C57BL/6, 6-8-week-old mice were purchased from the Jackson Laboratory. Mice

were housed in a temperature and light controlled environment. Mice were divided into

five groups as follows: saline control group (n =7), docetaxel (DTX) group (n =7), DTX

+ TGFRt15-TGFRs group (n =7), DTX + TGFRt15*-TGFRs group (n =7), and DTX + IL15SA group (n =7). B16F10 tumor cells (1 1 x 107 cells/mouse) were implanted in mice

on day 0. The mice were treated subcutaneously with 10 mg/kg docetaxel on days 1, 4,

and 7. On day 8, the mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3

mg/kg), TGFRt15*-TGFRs (3 mg/kg), or IL15SA (0.5 mg/kg). The mice were

euthanized day 17 post-treatment and liver and lungs were harvested in order to evaluate

the gene expression of senescence markers p21, IL-1a, and IL6 for liver and p21 and IL-

1a for lung by quantitative-PCR in tissues after treatment with TGFRt15-TGFRs or

TGFRt15*-TGFRs and control groups. Harvested organs were stored in liquid nitrogen

in 1.7 mL Eppendorf tubes. The samples were homogenized by using homogenizer in 1

mL of Trizol (Thermo Fischer). Homogenized tissues were transferred in fresh

Eppendorf tubes. Total RNA was extracted using RNeasy Mini Kit (Qiagen #74106)

according to the manufacturer's instructions. One ug of total RNA was used for cDNA

synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was

carried out with CFX96 Detection System (Bio-Rad) using FAM-labeled predesigned

primers purchased from Thermo Scientific. Reactions were run in triplicate for all the

genes examined. The housekeeping gene 18S ribosomal RNA was used as an internal

control to normalize the variability in expression levels. The expression of each target

mRNA relative to 18S rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Ct

target- Ct18S.

As shown in Figure 243, the senescence markers p21, IL-1a, and IL6 showed

decreased gene expression in liver (A) and lung (B) tissues in both TGFRt15-TGFRs and

TGFRt15*-TGFRs-treated tumor bearing mice, when compared to gene expression in

tissues of chemotherapy treated mice.

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Example 112: TGFRt15-TGFRs Treatment in Reducing Chemotherapy-induced

Senescent Tumor Cells in vivo

B16F10 melanoma cells were stably transduced with GFP lentiviral plasmid and

the GFP-expressing tumor cells (B16F10-GFP) were selected by growth in puromycin

containing media. Almost 95% B16F10 melanoma cells were GFP-positive as analyzed

by FACS. To induce senescence, B16F10-GFP cells were treated with 7.5 M docetaxel

(DTX) for 3 days followed by 4 days recovery in the normal growth media. To quantify

gene expression of senescence markers and NK cell ligands, docetaxel-treated B16F10

GFP cells (B16F10-GFP-SNC) were homogenized by using homogenizer in 1 mL of

Trizol (Thermo Fischer). Homogenized cells were transferred in fresh Eppendorf tubes.

Total RNA was extracted using RNeasy Mini Kit (Qiagen #74106) according to the

manufacturer's instructions. One ug of total RNA was used for cDNA synthesis using

the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with

CFX96 Detection System (Bio-Rad) using FAM-labeled predesigned primers purchased

from Thermo Scientific. The reactions were run in triplicate for all the genes examined.

The housekeeping gene 18S ribosomal RNA was used as an internal control to normalize

the variability in expression levels. The expression of each target mRNA relative to 18S

rRNA was calculated based on Ct as 2-A(ACt) in which ACt = Ct target- Ct18S. The

expression of different genes is plotted as fold-change in B16F10-GFP-SNC cells as

compared to untreated B16F10-GFP cells.

As shown in Figure 244, real time PCR analysis showed that B16F10-GFP cells

treated in vitro with docetaxel upregulated gene expression of senescence markers, p21,

H2AX, and IL6, and NK cell ligands, Rae-1e and ULBP-1, when compared to untreated

B16F10-GFP cells.

To determine whether chemotherapy-induced senescence tumor cells are reduced

by immunotherapy in vivo, B16F10 parental melanoma cells (0.75 x 106) were mixed

with B16F10-GFP-SNC cells (0.75 ) X 106) and injected the cell mixture subcutaneously in

C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls.

The B16F10 parent cells will grow to form tumor and B16F10-GFP-SNC cells will be the

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part of the tumor microenvironment. When tumors reached to approximately 350 mm³,

mice bearing the mixed tumors were divided into 2 groups. One group received PBS as

control and the other group received TGFRt15-TGFRs (3 mg/kg) with TA99 (200 ug)

subcutaneously. The mice were sacrificed day 4 post-immunotherapy treatment. The

tumor tissue was dissociated into single cell suspension by collagenase digestion to

determine the tumor-infiltrating immune cells. The single cell suspension was layered on

Ficoll-Paque media followed by density gradient centrifugation to separate the

lymphocytes and tumor cells. The cells were centrifuged at 1000 g for 20 minutes at 20

°C with slow acceleration and break turned off. After centrifugation the Ficoll-Paque

results in a distinct separation between two layers. The TILs are found on the interface

between the media and Ficoll-Paque, while the pellet consists of the tumor cells. The

TILs were carefully removed from the interface and washed with complete RPMI media.

After washing, the RBCs were lysed in ACK buffer for 5 minutes at room temperature.

The remaining cells were washed in FACS buffer (1X PBS (Hyclone) with 0.5% BSA

(EMD Millipore) and 0.001% Sodium Azide (Sigma)). To assess the different types of

immune cells in tumor, the cells were stained with antibodies specific to cell-surface

CD3, CD45, CD8, and NK1.1 (BioLegend) for 30 minutes at RT. After surface staining,

cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (1X

PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% Sodium Azide (Sigma)).

After two washes, the cells were resuspended in fixation buffer. After fixation, the cells

were washed and treated with permeabilization buffer (Invitrogen) for 20 minutes at 4 °C

followed by wash with permeabilization buffer (Invitrogen). The cells were then stained

for intracellular markers (Ki67) for proliferation for 30 minutes at RT. After two washes,

the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-

BD Bioscience).

As shown in Figure 245, the percentage of CD8+ T cells and natural killer (NK)

cells were increased after 4 days post-treatment in the tumor following TGFRt15-

TGFRs+TA99 treatment, compared to controls. These results demonstrate that

TGFRt15-TGFRs is able to stimulate infiltration of CD8+ T cells and NK cells in the

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tumor. Both CD8+ T cells and NK immune cells were also able to proliferate in the

tumor as measured by the Ki67 marker.

To determine whether chemotherapy-induced senescence tumor cells are reduced

by immunotherapy in vivo, B16F10 parental melanoma cells (0.75 x 106) were mixed

with B16F10-GFP-SNC cells (0.75 x 106) and injected the cell mixture subcutaneously in

C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls.

The B16F10 parent cells will grow to form tumor and B16F10-GFP-SNC cells will be the

part of the tumor microenvironment. When tumors reached to approximately 350 mm³,

mice bearing the mixed tumors were divided into 2 groups. One group received PBS as

control and the other group received TGFRt15-TGFRs (3 mg/kg) with TA99 (200 ug)

subcutaneously. The mice were sacrificed after day 4 and day 10 post-immunotherapy

treatment. The tumor tissue was dissociated into single cell suspension by collagenase

digestion to determine the tumor-infiltrating immune cells and GFP-positive cells in the

tumor. Flow cytometry analysis (Figure 246A) on tumor cells showed that mice which

received immunotherapy treatment showed lower number of GFP-positive cells 4 days

and 10 days post-treatment as compared to the PBS control group. Tumor cells were

plated in a 24-well plate to evaluate by fluorescence microscopy (Figure 246B).

Microscopic images also showed fewer GFP-positive cells in the tumor of

immunotherapy-treated mice as compared to the control PBS-treated group. The GFP

expression in the tumor is associated with the chemotherapy-induced B16F10-GFP

senescence cells, therefore reduction in the GFP expression after immunotherapy

treatment shows the successful elimination of senescence tumor cells in the tumor

bearing mice.

Example 113: TGFB Levels in Kidney after Inducing Kidney Injury by Cisplatin

and Treatment with TGFRt15-TGFRs by Tissue ELISA

The mouse kidney was harvested in order to evaluate changes in protein levels of

the senescence markers TGFß after inducing kidney injury by cisplatin and treatment

with TGFRt15-TGFRs. C57BL/6, 8-week-old mice were purchased from the Jackson

Laboratory. The mice were housed in a temperature and light controlled environment.

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The mice were injected with cisplatin (5 mg/kg, intraperitoneal) weekly for 3 weeks to

induce kidney injury. One week after cisplatin, the mice were treated with either PBS or

TGFRt15-TGFRs (3 mg/kg) (n =8/group). The mice were euthanized after 30 days of

immunotherapy treatment and kidney were harvested and stored in liquid nitrogen in 1.7

mL-Eppendorf tubes. The samples were homogenized by using homogenizer in 0.3 mL

of extraction buffer (Abcam). Homogenized tissues were transferred in fresh Eppendorf

tubes. Protein levels in homogenized tissue were quantified using BCA Protein Assay

Kit (Pierce). Mouse TGFB ELISA (R&D System) was performed in 200 ug of tissue.

The concentration of TGFB was calculated in per milligram of tissue.

As shown in Figure 247, the TGFB level decreased in TGFRt15-TGFRs treated

mice kidney compared to PBS control group. These results indicate that TGFRt15-

TGFRs treatment is capable of provide long lasting activity in reducing TGFB levels in

tissues of chemotherapy-treated mice.

Example 114: Toxicity of Subcutaneous Administration of TGFRt15-TGFRs in

Mice To further assess the dose-dependent toxicological effects of FRt15-TGFRs,

female C57BL/6 mice (N=3/group) were administered one or two (every two weeks)

subcutaneous doses of PBS or TGFRt15-TGFRs at 3, 10, 50, and 200 mg/kg. Animals

were monitored for signs of study drug-related toxicities, changes in body weight during

the study period and hematology and serum chemistry parameters at day 7 post-dosing.

Mice receiving 200 mg/kg TGFRt15-TGFRs exhibited significant body weight loss

beginning 4 days after the first injection (study day (SD) 0) and reaching a nadir between

SD6-9, before returning to pre-dose levels by SD11 (Figure 248A). Mortality was

observed in one mouse of the 200 mg/kg group on SD9. There were no apparent

treatment-mediated effects on body weight or other clinical signs in any other dose group

or after the second TGFRt15-TGFRs dose at 200 mg/kg. Spleen weights increased in a

dose dependent manner following one or two doses of TGFRt15-TGFRs (Figure 248B).

Compared to the PBS group, mice also exhibited a 25-fold increase in WBC counts 7

days after a single 200 mg/kg dose of TGFRt15-TGFRs, which remained 5-fold higher 7 days after the second 200 mg/kg dose (Figure 248C, Tables 3 and 4). WBC subset analysis showed a 16-fold increase in absolute lymphocyte counts and >50-fold increase in neutrophil, monocyte, eosinophil, and basophil counts at SD7 in the 200 mg/kg group.

These changes were not observed at lower TGFRt15-TGFRs dose levels but were similar

to those reported for C57BL/6 mice treated subcutaneously treatment with IL-15/IL-

15Ra complexes (Liu et al., Cytokine 107: 105-112, 2018). Other hematology and serum

chemistry parameters were similar in the TGFRt15-TGFRs and PBS treated animals and

were generally within expected ranges for C57BL/6 mice (Tables 3 and 4). TGFRt15-

TGFRs-mediated effects were greatest 7 days after the first dose and were reduced after

the second dose, consistent with previous studies showing decreased immune responses

in mice following repeat dosing with IL-15/IL-15Ra (Elpek et al., PNAS 107: 21647-

21652, 2010; Frutoso et al., J Immunol 201: 493-506, 2018). Overall, TGFRt15-TGFRs

was well tolerated by C57BL/6 mice at dose levels up to of 50 mg/kg.

Table 3. Hematology and serum chemistry parameters of C57BL/6 mice on Study Day 7 after single dose of TGFRt15-TGFRs.

TGFRt15-TGFRs Study Day 7 PBS 3 mg/kg 10 mg/kg 50 mg/kg 200 mg/kg Parameters Mean SD N Mean SD N Mean SD N Mean SD N Mean Mean SD N WBC count 1.53 6.53 1.80 3 6.63 1.37 3 5.07 3 11.57 2.99 3 165.37 2.20 3 (x 103/uL)

RBC count 7.59 0.90 3 6.44 0.34 3 7.03 0.34 3 6.56 0,68 0.68 3 6,25 6.25 0.84 3 (x 106/uL)

Hemoglobin (g/dL) 10.1 0.8 3 9.3 0.0 3 9.6 0.3 3 8.7 1.1 3 9.4 1.2 3

Hematocrit (%) 36.0 3.2 3 31.8 2.3 3 33,0 1.9 3 30.8 3.3 3 29.9 4.0 3

47.3 1.5 3 49.3 1.5 3 46,7 46.7 0.6 3 47.0 0.0 3 48.0 0.0 3 MCV(fL) MCH (pg) 13.3 13.3 0.6 3 14.3 0.6 3 13.7 0.6 3 13.3 0.6 3 15.0 1.0 3

28.0 0.0 3 29,7 2,1 3 29.0 1.0 3 28,3 1.5 3 31.3 1.5 3 MCHC (%) Neutrophils 0.82 0.42 3 0.91 0.28 3 0.53 0.11 3 1.32 0.43 3 51.25 0,97 3 (x 103/uL)

Lymphocytes 1,31 5.46 3 5,39 0,91 3 4.26 1.34 3 9.47 2.34 3 86.01 2.80 3 (x 103/uL)

Monocytes (x 0.18 0.08 3 0.24 0,21 3 0.24 0.07 3 0.69 0.20 3 18.17 2.68 3 103/uL)

Eosinophils 0.07 0,02 3 0.06 0,02 3 0.05 0.02 3 0.08 0.08 3 7,73 2.02 3 (x 103/uL)

Basophils (x 103/uL) 0.02 0.03 3 0.03 0,05 3 0.00 0.00 3 0.00 0.00 3 2.21 0.99 3

Platelet count 558.3 81.1 3 692.3 55.8 3 886.0 53.6 3 1004.3 60.2 3 467.3 32.5 3 (x 103/uL)

% Neutrophils 12.0 3.0 3 13,7 13.7 3.1 3 10.7 1.2 3 11.3 1.2 3 31.0 1.0 3

% Lymphocytes 84.0 3.0 3 81.7 3.8 3 83.7 1.5 3 82.0 1.0 3 52.0 1.0 3

% Monocytes 2.67 0.58 3 3.33 2.31 3 4.67 0.58 3 6.00 1.00 3 11.00 1.73 3

% Eosinophils 1.00 0.00 3 1.00 0.00 3 1.00 0.00 3 0.67 0.58 3 4.67 1.15 3

% Basophils 0,33 0,58 3 0,33 0,58 3 0.00 0.00 3 0.00 0.00 3 1,33 0.58 3

AST (U/L) 84.3 28.2 3 69.0 9.2 3 137,7 137.7 108.6 3 71.7 2.5 3 162.3 11.8 3

ALT (U/L) 41.3 10.0 3 47,3 1.5 3 38,3 5.5 3 56,3 11.2 3 121.0 52,8 3

Alkaline Phos. (U/L) 113.7 17.0 3 112.0 8.7 3 248.3 218.8 3 95.0 7.8 3 83.0 16.6 3

Total Bilirubin 0.87 0.47 3 0.33 0.15 3 0.45 0.07 2 0.20 0.00 2 (mg/dL) ND ND ND BUN (mg/dL) 23,0 2,6 3 21.0 3.5 3 24,7 4,6 3 21.3 2,9 3 18,7 7.2 3

Table 4. Hematology and serum chemistry parameters of C57BL/6 mice on Study Day 21 after two doses of TGFRt15-TGFRs.

TGFRt15-TGFRs Study Day 21 3 mg/kg 10 mg/kg 50 mg/kg 200 mg/kg Parameters Mean SD N Mean SD N Mean SD N Mean SD N WBC count 5.37 3.13 3 5,63 0,75 3 6,37 2.02 3 31.45 40.38 2 (x 103/uL)

RBC count 6,37 1.67 3 7.45 0.62 3 6.82 0.67 3 7.13 0.18 2 2 (x 106/uL)

Hemoglobin (g/dL) 9.0 2.1 3 10.1 0.8 3 9.7 0.7 3 10.5 0.8 2 Hematocrit (%) 30.3 7.2 3 35.6 3.5 3 33.7 2.2 3 36.2 3.5 2 47.7 2.3 3 47.7 1.2 3 49.7 2.1 3 50.5 3.5 2 MCV(fL) MCH (pg) 14.0 1.0 3 13.3 0.6 3 14.3 0.6 3 14.5 0.7 2 30.0 0.0 3 28.3 1.5 3 28,7 28.7 0.6 3 28.5 0.7 2 MCHC (%) Neutrophils 0.65 0.50 3 0.62 0.07 3 1.10 0.55 3 6.78 9.09 2 (x 103/uL)

Lymphocytes 4.58 2,62 3 4.81 0,61 3 4.88 1.20 3 20.75 25.82 2 (x 103/uL)

Monocytes (x 0.13 0.08 3 0.19 0.06 3 0.24 0.13 3 3.32 4.65 2 103/uL)

Eosinophils 0.01 0.01 3 0.02 0.03 3 0.12 0.12 3 0.62 0.83 2 (x 103/uL)

Basophils (x 103/uL) 0.00 0.00 3 0.00 0.00 3 0.03 0.05 3 0.00 0.00 2 Platelet count 531.3 413.1 3 806.3 125.2 3 778.0 34.9 3 711.5 44.5 2 (x 103/uL)

% Neutrophils 10.3 6.0 3 11.0 1.0 3 16.7 2.9 3 17.0 7.1 2 % Lymphocytes 87.0 6.0 3 85.3 1.5 3 77,7 5.1 3 75.5 14.8 2

% Monocytes 2.33 0.58 3 3.33 0.58 3 3.67 1.53 3 6.00 7.07 2 2 % Eosinophils 0.33 0.58 3 0.33 0.58 3 1.67 1.15 3 1.50 0.71 2 2 % Basophils 0.00 0.00 3 0.00 0.00 3 0.33 0.58 3 0.00 0.00 2 2 AST (U/L) 108.3 76.8 3 62.3 5.0 3 560.7ª 560.7 888.2 3 198.5 190.2 2 ALT (U/L) 49.3 17.7 3 51.0 12.5 3 57,7 57.7 3.5 3 48.0 9.9 2 Alkaline Phos. (U/L) 110.3 12.4 3 121.0 18.0 3 174.7 99,4 99.4 3 138.0 5,7 2 Total Bilirubin 0.57 0.12 3 0,47 0.47 0.15 3 0.45 0.07 2 0.65 0.07 2 (mg/dL)

BUN (mg/dL) 27.0 5.0 3 22.3 4.2 3 24,3 24.3 2.1 3 25.0 1.4 2 a One of three mice in 50 mg/kg TGFRt15-TGFRs group had an observed AST value of 1586 U/L (~6 X ULN). This

mouse did not show clinical signs and its ALT value (61 U/L) was within the normal range.

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Example 115: Sequestration of TGF-B by TGFRt15-TGFRs and TGFRt15*-TGFRs in Mice

Female C57BL/6 mice were injected subcutaneously with PBS or 3 mg/kg of

TGFRt15-TGFRs or TGFRt15*-TGFRs and plasma was collected at various times post-

treatment. Plasma levels of TGF-B1 and TGF-B2 were determined using the TGFB 3-Plex

assay (Eve Technologies, Calgary, AL, Canada). TGFRt15-TGFRs and TGFRt15*-

TGFRs were found to significantly decrease plasma TGF-B1 and TGF-B2 levels in

C57BL/6 mice 2 days after treatment (Figure 249), consistent with the activity of the

TGFßRII domains of these fusion proteins.

Example 116: Effects of TGFRt15-TGFRs and TGFRt15*-TGFRs on Immune Cell

Metabolism in vivo and in vitro

To assess treatment mediated effects on immune cell metabolism, extracellular

flux assays were performed on splenocytes isolated from mice 4 days after PBS,

TGFRt15-TGFRs, TGFRt15*-TGFRs or IL-15/IL-15R (IL15SA) administration.

Extracellular flux assays on mouse splenocytes were performed using a XFp Analyzer

(Seahorse Bioscience). As expected, TGFRt15-TGFRs and IL-15 increased the rates of

glycolytic capacity (ECAR) (Figure 250A) and mitochondrial respiratory capacity (OCR)

(Figure 250B) of the isolated splenocytes in a dose-level-dependent manner. In vivo

TGFRt15*-TGFRs treatment also increased ECAR and OCR of splenocytes. This

phenomenon was not observed when splenocytes from untreated C57BL/6 mice were

incubated 4 days with TGFRt15*-TGFRs in vitro. Only TGFRt15-TGFRs (but not

TGFRt15*-TGFRs] was capable of increasing splenocyte ECAR and OCR in vitro at

physiologically relevant concentrations (Figures 251A-251B). This suggests that both the

IL-15 and TGFßRII domains of TGFRt15-TGFRs have a role in stimulating immune cell

metabolism in vivo.

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Example 117: Antitumor efficacy of TGFRt15-TGFRs and TGFRt15*-TGFRs

Against B16F10 Melanoma in C57BL/6 Mice

To evaluate TGFRt15-TGFRs and TGFRt15*-TGFRs antitumor efficacy, the

murine B16F10 tumor model was selected as it is highly aggressive, poorly immunogenic

and devoid of immune infiltrates, expresses TGF-B which plays a role in its growth and is

resistant to cytokine and checkpoint blockade immunotherapies. B16F10 melanoma cells

(5 X 105 cells) (CRL-6475, ATCC) were subcutaneously injected into C57BL/6 mice

followed by subcutaneous injection of PBS, TGFRt15-TGFRs (3 or 20 mg/kg) or

TGFRt15*-TGFRs (3 or 20 mg/kg) on day 1 and 4 after tumor implantation. Tumor

volume was measured every other day and mice with tumors >4000 mm³ were sacrificed

per IACUC regulation. Mouse survival was also assessed throughout the study period.

When compared through SD15 (i.e., prior to animal mortality), treatment with TGFRt15-

TGFRs or TGFRt15*-TGFRs at 20 mg/kg resulted in significantly slower tumor growth

than was observed in the PBS treated mice (Figure 252A). Tumor-bearing mice treated

with 20 mg/kg TGFRt15-TGFRs also showed prolonged survival when compared to the 3

mg/kg TGFRt15-TGFRs and PBS treatment groups (Figure 252B). These results indicate

that TGFRt15-TGFRs and TGFRt15*-TGFRs have antitumor activity against solid

B16F10 melanoma tumors with the bifunctional TGFRt15-TGFRs complex exhibiting

the greater efficacy. Thus, both the TGFßRII and IL-15/IL-15RaSu domains play a role

in TGFRt15-TGFRs-mediated activity against B16F10 tumors.

TGFRt15-TGFRs treatment is capable of significantly increasing the number of

NK and T cells in vivo. To determine if these immune cells were responsible for

TGFRt15-TGFRs-mediated antitumor efficacy, Ab immunodepletion of CD8+ T cells

and NK1.1+ cells was conducted in tumor-bearing mice prior to TGFRt15-TGFRs

treatment. It was found that NK1.1+ cell depletion (alone or in combination with CD8+ T

cell depletion) eliminated the antitumor effects of TGFRt15-TGFRs in B16F10 tumor-

bearing mice during the first 2 weeks post-treatment (Figure 252C), whereas either

NK1.1+ cell depletion or CD8+ T cell depletion reduced the survival benefit seen with

TGFRt15-TGFRs (Figure 252D). Consistent with these findings, TGFRt15-TGFRs

treatment also promoted an increase in NK cell and CD8+ T cell infiltration into B16F10

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tumors (Figure 252E). These results support the conclusion that both CD8+ T cells and

NK cells play a major role in TGFRt15-TGFRs-mediated activity against melanoma

tumor cells in C57BL/6 mice.

Example 118: TGFRt15-TGFRs Improved the Glucose Control in db/db Mice

Five-week-old male BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice (Jackson Lab)

were fed with standard chow diet and maintained in the standard conditions. Mice (n =

5/group) were received subcutaneous injections of either PBS (control group) or

TGFRt15-TGFRs (3 mg/kg) (treatment group) at weeks 6 and 12 from the start of the

study. The fasting blood glucose and insulin were checked three weeks after the 1st dose.

The fasting glucose was significantly reduced (Figure 253A) after TGFRt15-TFGRs

treatment compared to controls but blood insulin levels were not changed (Figure 253B).

Example 119: TGFRt15-TGFRs Significantly Down-regulated Aging Index and

SASP Index Five-week-old male BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice were fed with

standard chow diet and received drinking water ad libitum. At the age of six weeks, mice

were randomly assigned to control and treatment groups (n = 5/group). The treatment

group received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12

from the start of the study, while the control group received vehicle (PBS) only. At end

of study (4-weeks post the 2nd dose), mice were euthanized and pancreas was collected.

The half of pancreas was homogenized with the TRIzol reagent (Invitrogen) and total

tissue RNA was purified with RNeasy Mini Kit (Qiagen). Synthesis of cDNA was

performed using a QuantiTect Reverse Transcription Kit (Qiagen) and quantitative PCR

was performed using a SsoAdvancedTM Universal SYBRR Green Supermix (BioRad)

and a QuantiStudio 3 Real-Time PCR System (Applied Biosystems) according to

comparative threshold cycle method following manufacturer's protocol. The

amplification reactions were performed in duplicate, and the fluorescence curves were

analyzed with the software included with the QuantiStudio 3 Real-Time PCR System.

The housekeeping gene 18s ribosomal RNA was used as an endogenous control

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reference. The expression of each target mRNA relative to 18s rRNA was calculated

based on Ct as 2-A(ACt) in which ACt = Cttarget Ctiss. As shown in Figure 254A,

TGFRt15-TGFRs treatment of db/db mice resulted in a reduction of pancreatic gene

expression for p16, p21, Igfrl, and Bamb1 of the Aging gene index and IL-1a, IL-6,

MCP-1, and TNFa of SASP gene index when compared to the control group. Generally,

pancreatic expression of genes of the SASP Index and Aging Index were significantly

reduced following TGFRt15-TGFRs treatment compared to controls, whereas pancreatic

gene expression of the beta cell index was not changed significantly in the TGFRt15-

TGFRs and PBS-treated db/db mice. (Figures 254B, 254C, 254D). The data suggested

TGFRt15-TGFRs has potent senolytic and senomorphic activities to reduce senescent

cells and SASP factors in the pancreas of db/db mice.

Example 120: TGFRt15-TGFRs Reduced Senescent Cells of Pancreatic Beta Cells

Five-week-old male BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice (Jackson Lab)

were fed with standard chow diet (Irradiated 2018 Teklad global 18% protein rodent diet,

Envigo) and received drinking water ad libitum. At the age of six weeks, mice were

randomly assigned to control and treatment groups (n = 5/group). The treatment group

received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12 from

the start of the study, while control group received vehicle (PBS) only. At end of study

(4-weeks post the 2nd dose), mice were euthanized and pancreata were removed en bloc,

immersion-fixed in 4% formaldehyde (4% formaldehyde in 0.1M phosphate buffer; PBS

pH 7.4) and stored at 4°C degrees until further processing. Dissected pancreata were

paraffinized, embedded, and sectioned, and three 10 mm sections (150 mm apart) were

cut from each block representing in total a systematic uniform random sample of the

whole pancreas from each animal.

Multispectral imaging was performed using the Akoya Vectra Polaris instrument.

This instrumentation allows for phenotyping, quantification, and spatial relationship

analysis of tissue infiltrate in formalin-fixed paraffin-imbedded biopsy sections. To

quantify levels of p21 in insulin islet regions of the pancreas, formalin-fixed paraffin-

embedded tissue sections were stained consecutively with specific primary antibodies

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according to standard protocols provided by Akoya and performed routinely by the

HIMSR. Briefly, the slides were deparaffinized, heat treated in antigen retrieval buffer,

blocked, and incubated with rabbit primary antibodies against insulin (#4590, Cell

Signaling Technology) and p21 (EPR362, Abcam), followed by horseradish peroxidase

(HRP)-conjugated secondary antibody polymer (anti-rabbit), and HRP-reactive OPAL

fluorescent reagents (OPAL-520 for insulin and OPAL-570 for p21, Akoya) that use TSA

chemistry to deposit dyes on the tissue immediately surrounding each HRP molecule. To

prevent further deposition of fluorescent dyes in subsequent staining steps, the slides

were stripped in between each stain with heat treatment in antigen retrieval buffer

(Citrate buffer for insulin and EDTA buffer for p21). Whole slide scans were collected

with the Akoya Vectra Polaris instrument using the 20x objective with a 0.5 micron

resolution. The 3 color images were analyzed with inForm software (Akoya) to unmix

adjacent fluorochromes, subtract autofluorescence, segment insulin regions of the tissue,

compare the frequency and location of cells, segment cellular cytoplasmic and nuclear

regions, and phenotype infiltrating cells according to cell marker expression.

As shown in Figure 255A-255D, p21 positive senescent cells (OPAL-570) were

accumulated more in insulin positive islet beta cells (OPAL-520) in pancreas of control

group (Figure 255A) and these senescent cells were reduced in pancreas of TGFRt15-

TGFRs treatment group (Figure 255B). The insulin positive islet cells were significantly

increased in TGFRt15-TGFRs treatment group compared with the control group

(p=0.0278, Figure 255C). The p21 positive senescent beta cells (insulin positive) were

reduced in TGFRt15-TGFRs treated group compared with the control group though the

difference was not statistically significant (Figure 255D). Overall, the data suggested

TGFR15-TGFRs has senolytic activity to remove senescent cells and promotes the

recovery of normal functional islet beta cells in the pancreas of db/db mice.

Example 121: TGFRt15-TGFRs Reduced Senescent Cells of Pancreatic Beta Cells

by Increasing NK, NKT, and CD8+ T cells

Five-week-old male BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice (Jackson Lab)

were fed with standard chow diet (Irradiated 2018 Teklad global 18% protein rodent diet,

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Envigo) and received drinking water ad libitum. At the age of six weeks, mice were

randomly assigned into control and treatment groups (n = 5/group). The treatment group

received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12 from

the start of the study, while control group received vehicle (PBS) only.

Four days after the 1st dose treatment, blood was collected and whole blood cells

(50 mL) were treated with ACK (Ammonium-Chloride-Potassium lysing buffer to lyse

red blood cells. The lymphocytes were then stained with PE-Cy7-anti-CD3, BV605-anti-

CD45, PerCP-Cy5.5-anti-CD8a, BV510-anti-CD4, and APC-anti-NKp46 antibodies (all

antibodies from BioLegend) to assess the population of T cells, NKT cells, and NK cells.

As shown in Figure 256A-256C, the percentages of CD8+ T cells, CD3 NKP46 NKT

cells, and CD3*NKP46 NK cells increased in the blood of db/db mice following

treatment with TGFRt15-TGFRs compared to the PBS-treated mice.

Example 122: Phenotyping of Immune Cell Subsets in Peripheral Blood of

Cynomolgus Monkeys Following Administration of TGFRt15-TGFRs

Cynomolgus monkeys (5M:5F per group) were treated subcutaneously with PBS

(vehicle) or TGFRt15-TGFRs at 1, 3 or 10 mg/kg on study days 1 and 15. Blood was

collected pre-day (day 1) and days 5, 22 and 29 post-treatment. PBMCs were prepared

and stained with a panel of fluor-conjugated antibodies to assess the phenotypes of B

cells, NK cells, NK-T cells, Treg cells and CD4+ and CD8+ T cells by flow cytometry.

Figure 257 shows that TGFRt15-TGFRs administration resulted in a significant increase

in the percentage of Ki67+ NK cells, NK-T cells, Treg cells and CD4+ and CD8+ T cells

on day 5 post-treatment. These findings indicate that TGFRt15-TGFRs treatment

induced proliferation of these lymphocyte subsets in non-human primates. No treatment

effects were observed on Ki67 expression in B cells.

Example 123: IL-15 Immunostimulatory and TGF-B Antagonist Activities of

TGFRt15-TGFRs Six-week-old (young) and 72-week-old (aged) C57BL/6 mice were

subcutaneously injected with single dose of PBS, TGFRt15-TGFRs (3 mg/kg) or

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TGFRt15*-TGFRs (3 mg/kg). On day 4 after treatment, mice were sacrificed, and the

spleens were harvested. The spleens were crushed with flat back end of the sterile

piston/plunger of 3 CC syringe to release the splenocytes. The splenocytes were passed

through a 70 cell strainer and homogenized into a single cell suspension. The RBCs

were lysed in ACK lysis buffer and the splenocytes were washed and counted. To

measure the glycolytic activity of the splenocytes, the cells were washed and resuspended

in Seahorse media and resuspended at 4 X 106 cells/mL. Cells were seeded at 50 uL/well

in Cell-Tak-coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse XF RPMI

medium, pH 7.4 supplemented with 2 mM L-glutamine for glycolysis stress test. The

cells were allowed to attach to the plate for 30 min at 37°C. Additionally, 130 uL of the

assay medium was added to each well of the plate (also the background wells). The plate

was incubated in 37°C, non-CO2 incubator for 1 hr. For glycolysis stress test the

calibration plate contained 10x solution of glucose/oligomycin/2DG prepared in Seahorse

assay media and 20 uL of glucose/oligomycin/2DG were added to each of the ports of the

extracellular flux plate that was calibrated overnight. The glycolysis stress test is based

on extracellular acidification rate (ECAR) and measures three key parameters of

glycolytic function including glycolysis, glycolytic capacity and glycolytic reserve.

Complete ECAR analysis consisted of four stages: non glycolytic acidification (without

drugs), glycolysis (10 mM glucose), maximal glycolysis induction/glycolytic capacity (2

uM oligomycin), and glycolysis reserve (100 mM 2-DG). At the end of the experiment

the data was exported as a Graph Pad Prism file. The XF glycolysis stress test report

generator automatically calculated the XF cell glycolysis stress test parameters from the

Wave data. The data was analyzed using the Wave software (Agilent).

As shown in Figure 258, the splenocytes isolated from aged mice on day 4 after

TGFRt15-TGFRs treatment showed enhanced basal glycolysis, glycolysis capacity, and

glycolysis reserve rates, when compared to splenocytes of the PBS or TGFRt15*-TGFRs

treatment groups. The glycolytic function of splenocytes of aged control mice was less

than that of the young control mice. Treatment of young and aged mice with TGFRt15*-

TGFRs was capable of increasing splenocyte glycolytic function. However, TGFRt15-

TGFRs treatment of aged mice was able to increase the rates of splenocyte basal

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glycolysis, glycolysis capacity, and glycolysis reserve to levels equivalent to those

observed in the splenocytes from TGFRt15-TGFRs treated young mice. These findings

suggest that the IL-15 immunostimulatory and TGF-B antagonist activities of TGFRt15-

TGFRs effectively stimulate and rejuvenate the diminished metabolic activity of immune

cells from aged mice.

Six-week-old (young) and 72-week-old (aged) C57BL/6 mice were

subcutaneously injected with single dose of PBS, TGFRt15-TGFRs (3 mg/kg) or

TGFRt15*-TGFRs (3 mg/kg). On day 4 after treatment, mice were sacrificed, and the

spleens were harvested. The spleens were crushed with flat back end of the sterile

piston/plunger of 3 cc syringe to release the splenocytes. The splenocytes were passed

through a 70 cell strainer and homogenized into a single cell suspension. The RBCs

were lysed in ACK lysis buffer and the splenocytes were washed and counted. To

measure the mitochondrial respiration of the splenocytes, the cells were washed and

resuspended in Seahorse media and resuspended at 4 X 106 cells/mL. Cells were seeded at

50 uL/well in Cell-Tak-coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse

XF RPMI medium, pH 7.4 supplemented with 2 mM L-glutamine for glycolysis stress

test. For mitochondrial stress test, the cells were seeded in Seahorse XF RPMI medium,

pH 7.4 supplemented with 10 mM glucose and 2 mM L-glutamine. The cells were

allowed to attach to the plate for 30 min at 37°C. Additionally, 130 uL of the assay

medium was added to each well of the plate (also the background wells). The plate was

incubated in 37°, non-CO2 incubator for 1 hr. For mitochondrial stress test, the

calibration plate contained 10x solution of oligomycin/FCCP/rotenone prepared in

Seahorse assay media and 20 uL of oligomycin, FCCP and rotenone was added to each

of the ports of the extracellular flux plate that was calibrated overnight. Oxygen

consumption rate (OCR) was measured using an XFe96 Extracellular Flux Analyzer.

Complete OCR analysis consisted of four stages: basal respiration (without drugs), ATP-

linked respiration/Proton leak (1.5 M oligomycin), maximal respiration (2 FCCP),

and spare respiration (0.5 uM rotenone). At the end of the experiment, the data was

exported as a Graph Pad Prism file. The XF mitochondrial stress test report generator

automatically calculates the XF mitochondrial stress test parameters from the Wave data

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that have been exported to Excel. The data was analyzed by using the Wave software

(Agilent).

As shown in Figure 259, the splenocytes isolated from aged mice on day 4 after

TGFRt15-TGFRs therapy showed enhanced basal respiration, ATP-linked respiration,

maximal respiration, and reserve capacity, when compared to splenocytes of the PBS or

TGFRt15*-TGFRs treatment groups. Treatment of young and aged mice with

TGFRt15*-TGFRs was capable of increasing splenocyte mitochondrial respiration.

However, TGFRt15-TGFRs treatment in aged mice able to increase the rates of basal

respiration, ATP-linked respiration, maximal respiration, and reserve capacity to levels

equivalent or higher to those observed in the splenocytes from TGFRt15-TGFRs treated

young mice. These findings suggest that the IL-15 immunostimulatory and TGF-B

antagonist activities of TGFRt15-TGFRs effectively stimulate and rejuvenate the

diminished metabolic activity of immune cells from aged mice.

Example 124: IL-15 Activity of TGFRt15-TGFRs Plays a Role in Increasing CD8+ T

Cells and NK Cells

Six-week-old (young) and 72-week-old (aged) C57BL/6 mice were purchased

from the Jackson Laboratory. Mice were housed in a temperature and light controlled

environment. Mice (n =6/group) were treated subcutaneously with PBS, TGFRt15-

TGFRs (3 mg/kg) and TGFRt15*-TGFRs (3 mg/kg). The mouse blood was collected

from submandibular vein on day 4 post treatment in tubes containing EDTA to evaluate

changes in the different subsets of immune cells. Whole blood RBCs were lysed in ACK

buffer for 5 minutes at room temperature. Remaining cells were washed in FACS buffer

(1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide

(Sigma)). To assess the different types of immune cells in blood, cells were stained with

antibodies specific to cell-surface CD3, CD4, CD45, CD8 and NK1.1 (BioLegend) for 30

min at room temperature (RT). After surface staining, cells were washed (1500 RPM for

5 min at RT) in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and

0.001% sodium azide (Sigma)). After two washes, cells were resuspended in fixation

buffer and analyzed by flow cytometry (Celesta-BD Bioscience).

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As shown in Figure 260, the results indicate that treatment of aged mice with

TGFRt15-TGFRs induced an increase in the percentages of CD3*CD45*, CD3+CD8*,

and CD3*NK1.1+ immune cells in the blood, whereas treatment of aged mice with

TGFRt15*-TGFRs had no effect on the percentage of these blood cell populations.

These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing

CD8+ T cells and NK cells in the blood of aged mice. The percentage of blood T cells

and NK cells in aged control mice was less than that of the young control mice. However,

treatment of aged mice with TGFRt15-TGFRs increased the percentages of CD3*CD45+,

CD3+CD8*, and CD3 NK1.1+ immune cells in the blood to levels similar to those

observed in the blood of TGFRt15-TGFRs treated young mice.

Six-week-old (young) and 72-week-old (aged) C57BL/6 mice were purchased

from the Jackson Laboratory. Mice were housed in a temperature and light controlled

environment. Mice (n =6/group) were treated subcutaneously with PBS, TGFRt15-

TGFRs (3 mg/kg) and TGFRt15*-TGFRs (3 mg/kg). Four days after treatment, the mice

were euthanized, and spleen was harvested and processed to a single cell suspension.

Single cells suspension was prepared in order to evaluate the different subsets of immune

cells. RBCs were lysed in ACK buffer for 5 min at room temperature. The remaining

cells were washed in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore)

and 0.001% sodium azide (Sigma)). To assess the different types of immune cells in

spleen, cells were stained with antibodies specific to cell-surface CD3, CD45, CD8 and

NK1.1 (BioLegend) for 30 minutes at RT. After surface staining, cells were washed

(1500 RPM for 5 min at room temperature) in FACS buffer (1X PBS (Hyclone) with

0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, cells

were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD

Bioscience).

As shown in Figure 261, the results indicate that treatment of aged mice with

TGFRt15-TGFRs induced an increase in the percentages of CD3*CD45*, CD3*CD8*,

and CD3*NK1.1+ immune cells in the spleen, whereas treatment of aged mice with

TGFRt15*-TGFRs had no effect on the percentage of these splenocyte populations.

These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing

CD8+ T cells and NK cells in the blood of aged mice. The percentage of spleen T cells

and NK cells in aged control mice was less than that of the young control mice. However,

treatment of aged mice with TGFRt15-TGFRs increased the percentages of CD3*CD45*,

CD3+CD8*, and CD3`NK1.1+ immune cells in the spleen to levels similar to those

observed in the spleen of TGFRt15-TGFRs treated young mice.

Example 125: TGFRt15-TGFRs-associated Decrease in Naturally-occurring

Senescent Cells in the Liver

Seventy-two-week-old (aged) C57BL/6 mice were purchased from the Jackson

Laboratory. Mice were housed in a temperature and light controlled environment. Mice

(n =8/group) were treated subcutaneously with either PBS or one dose or two doses (at

day 0 and 60) of TGFRt15-TGFRs (3 mg/kg). On day 71 post treatment, mice were

euthanized and the livers were harvested and stored in liquid nitrogen in 1.7 mL

Eppendorf tubes. Tissue samples were homogenized by using homogenizer in 1 mL of

Trizol (Thermo Fischer). Homogenized tissues were transferred in fresh Eppendorf tubes

and total RNA was extracted using RNeasy Mini Kit (Qiagen #74106) according to the

manufacturer's instructions. One ug of total RNA was used for cDNA synthesis using the

QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with

CFX96 Detection System (Bio-Rad) using FAM labeled predesigned primers purchased

from Thermo Scientific. Reactions were run in triplicate for all the genes examined. The

housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the

variability in gene expression levels. The expression of each target mRNA relative

to 18S rRNA was calculated based on Ct as 2-A(ACt)_ in which ACt = Cttarget Ctiss.

Untreated 6-week-old mice were used as a control to compare the gene expression level

to aged mice. The results showed that gene expression of IL-1a, IL-1B, IL-6, p21 and

PAI-1 in liver increased with the age of the mice as expected with the age-dependent

increase in cellular senescence-associated transcripts. Treatment of 72-week-old mice

with a single dose or two doses of TGFRt15-TGFRs resulted in a significant reduction in

gene expression of senescence markers IL-1a, IL-1ß, IL-6, p21 and PAI-1 in liver when

compared to the PBS control group (Figure 262). These findings suggest a TGFRt15-

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TGFRs-associated decrease in naturally-occurring senescent cells in the liver of aged

mice.

Example 126: TGFRt15-TGFRs Treatment is Capable of Reducing Inflammation in

Liver Tissues

Seventy-two-week-old (aged) C57BL/6 mice were purchased from the Jackson

Laboratory. Mice were housed in a temperature and light controlled environment. Mice

(n : =10/group) were treated subcutaneously with either PBS or one or two doses of

TGFRt15-TGFRs (3 mg/kg). On day 120 after treatment, mice were euthanized and the

mouse liver was prepared to evaluate by histochemistry. Liver tissue specimens were

fixed in 10% formaldehyde and after a paraffin blocking procedure, cross-sections were

stained with hematoxylin-eosin. The extent of liver injury was evaluated histologically in

a blinded manner. Histological sections of whole liver areas were scores for inflammation

using a scale from 0 to 4 (0, absent and appearing to be normal; 1, light; 2, moderate; 3,

strong; and 4, intense). As shown in Figure 263, two doses of TGFRt15-TGFRs decrease

the liver inflammation score in liver of aged mice compared to single dose TGFRt15-

TGFRs or PBS control groups. These results suggest that TGFRt15-TGFRs treatment is

capable of reducing inflammation in liver tissues of aged mice.

Example 127: TGFRt15-TGFRs Treatment can Reduce IL1-a, IL-6, IL-8, PAI-1

and Fibronectin Protein Levels

Seventy-two-week-old (aged) C57BL/6 mice were purchased from the Jackson

Laboratory. Mice were housed in a temperature and light controlled environment. Mice

(n = =10/group) were treated with either PBS or one dose or two doses (at day 0 and 60) of

TGFRt15-TGFRs (3 mg/kg). On day 120 after treatment, mice were euthanized and liver

were harvested and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Tissue samples

were homogenized by using homogenizer in 0.3 mL of extraction buffer (Abcam).

Homogenized tissues were transferred in fresh Eppendorf tubes. Protein levels in

homogenized tissue were quantified using BCA Protein Assay Kit (Pierce). An ELISA to

detect IL-1a, IL-1B, IL-6, IL-8, TGF-B, PAI-1, collagen and fibronectin (R&D System)

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was performed using 25 ug of tissue homogenize. As shown in Figure 264, protein levels

of IL-1a, IL-6, IL-8, PAI-1 and fibronectin were reduced in liver of mice treated with 2

doses of TGFRt15-TGFRs compared to PBS control or one dose TGFRt15-TGFRs

treatment groups. These results indicate that 2 doses of TGFRt15-TGFRs treatment can

reduce IL-1a, IL-6, IL-8, PAI-1 and fibronectin protein levels in liver of aged mice.

Protein levels of IL-1B, TGF-B and collagen were also lower in liver of mice treated with

2 doses of TGFRt15-TGFRs compared to PBS controls; however, these changes did not

reach statistical significance.

Example 128: TGFRt15-TGFRs Reduces Senescence Cells

Seventy-two-week-old (aged) C57BL/6 aged mice which were purchased from

the Jackson Laboratory. Mice were housed in a temperature and light controlled

environment. Mice (n =5/group) were treated subcutaneously with either PBS or

TGFRt15-TGFRs (3 mg/kg). On day 4 after treatment, mice were euthanized and livers

were harvested, homogenized in PBS containing 2% FBS, and filtered in 70-micron filter

to obtain a single cell suspension. Cells were spun down then resuspended in 5 mL

RPMI containing 0.5 mg/mL collagenase IV and 0.02 mg/mL DNAse in 14 mL round

bottom tubes. Cells were then shaken on orbital shaker for 1 hr at 37°C and washed twice

with RPMI. Cells were resuspended at 2 X 106/mL in 24 wells flat bottom plate in 2 mL

of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo

Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life

Technologies), and 10% FBS (Hyclone)) and cultured for 48 hr at 37°C, 5% CO2. Cells

were harvested, washed once in warm complete media at 1000 rpm for 10 minutes at

room temperature. Cell pellet was resuspended in 500 uL of fresh media containing 1.5

uL of Senescence Dye per tube (Abcam). Cells were further incubated for 1-2 hr at 37°,

5% CO2 and wash twice with 500 uL wash buffer. Cell pellet was resuspended in 500 uL

of wash buffer and was analyzed immediately by flow cytometry (Celesta-BD

Bioscience). As shown in Figure 265, the percentage of senescence marker B-gal+ cells

were decreased 4 days after in vivo treatment with TGFRt15-TGFR. These results

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demonstrate that TGFRt15-TGFRs is capable of reducing senescence cells (based on the

B-gal marker) in liver of aged mice.

Example 129: Effects of TGFRt15-TGFRs on Survival of Aged Mice

Seventy-two-week-old C57BL/6 mice were purchased from the Jackson

Laboratory. Mice were housed in a temperature and light controlled environment. Mice

were treated subcutaneously with either PBS or one dose of TGFRt15-TGFRs (3 mg/kg)

(n = =20/group). Mice were monitored every day for survival up to 120 weeks post

treatment. The survival probability of the treatment groups based on the Mantel-Cox log-

rank test is shown in Figure 266. Compared with TGFRt15-TGFRs, higher mortality rates

were found in control mice which was represented by a decline in the survival rates of the

mice. By week 120 post treatment, there was a 70% mortality rate in PBS control mice

compared to a 45% mortality rate in the TGFRt15-TGFRs-treated mice.

Example 130: Effects of TGFRt15-TGFRs in Reducing SASP Factors in Liver of

B16F10 Tumor-bearing Mice Following Chemotherapy

The effects of TGFRt15-TGFRs treatment in reducing protein levels of SASP

factors in B16F10 tumor-bearing mice following chemotherapy were further assessed.

B16F10 tumor cells (1 x107 cells/mouse) were implanted in mice on day 0. The mice

were treated subcutaneously with 10 mg/kg docetaxel on days 1, 4, and 7. On day 8, the

mice were treated subcutaneously with PBS or TGFRt15-TGFRs (3 mg/kg). Mice were

euthanized on day 17 post-tumor inoculation and livers were collected and homogenized.

Protein levels of SASP factors in the liver homogenates was determined by ELISA. As

shown in Figure 267, in vivo treatment with TGFRt15-TGFRs resulted in a significant

reduction in levels of liver IL-1a, IL-6, TNFa and IL-8 SASP factors in B16F10 tumor

bearing mice following chemotherapy.

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Example 131: Role of Immune Cell Subsets in TGFRt15-TGFRs-mediated

Elimination of Senescent Tumor Cells in B16F10 Melanoma Mouse Model

To assess the role of immune cell subsets in TGFRt15-TGFRs-mediated

senescent-tumor-cell elimination, in vitro-docetaxel induced senescent B16F10-GFP

tumor cells were mixed with parental B16F10 cells were implanted subcutaneously in

mice following treatment with anti-NK1.1 or anti-CD8a antibodies. When tumors

reached to approximately 350 mm³, mice were randomized to receive subcutaneous

treatment with PBS or TGFRt15-TGFRs (3 mg/kg) + TA99 (200 ug). The mice were

sacrificed day 4 post-therapy and tumors were collected and analyzed. The level of GFP-

positive B16F10-GFP TIS cells and NK and CD8+ T cells in the tumors were assess by

flow cytometry. As shown in Figure 268A, TGFRt15-TGFRs-treated mixed tumors

without immunodepletion or depleted for CD8+ T immune cells contained significantly

fewer GFP-expressing senescence tumor cells than that of control treated mice. It was

also observed that the tumors of CD8+ depleted mice were significantly infiltrated with

NK cells and tumors of NK depleted mice were significantly infiltrated with CD8+ T cells

(Figure 268B). These results suggested that both NK and CD8+ T cells play a role in

controlling tumor growth with NK cells predominately mediating the activity of

TGFRt15-TGFRs to deplete TIS tumor cells.

Example 132: Anti-PD-L1 Antibody in Combination with TGFRt15-TGFRs+TA99

and Chemotherapy in B16F10 Melanoma Mouse Model To further assess a sequential TGFRt15-TGFRs-immune checkpoint inhibitor

treatment regimen (described in Example 109), B16F10 tumor-bearing mice were first

treated with doxetaxel (DTX) and then either TGFRt15-TGFRs+TA99 followed by anti-

PD-L1 antibody or anti-PD-L1 antibody followed by TGFRt15-TGFRs+TA99 (Figure

269A). Tumor growth curves and end point tumor volume at day 18 indicated that both

combination strategies (TGFRt15-TGFRs+TA99 followed by anti-PD-L1 and vice versa)

showed significant tumor volume reduction as compared to the individual

immunotherapies (either TGFRt15-TGFRs+TA99 or anti PD-L1 alone) or DTX alone

(Figure 269B). Interestingly, TGFRt15-TGFRs +TA99-treated tumors showed

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significantly lower tumor volume at day 13 prior to start of combination treatments as

compared to anti-PD-L1-treated tumors, showing the effect of TGFRt15-TGFRs+TA99

in initial control of tumor growth. End point analysis also showed that tumors treated

with the combination of TGFRt15-TGFRs+TA99 and anti-PD-L1 antibody led to

significantly increased levels of tumor infiltrating CD8+ T cells and NK cells as

compared to single treatment groups. Combination treatment increased the expression of

costimulatory receptor CD28 on CD8+ TILs compared to single treatment suggesting that

checkpoint blockade could rescue dysfunctional CD8+ TILs that are further activated by

IL-15 activity of TGFRt15-TGFRs within the tumor microenvironment (Figure 269C).

This was concomitant with enhanced activation phenotype (IFNy secretion) of splenic

CD8+ T cells from combination treatment group following stimulation with

PMA/ionomycin (Figure 269D). Combination treatment also showed increased NKG2D

expression on total CD8+ T cells and CD44hi CD8+ T cells in the tumors compared to the

individual immunotherapy treatment (Figure 269E). These data collectively shows that

combination therapy of TGFRt15-TGFRs+TA99 and anti-PD-L1 antibody led to

activation and infiltration of CD8+ T cells that may contributed to effective tumor control.

Example 133: Antitumor Efficacy of TGFRt15-TGFRs in Combination with

Chemotherapy against SW1990 Human Pancreatic Tumors in C57BL/6 SCID Mice

To further assess the anti-tumor activity of TGFRt15-TGFRs in combination with

chemotherapy, SW 1990 human pancreatic cancer cells (2x106 cells/mouse) were

subcutaneously (s.c.) injected into C57BL/6 scid mice. Nine days after tumor cell

implantation, gemcitabine (40 mg/kg, i.p.) and nab-paclitaxel (Abraxane) (5 mg/kg, i.p.)

chemotherapy was initiated followed 2 days later by TGFRt15-TGFRs (3 mg/kg, S.C.).

This was considered one treatment cycle and was repeated for another 3 cycles (1

cycle/week) (Figure 270A). Tumor-bearing control groups received PBS, chemotherapy,

or TGFRt15-TGFRs treatment alone. During and after the study treatment, tumor

volumes were measured and animal survival based on tumor volume < 4000 mm³ was

assessed. The results indicated that the animals receiving a combination of TGFRt15-

TGFRs and chemotherapy had significantly slower SW1990 tumor growth comparing to

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the PBS group (Figure 270B-270C). TGFRt15-TGFRs + chemotherapy also prolonged

survival of SW1990 tumor-bearing mice (Figure 270D). These results confirm that

TGFRt15-TGFRs enhanced the efficacy of standard of care chemotherapy against human

pancreatic tumors in a mouse xenograft tumor model.

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction

with the detailed description thereof, the foregoing description is intended to illustrate

and not limit the scope of the invention, which is defined by the scope of the appended

claims. Other aspects, advantages, and modifications are within the scope of the

following claims.

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Exemplary Embodiments

Embodiment A1. A single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain.

Embodiment A2. The single-chain chimeric polypeptide of embodiment A1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other.

Embodiment A3. The single-chain chimeric polypeptide of embodiment A1,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the first target-binding domain and the soluble tissue factor domain.

Embodiment A4. The single-chain chimeric polypeptide of any one of

embodiments A1-A3, wherein the soluble tissue factor domain and the second target-

binding domain directly abut each other.

Embodiment A5. The single-chain chimeric polypeptide of any one of

embodiments A1-A3, wherein the single-chain chimeric polypeptide further comprises a

linker sequence between the soluble tissue factor domain and the second target-binding

domain.

Embodiment A6. The single-chain chimeric polypeptide of embodiment A1,

wherein the first target-binding domain and the second target-binding domain directly

abut each other.

Embodiment A7. The single-chain chimeric polypeptide of embodiment A1,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the first target-binding domain and the second target-binding domain.

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Embodiment A8. The single-chain chimeric polypeptide of embodiment A6 or

A7, wherein the second target-binding domain and the soluble tissue factor domain

directly abut each other.

Embodiment A9. The single-chain chimeric polypeptide of embodiment A6 or

A7, wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the second target-binding domain and the soluble tissue factor domain.

Embodiment A10. The single-chain chimeric polypeptide of any one of

embodiments A1-A9, wherein the first target-binding domain and the second target-

binding domain bind specifically to the same antigen.

Embodiment A11. The single-chain chimeric polypeptide of embodiment A10,

wherein the first target-binding domain and the second target-binding domain bind

specifically to the same epitope.

Embodiment A12. The single-chain chimeric polypeptide of embodiment A11,

wherein the first target-binding domain and the second target-binding domain comprise

the same amino acid sequence.

Embodiment A13. The single-chain chimeric polypeptide of any one of

embodiments A1-A9, wherein the first target-binding domain and the second target-

binding domain bind specifically to different antigens.

Embodiment A14. The single-chain chimeric polypeptide of any one of

embodiments A1-A13, wherein one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain.

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Embodiment A15. The single-chain chimeric polypeptide of embodiment A14,

wherein the first target-binding domain and the second target-binding domain are each an

antigen-binding domain.

Embodiment A16. The single-chain chimeric polypeptide of embodiment A13,

wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment A17. The single-chain chimeric polypeptide of any one of

embodiments A1-A16, wherein one or both of the first target-binding domain and the

second target-binding domain bind to a target selected from the group consisting of:

CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,

IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa,

CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET,

EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,

CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a

ligand of DNAMI, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand

of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a

receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor

for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for stem

cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a

receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a

receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment A18. The single-chain chimeric polypeptide of any one of

embodiments A1-A16, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble interleukin or cytokine protein.

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Embodiment A19. The single-chain chimeric polypeptide of embodiment A18,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-D, and SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment A20 The single-chain chimeric polypeptide of any one of

embodiments A1-A16, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble interleukin or cytokine receptor.

Embodiment A21. The single-chain chimeric polypeptide of embodiment A20,

wherein the soluble interleukin or cytokine receptor is a soluble TGF-B receptor II (TGF-

(BRII), a soluble TGF-BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a

soluble NKp46, a soluble DNAMI, a scMHCI, a scMHCII, a scTCR, a soluble CD155,

or a soluble CD28.

Embodiment A22. The single-chain chimeric polypeptide of any one of

embodiments A1-A21, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment A23. The single-chain chimeric polypeptide of embodiment A22,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment A24. The single-chain chimeric polypeptide of embodiment A23,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment A25. The single-chain chimeric polypeptide of embodiment A24,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

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Embodiment A26. The single-chain chimeric polypeptide of any one of

embodiments A22-A25, wherein the soluble human tissue factor domain does not

comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment A27. The single-chain chimeric polypeptide of embodiment A26,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

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an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment A28 The single-chain chimeric polypeptide of any one of

embodiments A1-A27, wherein the soluble tissue factor domain is not capable of binding

Factor VIIa.

Embodiment A29. The single-chain chimeric polypeptide of any one of

embodiments A1-A28, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment A30. The single-chain chimeric polypeptide of any one of

embodiments A1-A29, wherein the single-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment A31. The single-chain chimeric polypeptide of any one of

embodiments A1-A30, wherein the single-chain chimeric polypeptide further comprises

one or more additional target-binding domains at its N- and/or C-terminus.

Embodiment A32. The single-chain chimeric polypeptide of embodiment A31,

wherein the single-chain chimeric polypeptide comprises one or more additional target-

binding domains at its N-terminus.

Embodiment A33. The single-chain chimeric polypeptide of embodiment A32,

wherein one or more additional target-binding domains directly abuts the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

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Embodiment A34. The single-chain chimeric polypeptide of embodiment A33,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the at least one additional target-binding domains and the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment A35. The single-chain chimeric polypeptide of embodiment A31,

wherein the single-chain chimeric polypeptide comprises one or more additional target-

binding domains at its C-terminus.

Embodiment A36. The single-chain chimeric polypeptide of embodiment A35,

wherein one of the one or more additional target-binding domains directly abuts the first

target-binding domain, the second target-binding domain, or the soluble tissue factor

domain.

Embodiment A37. The single-chain chimeric polypeptide of embodiment A35,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the at least one additional target-binding domains and the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment A38. The single-chain chimeric polypeptide of embodiment A31,

wherein the single-chain chimeric polypeptide comprises one or more additional target

binding domains at its N-terminus and the C-terminus.

Embodiment A39. The single-chain chimeric polypeptide of embodiment A38,

wherein one of the one or more additional antigen binding domains at the N-terminus

directly abuts the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain.

Embodiment A40. The single-chain chimeric polypeptide of embodiment A38,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

791

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between one of the one or more additional antigen-binding domains at the N-terminus

and the first target-binding domain, the second target-binding domain, or the soluble

tissue factor domain.

Embodiment A41. The single-chain chimeric polypeptide of embodiment A38,

wherein one of the one or more additional antigen binding domains at the C-terminus

directly abuts the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain.

Embodiment A42. The single-chain chimeric polypeptide of embodiment A38,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the one or more additional antigen-binding domains at the C-terminus

and the first target-binding domain, the second target-binding domain, or the soluble

tissue factor domain.

Embodiment A43. The single-chain chimeric polypeptide of any one of

embodiments A31-A42, wherein two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

bind specifically to the same antigen.

Embodiment A44. The single-chain chimeric polypeptide of embodiment A43,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope.

Embodiment A45. The single-chain chimeric polypeptide of embodiment A44,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains comprise the same amino

acid sequence.

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Embodiment A46. The single-chain chimeric polypeptide of embodiment A43,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same antigen.

Embodiment A47. The single-chain chimeric polypeptide of embodiment A46,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same epitope.

Embodiment A48. The single-chain chimeric polypeptide of embodiment A47,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each comprise the same amino acid sequence.

Embodiment A49. The single-chain chimeric polypeptide of any one of

embodiments A31-A42, wherein the first target-binding domain, the second target-

binding domain, and the one or more additional target-binding domains bind specifically

to different antigens.

Embodiment A50. The single-chain chimeric polypeptide of any one of

embodiments A31-A49, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more target-binding domains is an antigen-

binding domain.

Embodiment A51. The single-chain chimeric polypeptide of embodiment A50,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains are each an antigen-binding domain.

Embodiment A52. The single-chain chimeric polypeptide of embodiment A51,

wherein antigen-binding domain comprises a scFv or a single domain antibody.

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Embodiment A53. The single-chain chimeric polypeptide of any one of

embodiments A31-A52, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more target-binding domains bind

specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33,

CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT,

PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30,

CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein,

HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B

receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46,

a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a

ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a

receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor

for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for

IL-21, a receptor for PDGF-D, a receptor for stem cell factor (SCF), a receptor for stem

cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a

receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a

receptor for CD28.

Embodiment A54. The single-chain chimeric polypeptide of any one of

embodiments A31-A52, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains is a

soluble interleukin or cytokine protein.

Embodiment A55. The single-chain chimeric polypeptide of embodiment A54,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-D, and SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

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Embodiment A56. The single-chain chimeric polypeptide of any one of

embodiments A31-A52, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains is a

soluble interleukin or cytokine receptor.

Embodiment A57. The single-chain chimeric polypeptide of embodiment A56,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-BRII), a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAMI, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble

CD3, or a soluble CD28.

Embodiment A58. The single-chain chimeric polypeptide of any one of

embodiments A1-A57, wherein the single-chain chimeric polypeptide further comprises a

signal sequence at its N-terminal end.

Embodiment A59. The single-chain chimeric polypeptide of any one of

embodiments A1-A58, wherein the single-chain chimeric polypeptide further comprises a

peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain

chimeric polypeptide.

Embodiment A60. A composition comprising any of the single-chain chimeric

polypeptides of embodiments A1-A59.

Embodiment A61. The composition of embodiment A60, wherein the

composition is a pharmaceutical composition.

Embodiment A62. A kit comprising at least one dose of the composition of

embodiment A60 or A61.

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Embodiment A63. Nucleic acid encoding any of the single-chain chimeric

polypeptides of any one of embodiments A1-A59.

Embodiment A64. A vector comprising the nucleic acid of embodiment A63.

Embodiment A65. The vector of embodiment A64, wherein the vector is an

expression vector.

Embodiment A66. A cell comprising the nucleic acid of embodiment A63 or the

vector of embodiment A64 or A65.

Embodiment A67. A method of producing a single-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment A66 in a culture medium under conditions

sufficient to result in the production of the single-chain chimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment A68. A single-chain chimeric polypeptide produced by the method

of embodiment A67.

Embodiment A69. The single-chain chimeric polypeptide of embodiment A26,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

Embodiment A70. The single-chain chimeric polypeptide of embodiment A69,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

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Embodiment A71. The single-chain chimeric polypeptide of embodiment A70,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment A72. The single-chain chimeric polypeptide of embodiment A71,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

Embodiment A73. The single-chain chimeric polypeptide of embodiment A26,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment A74. The single-chain chimeric polypeptide of embodiment A73,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment A75. The single-chain chimeric polypeptide of embodiment A74,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment A76. The single-chain chimeric polypeptide of embodiment A75,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

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Embodiment B1. A single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain,

wherein:

the first target-binding domain and the second target-binding domain each

specifically bind to an IL-2 receptor; or

the first target-binding domain and the second target-binding domain each

specifically bind to an IL-15 receptor.

Embodiment B2. The single-chain chimeric polypeptide of embodiment B1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other.

Embodiment B3. The single-chain chimeric polypeptide of embodiment B1,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the first target-binding domain and the soluble tissue factor domain.

Embodiment B4. The single-chain chimeric polypeptide of any one of

embodiments B1-B3, wherein the soluble tissue factor domain and the second target-

binding domain directly abut each other.

Embodiment B5. The single-chain chimeric polypeptide of any one of

embodiments B1-B3, wherein the single-chain chimeric polypeptide further comprises a

linker sequence between the soluble tissue factor domain and the second target-binding

domain.

Embodiment B6. The single-chain chimeric polypeptide of embodiment B1,

wherein the first target-binding domain and the second target-binding domain directly

abut each other.

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Embodiment B7. The single-chain chimeric polypeptide of embodiment B1,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the first target-binding domain and the second target-binding domain.

Embodiment B8. The single-chain chimeric polypeptide of embodiment B6 or B7,

wherein the second target-binding domain and the soluble tissue factor domain directly

abut each other.

Embodiment B9. The single-chain chimeric polypeptide of embodiment B6 or B7,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between the second target-binding domain and the soluble tissue factor domain.

Embodiment B10. The single-chain chimeric polypeptide of any one of

embodiments B1-B9, wherein both the first target-binding domain and the second target-

binding domain is a soluble interleukin protein.

Embodiment B11. The single-chain chimeric polypeptide of embodiment B10,

wherein the first target-binding domain and the second target-binding domain is a soluble

IL-2 protein.

Embodiment B12. The single-chain chimeric polypeptide of embodiment B11,

wherein the soluble IL-2 protein is a soluble human IL-2 protein.

Embodiment B13. The single-chain chimeric polypeptide of embodiment B12,

wherein the soluble human IL-2 protein comprises SEQ ID NO: 78.

Embodiment B14. The single-chain chimeric polypeptide of embodiment B10,

wherein the first target-binding domain and the second target-binding domain is a soluble

IL-15 protein.

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Embodiment B15. The single-chain chimeric polypeptide of embodiment B14,

wherein the soluble IL-15 protein is a soluble human IL-15 protein.

Embodiment B16. The single-chain chimeric polypeptide of embodiment B15,

wherein the soluble human IL-15 protein comprises SEQ ID NO: 82.

Embodiment B17. The single-chain chimeric polypeptide of any one of

embodiments B1-B16, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment B18. The single-chain chimeric polypeptide of embodiment B17,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment B19. The single-chain chimeric polypeptide of embodiment B18,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment B20. The single-chain chimeric polypeptide of embodiment B19,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

Embodiment B21. The single-chain chimeric polypeptide of any one of

embodiments B17-B20, wherein the soluble human tissue factor domain does not

comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

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a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment B22. The single-chain chimeric polypeptide of embodiment B21,

wherein the soluble human tissue factor domain does not comprise any of:

lysine at an amino acid position that corresponds to amino acid position 20 of a

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

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Embodiment B23. The single-chain chimeric polypeptide of any one of

embodiments B1-B22, wherein the soluble tissue factor domain is not capable of binding

Factor VIIa.

Embodiment B24. The single-chain chimeric polypeptide of any one of

embodiments B1-B23, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment B25. The single-chain chimeric polypeptide of any one of

embodiments B1-B24, wherein the single-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment B26. The single-chain chimeric polypeptide of any one of

embodiments B1-B25, wherein the single-chain chimeric polypeptide further comprises

one or more additional target-binding domains at its N- and/or C-terminus.

Embodiment B27. The single-chain chimeric polypeptide of embodiment B26,

wherein the single-chain chimeric polypeptide comprises one or more additional target-

binding domains at its N-terminus.

Embodiment B28. The single-chain chimeric polypeptide of embodiment B27,

wherein one or more additional target-binding domains directly abuts the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B29. The single-chain chimeric polypeptide of embodiment B28,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the at least one additional target-binding domains and the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

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Embodiment B30. The single-chain chimeric polypeptide of embodiment B26,

wherein the single-chain chimeric polypeptide comprises one or more additional target-

binding domains at its C-terminus.

Embodiment B31. The single-chain chimeric polypeptide of embodiment B30,

wherein one of the one or more additional target-binding domains directly abuts the first

target-binding domain, the second target-binding domain, or the soluble tissue factor

domain.

Embodiment B32. The single-chain chimeric polypeptide of embodiment B30,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the at least one additional target-binding domains and the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment B33. The single-chain chimeric polypeptide of embodiment B26,

wherein the single-chain chimeric polypeptide comprises one or more additional target

binding domains at its N-terminus and the C-terminus.

Embodiment B34. The single-chain chimeric polypeptide of embodiment B33,

wherein one of the one or more additional antigen binding domains at the N-terminus

directly abuts the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain.

Embodiment B35. The single-chain chimeric polypeptide of embodiment B33,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the one or more additional antigen-binding domains at the N-terminus

and the first target-binding domain, the second target-binding domain, or the soluble

tissue factor domain.

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Embodiment B36. The single-chain chimeric polypeptide of embodiment B33,

wherein one of the one or more additional antigen binding domains at the C-terminus

directly abuts the first target-binding domain, the second target-binding domain, or the

soluble tissue factor domain.

Embodiment B37. The single-chain chimeric polypeptide of embodiment B33,

wherein the single-chain chimeric polypeptide further comprises a linker sequence

between one of the one or more additional antigen-binding domains at the C-terminus

and the first target-binding domain, the second target-binding domain, or the soluble

tissue factor domain.

Embodiment B38, The single-chain chimeric polypeptide of any one of

embodiments B26-B37, wherein each of the first target-binding domain, the second

target-binding domain, and the one or more additional target-binding domains bind

specifically to an IL-2 receptor or an IL-15 receptor.

Embodiment B39. The single-chain chimeric polypeptide of embodiment B38,

wherein each of the first target-binding domain, the second target-binding domain, and

the one or more additional target-binding domains comprise the same amino acid

sequence.

Embodiment B40 The single-chain chimeric polypeptide of any one of

embodiments B26-B37, wherein the one or more additional target-binding domains is an

antigen-binding domain.

Embodiment B41. The single-chain chimeric polypeptide of embodiment B40,

wherein the antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment B42. The single-chain chimeric polypeptide of any one of

embodiments B26-B37, B40, and B41, wherein the one or more additional target-binding

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domains bind specifically to a target selected from the group consisting of: CD16a,

CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-

10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26,

CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR,

HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-

DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of

DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of

NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor

for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a

receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a

receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell

factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor

for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for

CD155, a receptor for CD122, and a receptor for CD28.

Embodiment B43. The single-chain chimeric polypeptide of any one of

embodiments B6-B37, B40, and B41, wherein the one or more additional target-binding

domains is a soluble interleukin or cytokine protein.

Embodiment B44. The single-chain chimeric polypeptide of embodiment B43,

wherein the soluble interleukin or cytokine protein is selected from the group consisting

of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and

SCF.

Embodiment B45. The single-chain chimeric polypeptide of any one of

embodiments B6-B37, B40, and B41, wherein the one or more additional target-binding

domains is a soluble interleukin or cytokine receptor.

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Embodiment B46. The single-chain chimeric polypeptide of embodiment B45,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-BRII) and a soluble

TGF-BRIII.

Embodiment B47. The single-chain chimeric polypeptide of any one of

embodiments B1-B46, wherein the single-chain chimeric polypeptide further comprises a

signal sequence at its N-terminal end.

Embodiment B48. The single-chain chimeric polypeptide of any one of

embodiments B1-B47, wherein the single-chain chimeric polypeptide further comprises a

peptide tag positioned at the N-terminal end or the C-terminal end of the single-chain

chimeric polypeptide.

Embodiment B49. A composition comprising any of the single-chain chimeric

polypeptides of embodiments B1-B48

Embodiment B50. The composition of embodiment B49, wherein the composition

is a pharmaceutical composition.

Embodiment B51. A kit comprising at least one dose of the composition of

embodiment B49 or B50.

Embodiment B52. A nucleic acid encoding any of the single-chain chimeric

polypeptides of any one of embodiments B1-B48.

Embodiment B53. A vector comprising the nucleic acid of embodiment B52.

Embodiment B54. The vector of embodiment B53, wherein the vector is an

expression vector.

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Embodiment B55. A cell comprising the nucleic acid of embodiment B52 or the

vector of embodiment B53 or B54.

Embodiment B56. A method of producing a single-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment B55 in a culture medium under conditions

sufficient to result in the production of the single-chain chimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment B57. A single-chain chimeric polypeptide produced by the method

of embodiment B56.

Embodiment B58. The single-chain chimeric polypeptide of embodiment B21,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

Embodiment B59. The single-chain chimeric polypeptide of embodiment B58,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment B60. The single-chain chimeric polypeptide of embodiment B59,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment B61. The single-chain chimeric polypeptide of embodiment B60,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

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Embodiment B62. The single-chain chimeric polypeptide of embodiment B21,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment B63. The single-chain chimeric polypeptide of embodiment B62,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment B64. The single-chain chimeric polypeptide of embodiment B63,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment B65. The single-chain chimeric polypeptide of embodiment B64,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

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Embodiment C1. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide

associate through the binding of the first domain and the second domain of the pair of

affinity domains.

Embodiment C2. The multi-chain chimeric polypeptide of embodiment C1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other in the first chimeric polypeptide.

Embodiment C3. The multi-chain chimeric polypeptide of embodiment C1,

wherein the first chimeric polypeptide further comprises a linker sequence between the

first target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

Embodiment C4. The multi-chain chimeric polypeptide of any one of

embodiments C1-C3, wherein the soluble tissue factor domain and the first domain of the

pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment C5. The multi-chain chimeric polypeptide of any one of

embodiments C1-C3, wherein the first chimeric polypeptide further comprises a linker

sequence between the soluble tissue factor domain and the first domain of the pair of

affinity domains in the first chimeric polypeptide.

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Embodiment C6. The multi-chain chimeric polypeptide of any one of

embodiments C1-C5, wherein the second domain of the pair of affinity domains and the

second target-binding domain directly abut each other in the second chimeric

polypeptide.

Embodiment C7. The multi-chain chimeric polypeptide of any one of

embodiments C1-C5, wherein second chimeric polypeptide further comprises a linker

sequence between the second domain of the pair of affinity domains and the second

target-binding domain in the second chimeric polypeptide.

Embodiment C8. The multi-chain chimeric polypeptide of any one of

embodiments C1-C7, wherein the first target-binding domain and the second target-

binding domain bind specifically to the same antigen.

Embodiment C9. The multi-chain chimeric polypeptide of embodiment C8,

wherein the first target-binding domain and the second target-binding domain bind

specifically to the same epitope.

Embodiment C10. The multi-chain chimeric polypeptide of embodiment C9,

wherein the first target-binding domain and the second target-binding domain comprise

the same amino acid sequence.

Embodiment C11. The multi-chain chimeric polypeptide of any one of

embodiments C1-C7, wherein the first target-binding domain and the second target-

binding domain bind specifically to different antigens.

Embodiment C12. The multi-chain chimeric polypeptide of any one of

embodiments C1-C11, wherein one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain.

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Embodiment C13. The multi-chain chimeric polypeptide of embodiment C12,

wherein the first target-binding domain and the second target-binding domain are each

antigen-binding domains.

Embodiment C14. The multi-chain chimeric polypeptide of embodiment C12 or

C13, wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment C15. The multi-chain chimeric polypeptide of any one of

embodiments C1-C14, wherein one or both of the first target-binding domain and the

second target-binding domain bind specifically to a target selected from the group

consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1,

VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6,

IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC,

Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P- cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER,

CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-

BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D,

a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR,

a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor

for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-

17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for

stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a

receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a

receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment C16. The multi-chain chimeric polypeptide of any one of

embodiments C1-C14, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble interleukin or cytokine protein.

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Embodiment C17. The multi-chain chimeric polypeptide of embodiment C16,

wherein the soluble interleukin or cytokine protein is selected from the group consisting

of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD,

SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment C18. The multi-chain chimeric polypeptide of any one of

embodiments C1-C14, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble interleukin or cytokine receptor.

Embodiment C19. The multi-chain chimeric polypeptide of embodiment C18,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-B RII), a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment C20. The multi-chain chimeric polypeptide of any one of

embodiments C1-C19, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domain(s), where at least one of the one or more

additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains.

Embodiment C21. The multi-chain chimeric polypeptide of embodiment C20,

wherein the first chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the at least one of the one or more additional antigen-

binding domain(s), and/or a linker sequence between the at least one of the one or more

additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment C22. The multi-chain chimeric polypeptide of any one of

embodiments C1-C19, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal and/or C-terminal end of the

first chimeric polypeptide.

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Embodiment C23. The multi-chain chimeric polypeptide of embodiment C22,

wherein at least one of the one or more additional target-binding domains directly abuts

the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C24. The multi-chain chimeric polypeptide of embodiment C22,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first domain of the

pair of affinity domains.

Embodiment C25. The multi-chain chimeric polypeptide of embodiment C22,

wherein the at least one of the one or more additional target-binding domains directly

abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment C26. The multi-chain chimeric polypeptide of embodiment C22,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first target-binding

domain.

Embodiment C27. The multi-chain chimeric polypeptide of embodiment C22,

wherein at least one of the one or more additional target-binding domains is disposed at

the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment C28. The multi-chain chimeric polypeptide of embodiment C27,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the N-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment C29. The multi-chain chimeric polypeptide of embodiment C27,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment C30, The multi-chain chimeric polypeptide of embodiment C27,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the C-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment C31. The multi-chain chimeric polypeptide of embodiment C27,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment C32. The multi-chain chimeric polypeptide of embodiment C27,

wherein the at least one of the one or more additional target-binding domains positioned

between the soluble tissue factor domain and the first domain of the pair of affinity

domains, directly abuts the soluble tissue factor domain and/or the first domain of the

pair of affinity domains.

Embodiment C33. The multi-chain chimeric polypeptide of embodiment C27,

wherein the first chimeric polypeptide further comprises a linker sequence disposed (i)

between the soluble tissue factor domain and the at least one of the one or more

additional target-binding domains positioned between the soluble tissue factor domain

and the first domain of the pair of affinity domains, and/or (ii) between the first domain

of the pair of affinity domains and the at least one of the one or more additional target-

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binding domains positioned between the soluble tissue factor domain and the first domain

of the pair of affinity domains.

Embodiment C34. The multi-chain chimeric polypeptide of any one of

embodiments C1-C33, wherein the second chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal end or the C-terminal end of

the second chimeric polypeptide.

Embodiment C35. The multi-chain chimeric polypeptide of embodiment C34,

wherein at least one of the one or more additional target-binding domains directly abuts

the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment C36. The multi-chain chimeric polypeptide of embodiment C34,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second domain of

the pair of affinity domains in the second chimeric polypeptide.

Embodiment C37. The multi-chain chimeric polypeptide of embodiment C34,

wherein at least one of the one or more additional target-binding domains directly abuts

the second target-binding domain in the second chimeric polypeptide.

Embodiment C38. The multi-chain chimeric polypeptide of embodiment C34,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second target-

binding domain in the second chimeric polypeptide.

Embodiment C39. The multi-chain chimeric polypeptide of any one of

embodiments C20-C38, wherein two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

bind specifically to the same antigen.

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Embodiment C40. The multi-chain chimeric polypeptide of embodiment C39,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope.

Embodiment C41. The multi-chain chimeric polypeptide of embodiment C40,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains comprise the same amino

acid sequence.

Embodiment C42. The multi-chain chimeric polypeptide of embodiment C39,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same antigen.

Embodiment C43. The multi-chain chimeric polypeptide of embodiment C42,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each bind specifically to the same epitope.

Embodiment C44. The multi-chain chimeric polypeptide of embodiment C43,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains each comprise the same amino acid sequence.

Embodiment C45. The multi-chain chimeric polypeptide of any one of

embodiments C20-C38, wherein the first target-binding domain, the second target-

binding domain, and the one or more additional target-binding domains bind specifically

to different antigens.

Embodiment C46. The multi-chain chimeric polypeptide of any one of

embodiments C20-C45, wherein one or more of the first target-binding domain, the

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second target-binding domain, and the one or more target-binding domains is an antigen-

binding domain.

Embodiment C47 The multi-chain chimeric polypeptide of embodiment C46,

wherein the first target-binding domain, the second target-binding domain, and the one or

more additional target-binding domains are each an antigen-binding domain.

Embodiment C48. The multi-chain chimeric polypeptide of embodiment C47,

wherein antigen-binding domain comprises a scFv.

Embodiment C49. The multi-chain chimeric polypeptide of any one of

embodiments C20-C48, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more target-binding domains bind

specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33,

CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT,

PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30,

CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein,

HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B

receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46,

a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a

ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a

receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor

for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for

IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem

cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a

receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a

receptor for CD3, and a receptor for CD28.

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Embodiment C50. The multi-chain chimeric polypeptide of any one of

embodiments C20-C48, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains is a

soluble interleukin or cytokine protein.

Embodiment C51. The multi-chain chimeric polypeptide of embodiment C50,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, and SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment C52. The multi-chain chimeric polypeptide of any one of

embodiments C20-C48, wherein one or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains is a

soluble interleukin or cytokine receptor.

Embodiment C53. The multi-chain chimeric polypeptide of embodiment C52,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-B RII), a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble

CD3, or a soluble CD28.

Embodiment C54. The multi-chain chimeric polypeptide of any one of

embodiments C1-C53, wherein the first chimeric polypeptide further comprises a peptide

tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment C55. The multi-chain chimeric polypeptide of any one of

embodiments C1-C53, wherein the second chimeric polypeptide further comprises a

peptide tag at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

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Embodiment C56. The multi-chain chimeric polypeptide of any one of

embodiments C1-C55, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment C57. The multi-chain chimeric polypeptide of embodiment C56,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment C58. The multi-chain chimeric polypeptide of embodiment C57,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment C59. The multi-chain chimeric polypeptide of embodiment C58,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

Embodiment C60. The multi-chain chimeric polypeptide of any one of

embodiments C56-C59, wherein the soluble human tissue factor domain does not

comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

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an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment C61. The multi-chain chimeric polypeptide of embodiment C60,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment C62. The multi-chain chimeric polypeptide of any one of

embodiments C1-C61, wherein the soluble tissue factor domain is not capable of binding

to Factor VIIa.

Embodiment C63. The multi-chain chimeric polypeptide of any one of

embodiments C1-C62, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

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Embodiment C64. The multi-chain chimeric polypeptide of any one of

embodiments C1-C63, wherein the multi-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment C65. The multi-chain chimeric polypeptide of any one of

embodiments C1-C64, wherein the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra) and a soluble IL-15.

Embodiment C66. The multi-chain chimeric polypeptide of embodiment C65,

wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment C67. The multi-chain chimeric polypeptide of embodiment C65 or

C66, wherein the human IL-15Ra is a mature full-length IL-15Ra.

Embodiment C68. The multi-chain chimeric polypeptide of any one of

embodiments C1-C64, wherein the pair of affinity domains is selected from the group

consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules

based on mutated RNase I fragments, and SNARE modules based on interactions of the

proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment C69. The multi-chain chimeric polypeptide of any one of

embodiments C1-C68, wherein the first chimeric polypeptide and/or the second chimeric

polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment C70. A composition comprising any of the multi-chain chimeric

polypeptides of embodiments C1-C69.

Embodiment C71. The composition of embodiment C70, wherein the composition

is a pharmaceutical composition.

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Embodiment C72. A kit comprising at least one dose of the composition of

embodiment C70 or C71.

Embodiment C73. Nucleic acid encoding any of the multi-chain chimeric

polypeptides of any one of embodiments C1-C69.

Embodiment C74. A vector comprising the nucleic acid of embodiment C73.

Embodiment C75. The vector of embodiment C74, wherein the vector is an

expression vector.

Embodiment C76. A cell comprising the nucleic acid of embodiment C73 or the

vector of embodiment C74 or C75.

Embodiment C77. A method of producing a multi-chain chimeric polypeptide, the

method comprising:

culturing the cell of embodiment C76 in a culture medium under conditions

sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment C78. A multi-chain chimeric polypeptide produced by the method of

embodiment C77.

Embodiment C79. The multi-chain chimeric polypeptide of embodiment A56,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

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Embodiment C80. The multi-chain chimeric polypeptide of embodiment C79,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment C81. The multi-chain chimeric polypeptide of embodiment C80,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment C82. The multi-chain chimeric polypeptide of embodiment C81,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

Embodiment C83. The multi-chain chimeric polypeptide of embodiment C56,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment C84. The multi-chain chimeric polypeptide of embodiment C83,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment C85. The multi-chain chimeric polypeptide of embodiment C84,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment C86. The multi-chain chimeric polypeptide of embodiment C85,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

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Embodiment D1. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate

through the binding of the first domain and the second domain of the pair of affinity

domains;

the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment D2. The multi-chain chimeric polypeptide of embodiment D1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other in the first chimeric polypeptide.

Embodiment D3. The multi-chain chimeric polypeptide of embodiment D1,

wherein the first chimeric polypeptide further comprises a linker sequence between the

first target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

Embodiment D4. The multi-chain chimeric polypeptide of any one of

embodiments D1-D3, wherein the soluble tissue factor domain and the first domain of the

pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment D5. The multi-chain chimeric polypeptide of any one of

embodiments D1-D3, wherein the first chimeric polypeptide further comprises a linker

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sequence between the soluble tissue factor domain and the first domain of the pair of

affinity domains in the first chimeric polypeptide.

Embodiment D6. The multi-chain chimeric polypeptide of any one of

embodiments D1-D5, wherein the second domain of the pair of affinity domains and the

second target-binding domain directly abut each other in the second chimeric

polypeptide.

Embodiment D7. The multi-chain chimeric polypeptide of any one of

embodiments D1-D5, wherein second chimeric polypeptide further comprises a linker

sequence between the second domain of the pair of affinity domains and the second

target-binding domain in the second chimeric polypeptide.

Embodiment D8. The multi-chain chimeric polypeptide of any one of

embodiments D1-D7, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment D9. The multi-chain chimeric polypeptide of embodiment D8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment D10. The multi-chain chimeric polypeptide of embodiment D9,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment D11. The multi-chain chimeric polypeptide of embodiment D10,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

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Embodiment D12. The multi-chain chimeric polypeptide of any one of

embodiments D8-D11, wherein the soluble human tissue factor domain does not

comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment D13. The multi-chain chimeric polypeptide of embodiment D12,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

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an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment D14. The multi-chain chimeric polypeptide of any one of

embodiments D1-D13, wherein the soluble tissue factor domain is not capable of binding

to Factor VIIa.

Embodiment D15. The multi-chain chimeric polypeptide of any one of

embodiments D1-D14, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment D16. The multi-chain chimeric polypeptide of any one of

embodiments D1-D15, wherein the multi-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment D17. The multi-chain chimeric polypeptide of any one of

embodiments D1-D16, wherein the first chimeric polypeptide further comprises a peptide

tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment D18. The multi-chain chimeric polypeptide of any one of

embodiments D1-D17, wherein the second chimeric polypeptide further comprises a

peptide tag at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

Embodiment D19. The multi-chain chimeric polypeptide of any one of

embodiments D1-D18, wherein the first chimeric polypeptide and/or the second chimeric

polypeptide further comprises a signal sequence at its N-terminal end.

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Embodiment D20. The multi-chain chimeric polypeptide of embodiment D19,

wherein the signal sequence comprises SEQ ID NO: 117.

Embodiment D21. The multi-chain chimeric polypeptide of embodiment D20,

wherein the signal sequence is SEQ ID NO: 117.

Embodiment D22. The multi-chain chimeric polypeptide of any one of

embodiments D1-D21, wherein the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra) and a soluble IL-15.

Embodiment D23. The multi-chain chimeric polypeptide of embodiment D22,

wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment D24. The multi-chain chimeric polypeptide of embodiment D22,

wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 82.

Embodiment D25. The multi-chain chimeric polypeptide of embodiment D24,

wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 82.

Embodiment D26. The multi-chain chimeric polypeptide of embodiment D25,

wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 82.

Embodiment D27. The multi-chain chimeric polypeptide of embodiment D26,

wherein the soluble IL-15 comprises SEQ ID NO: 82.

Embodiment D28. The multi-chain chimeric polypeptide of any one of

embodiments D22-D27, wherein the sushi domain of IL-15Ra comprises a sushi domain

from human IL-15Ra.

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Embodiment D29. The multi-chain chimeric polypeptide of embodiment D28,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 80%

identical to SEQ ID NO: 113.

Embodiment D30. The multi-chain chimeric polypeptide of embodiment D29,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 90%

identical to SEQ ID NO: 113.

Embodiment D31. The multi-chain chimeric polypeptide of embodiment D30,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 95%

identical to SEQ ID NO: 113.

Embodiment D32. The multi-chain chimeric polypeptide of embodiment D31,

wherein the sushi domain from human IL-15Ra comprises SEQ ID NO: 113.

Embodiment D33. The multi-chain chimeric polypeptide of embodiment D28,

wherein the sushi domain from human IL-15Ra is a mature full-length IL-15Ra.

Embodiment D34. The multi-chain chimeric polypeptide of any one of

embodiments D1-D21, wherein the pair of affinity domains is selected from the group

consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules

based on mutated RNase I fragments, and SNARE modules based on interactions of the

proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment D35. The multi-chain chimeric polypeptide of any one of

embodiments D1-D34, wherein one or both of the first target-binding domain and the

second target-binding domain is an agonistic antigen-binding domain.

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Embodiment D36. The multi-chain chimeric polypeptide of embodiment D35,

wherein the first target-binding domain and the second target-binding domain are each

agonistic antigen-binding domains.

Embodiment D37. The multi-chain chimeric polypeptide of embodiment D35 or

D36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment D38. The multi-chain chimeric polypeptide of any one of

embodiments D1-D34, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble IL-15 or a soluble IL-18.

Embodiment D39. The multi-chain chimeric polypeptide of embodiment D38,

wherein the first target-binding domain and the second target-binding domain are each

independently a soluble IL-15 or a soluble IL-18.

Embodiment D40. The multi-chain chimeric polypeptide of any one of

embodiments D1-D39, wherein the first target-binding domain and the second target-

binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment D41. The multi-chain chimeric polypeptide of embodiment B40,

wherein the first target-binding domain and the second target-binding domain bind

specifically to the same epitope.

Embodiment D42. The multi-chain chimeric polypeptide of embodiment D41,

wherein the first target-binding domain and the second target-binding domain comprise

the same amino acid sequence.

Embodiment D43. The multi-chain chimeric polypeptide of any one of

embodiments D1-D39, wherein the first target-binding domain binds specifically to a

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receptor for IL-12, and the second target-binding domain binds specifically to a receptor

for IL-18.

Embodiment D44. The multi-chain chimeric polypeptide of any one of

embodiments D1-D39, wherein the first target-binding domain binds specifically to a

receptor for IL-18, and the second target-binding domain bind specifically to a receptor

for IL-12.

Embodiment D45. The multi-chain chimeric polypeptide of embodiment D44,

wherein the first target-binding domain comprises a soluble IL-18.

Embodiment D46. The multi-chain chimeric polypeptide of embodiment D45,

wherein the soluble IL-18 is a soluble human IL-18.

Embodiment D47. The multi-chain chimeric polypeptide of embodiment D46,

wherein the soluble human IL-18 comprises a sequence at least 80% identical to SEQ ID

NO: 109.

Embodiment D48. The multi-chain chimeric polypeptide of embodiment D47,

wherein the soluble human IL-18 comprises a sequence at least 90% identical to SEQ ID

NO: 109.

Embodiment D49. The multi-chain chimeric polypeptide of embodiment D48,

wherein the soluble human IL-18 comprises a sequence at least 95% identical to SEQ ID

NO: 109.

Embodiment D50. The multi-chain chimeric polypeptide of embodiment D49,

wherein the soluble human IL-18 comprises a sequence of SEQ ID NO: 109.

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Embodiment D51. The multi-chain chimeric polypeptide of any one of

embodiments D44-D50, wherein the second target-binding domain comprises a soluble

IL-12.

Embodiment D52. The multi-chain chimeric polypeptide of embodiment D51,

wherein the soluble IL-18 is a soluble human IL-12.

Embodiment D53. The multi-chain chimeric polypeptide of embodiment D52,

wherein the soluble human IL-15 comprises a sequence of soluble human IL-12B (p40)

and a sequence of soluble human IL-12a (p35).

Embodiment D54. The multi-chain chimeric polypeptide of embodiment D53,

wherein the soluble human IL-15 further comprises a linker sequence between the

sequence of soluble IL-12B (p40) and the sequence of soluble human IL-12a (p35).

Embodiment D55. The multi-chain chimeric polypeptide of embodiment D54,

wherein the linker sequence comprises SEQ ID NO: 102.

Embodiment D56. The multi-chain chimeric polypeptide of any one of

embodiments D53-D55, wherein the sequence of soluble human IL-12B (p40) comprises

a sequence that is at least 80% identical to SEQ ID NO: 81.

Embodiment D57. The multi-chain chimeric polypeptide of embodiment D56,

wherein the sequence of soluble human IL-12B (p40) comprises a sequence that is at least

90% identical to SEQ ID NO: 81.

Embodiment D58. The multi-chain chimeric polypeptide of embodiment D57,

wherein the sequence of soluble human IL-12B (p40) comprises a sequence that is at least

95% identical to SEQ ID NO: 81.

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Embodiment D59. The multi-chain chimeric polypeptide of embodiment D58,

wherein the sequence of soluble human IL-12B (p40) comprises SEQ ID NO: 81.

Embodiment D60. The multi-chain chimeric polypeptide of any one of

embodiments D53-D59, wherein the sequence of soluble human IL-12a (p35) comprises

a sequence that is at least 80% identical to SEQ ID NO: 80.

Embodiment D61. The multi-chain chimeric polypeptide of embodiment D60,

wherein the sequence of soluble human IL-12a (p35) comprises a sequence that is at least

90% identical to SEQ ID NO: 80.

Embodiment D62. The mule-chain chimeric polypeptide of embodiment D61,

wherein the sequence of soluble human IL-12a (p35) comprises a sequence that is at least

95% identical to SEQ ID NO: 80.

Embodiment D63. The multi-chain chimeric polypeptide of embodiment D62,

wherein the sequence of soluble human IL-12a (p35) comprises SEQ ID NO: 80.

Embodiment D64. The multi-chain chimeric polypeptide of embodiment D1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 174.

Embodiment D65. The multi-chain chimeric polypeptide of embodiment D64,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 174.

Embodiment D66. The multi-chain chimeric polypeptide of embodiment D65,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 174.

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Embodiment D67. The multi-chain chimeric polypeptide of embodiment D66,

wherein the first chimeric polypeptide comprises SEQ ID NO: 174.

Embodiment D68. The multi-chain chimeric polypeptide of embodiment D67,

wherein the first chimeric polypeptide comprises SEQ ID NO: 176.

Embodiment D69. The multi-chain chimeric polypeptide of any one of

embodiments D1 and D64-D68, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 178.

Embodiment D70. The multi-chain chimeric polypeptide of embodiment D69,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 178.

Embodiment D71. The multi-chain chimeric polypeptide of embodiment D70,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 178.

Embodiment D72. The multi-chain chimeric polypeptide of embodiment D71,

wherein the second chimeric polypeptide comprises SEQ ID NO: 178.

Embodiment D73. The multi-chain chimeric polypeptide of embodiment D72,

wherein the second chimeric polypeptide comprises SEQ ID NO: 180.

Embodiment D74. The multi-chain chimeric polypeptide of any one of

embodiments D1-D63, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domain(s), where at least one of the one or more

additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains.

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Embodiment D75. The multi-chain chimeric polypeptide of embodiment D74,

wherein the first chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the at least one of the one or more additional antigen-

binding domain(s), and/or a linker sequence between the at least one of the one or more

additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment D76. The multi-chain chimeric polypeptide of any one of

embodiments D1-D63, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal and/or C-terminal end of the

first chimeric polypeptide.

Embodiment D77. The multi-chain chimeric polypeptide of embodiment D76,

wherein at least one of the one or more additional target-binding domains directly abuts

the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D78. The multi-chain chimeric polypeptide of embodiment D76,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first domain of the

pair of affinity domains.

Embodiment D79. The multi-chain chimeric polypeptide of embodiment D76,

wherein the at least one of the one or more additional target-binding domains directly

abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment D80. The multi-chain chimeric polypeptide of embodiment D76,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first target-binding

domain.

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Embodiment D81. The multi-chain chimeric polypeptide of embodiment D76,

wherein at least one of the one or more additional target-binding domains is disposed at

the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment D82. The multi-chain chimeric polypeptide of embodiment D81,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the N-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment D83. The multi-chain chimeric polypeptide of embodiment D81,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment D84. The multi-chain chimeric polypeptide of embodiment D81,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the C-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment D85. The multi-chain chimeric polypeptide of embodiment D81,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

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Embodiment D86. The multi-chain chimeric polypeptide of embodiment D81,

wherein the at least one of the one or more additional target-binding domains positioned

between the soluble tissue factor domain and the first domain of the pair of affinity

domains, directly abuts the soluble tissue factor domain and/or the first domain of the

pair of affinity domains.

Embodiment D87. The multi-chain chimeric polypeptide of embodiment D81,

wherein the first chimeric polypeptide further comprises a linker sequence disposed (i)

between the soluble tissue factor domain and the at least one of the one or more

additional target-binding domains positioned between the soluble tissue factor domain

and the first domain of the pair of affinity domains, and/or (ii) between the first domain

of the pair of affinity domains and the at least one of the one or more additional target-

binding domains positioned between the soluble tissue factor domain and the first domain

of the pair of affinity domains.

Embodiment D88. The multi-chain chimeric polypeptide of any one of

embodiments D1-D63 and D74-D87, wherein the second chimeric polypeptide further

comprises one or more additional target-binding domains at the N-terminal end or the C-

terminal end of the second chimeric polypeptide.

Embodiment D89. The multi-chain chimeric polypeptide of embodiment D88,

wherein at least one of the one or more additional target-binding domains directly abuts

the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment D90. The multi-chain chimeric polypeptide of embodiment D88,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second domain of

the pair of affinity domains in the second chimeric polypeptide.

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Embodiment D91. The multi-chain chimeric polypeptide of embodiment D88,

wherein at least one of the one or more additional target-binding domains directly abuts

the second target-binding domain in the second chimeric polypeptide.

Embodiment D92. The multi-chain chimeric polypeptide of embodiment B88,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second target-

binding domain in the second chimeric polypeptide.

Embodiment D93. The multi-chain chimeric polypeptide of any one of

embodiments D74-D92, wherein two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

bind specifically to the same antigen.

Embodiment D94. The multi-chain chimeric polypeptide of embodiment B93,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope.

Embodiment D95. The multi-chain chimeric polypeptide of embodiment B94,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains comprise the same amino

acid sequence.

Embodiment D96. The multi-chain chimeric polypeptide of any one of

embodiments D74-D92, wherein the first target-binding domain, the second target-

binding domain, and the one or more additional target-binding domains bind specifically

to different antigens.

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Embodiment D97. The multi-chain chimeric polypeptide of any one of

embodiments D74-D96, wherein the one or more additional antigen-binding domains

bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3,

CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAMI, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a

scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for

IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a

receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a

receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a

receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a

receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, and a

receptor for CD28.

Embodiment D98. The multi-chain chimeric polypeptide of any one of

embodiments D74-D96, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine protein.

Embodiment D99. The multi-chain chimeric polypeptide of embodiment B98,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment D100. The multi-chain chimeric polypeptide of any one of

embodiments D74-D96, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine receptor.

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Embodiment D101. The multi-chain chimeric polypeptide of embodiment B100,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-B RII), a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a

soluble CD28.

Embodiment D102. A composition comprising any of the multi-chain chimeric

polypeptides of embodiments D1-D101.

Embodiment D103. The composition of embodiment D102, wherein the

composition is a pharmaceutical composition.

Embodiment D104. A kit comprising at least one dose of the composition of

embodiment D 102 or D103.

Embodiment D105. Nucleic acid encoding any of the multi-chain chimeric

polypeptides of any one of embodiments D1-D101.

Embodiment D106. A vector comprising the nucleic acid of embodiment D105.

Embodiment D107. The vector of embodiment D106, wherein the vector is an

expression vector.

Embodiment D108. A cell comprising the nucleic acid of embodiment D 105 or

the vector of embodiment D106 or D107.

Embodiment D109. A method of producing a multi-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment D108 in a culture medium under conditions

sufficient to result in the production of the multi-chain chimeric polypeptide; and

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recovering the multi-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment D110. A multi-chain chimeric polypeptide produced by the method

of embodiment D109.

Embodiment D111. The multi-chain chimeric polypeptide of embodiment D8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

Embodiment D112. The multi-chain chimeric polypeptide of embodiment D111,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment D113. The multi-chain chimeric polypeptide of embodiment D112,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment D114. The multi-chain chimeric polypeptide of embodiment D113,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

Embodiment D115. The multi-chain chimeric polypeptide of embodiment D8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment D116. The multi-chain chimeric polypeptide of embodiment D115,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

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Embodiment D117. The multi-chain chimeric polypeptide of embodiment D116,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment D118. The multi-chain chimeric polypeptide of embodiment D117,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

Embodiment E1. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate

through the binding of the first domain and the second domain of the pair of affinity

domains; and

the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-21 or a ligand of tumor growth factor

receptor II (TGFBRII).

Embodiment E2. The multi-chain chimeric polypeptide of embodiment E1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other in the first chimeric polypeptide.

Embodiment E3. The multi-chain chimeric polypeptide of embodiments E1,

wherein the first chimeric polypeptide further comprises a linker sequence between the

first target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

Embodiment E4. The multi-chain chimeric polypeptide of any one of

embodiments E1-E3, wherein the soluble tissue factor domain and the first domain of the

pair of affinity domains directly abut each other in the first chimeric polypeptide.

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Embodiment E5. The multi-chain chimeric polypeptide of any one of

embodiments E1-E3, wherein the first chimeric polypeptide further comprises a linker

sequence between the soluble tissue factor domain and the first domain of the pair of

affinity domains in the first chimeric polypeptide.

Embodiment E6. The multi-chain chimeric polypeptide of any one of

embodiments E1-E5, wherein the second domain of the pair of affinity domains and the

second target-binding domain directly abut each other in the second chimeric

polypeptide.

Embodiment E7. The multi-chain chimeric polypeptide of any one of

embodiments E1-E5, wherein second chimeric polypeptide further comprises a linker

sequence between the second domain of the pair of affinity domains and the second

target-binding domain in the second chimeric polypeptide.

Embodiment E8 The multi-chain chimeric polypeptide of any one of

embodiments E1-E7, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment E9. The multi-chain chimeric polypeptide of embodiment E8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment E10. The multi-chain chimeric polypeptide of embodiment E9,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment E11. The multi-chain chimeric polypeptide of embodiment E10,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

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Embodiment E12. The multi-chain chimeric polypeptide of any one of

embodiments E8-E11, wherein the soluble human tissue factor domain does not comprise

one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment E13. The multi-chain chimeric polypeptide of embodiment E12,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

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an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment E14. The multi-chain chimeric polypeptide of any one of

embodiments E1-E13, wherein the soluble tissue factor domain is not capable of binding

to Factor VIIa.

Embodiment E15. The multi-chain chimeric polypeptide of any one of

embodiments E1-E14, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment E16. The multi-chain chimeric polypeptide of any one of

embodiments E1-E15, wherein the multi-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment E17. The multi-chain chimeric polypeptide of any one of

embodiments E1-E16, wherein the first chimeric polypeptide further comprises a peptide

tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment E18. The multi-chain chimeric polypeptide of any one of

embodiments E1-E17, wherein the second chimeric polypeptide further comprises a

peptide tag at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

Embodiment E19. The multi-chain chimeric polypeptide of any one of

embodiments E1-E18, wherein the first chimeric polypeptide and/or the second chimeric

polypeptide further comprises a signal sequence at its N-terminal end.

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Embodiment E20. The multi-chain chimeric polypeptide of embodiment E19,

wherein the signal sequence comprises SEQ ID NO: 117.

Embodiment E21. The multi-chain chimeric polypeptide of embodiment E20,

wherein the signal sequence is SEQ ID NO: 117.

Embodiment E22. The multi-chain chimeric polypeptide of any one of

embodiments E1-E21, wherein the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra and a soluble IL-15.

Embodiment E23. The multi-chain chimeric polypeptide of embodiment E22,

wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment E24. The multi-chain chimeric polypeptide of embodiment E22,

wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 82.

Embodiment E25. The multi-chain chimeric polypeptide of embodiment E24,

wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 82.

Embodiment E26. The multi-chain chimeric polypeptide of embodiment E25,

wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 82.

Embodiment E27. The multi-chain chimeric polypeptide of embodiment E26,

wherein the soluble IL-15 comprises SEQ ID NO: 82.

Embodiment E28. The multi-chain chimeric polypeptide of any one of

embodiments E22-E27, wherein the sushi domain of IL-15Ra comprises a sushi domain

from human IL-15Ra.

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Embodiment E29. The multi-chain chimeric polypeptide of embodiment E28,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 80%

identical to SEQ ID NO: 113.

Embodiment E30. The multi-chain chimeric polypeptide of embodiment E29,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 90%

identical to SEQ ID NO: 113.

Embodiment E31. The multi-chain chimeric polypeptide of embodiment E30,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 95%

identical to SEQ ID NO: 113.

Embodiment E32. The multi-chain chimeric polypeptide of embodiment E31,

wherein the sushi domain from human IL-15Ra comprises SEQ ID NO: 113.

Embodiment E33. The multi-chain chimeric polypeptide of embodiment E28,

wherein the sushi domain from human IL-15Ra is a mature full-length IL-15Ra.

Embodiment E34. The multi-chain chimeric polypeptide of any one of

embodiments E1-E21, wherein the pair of affinity domains is selected from the group

consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules

based on mutated RNase I fragments, and SNARE modules based on interactions of the

proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment E35. The multi-chain chimeric polypeptide of any one of

embodiments E1-E34, wherein one or both of the first target-binding domain and the

second target-binding domain is an antigen-binding domain.

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Embodiment E36. The multi-chain chimeric polypeptide of embodiment E35,

wherein the first target-binding domain and the second target-binding domain are

antigen-binding domains.

Embodiment E37. The multi-chain chimeric polypeptide of embodiment E35 or

E36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment E38. The multi-chain chimeric polypeptide of any one of

embodiments E1-E34, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble IL-21 or a soluble TGFßRII.

Embodiment E39. The multi-chain chimeric polypeptide of any one of

embodiments E1-E38, wherein the first target-binding domain and the second target-

binding domain both bind specifically to a receptor of IL-21 or a ligand of TGFßRII.

Embodiment E40. The multi-chain chimeric polypeptide of embodiment E39,

wherein the first target-binding domain and the second target-binding domain bind

specifically to the same epitope.

Embodiment E41. The multi-chain chimeric polypeptide of embodiment E40,

wherein the first target-binding domain and the second target-binding domain comprise

the same amino acid sequence.

Embodiment E42. The multi-chain chimeric polypeptide of any one of

embodiments E1-E38, wherein the first target-binding domain binds specifically to a

ligand of TGFßRII, and the second target-binding domain binds specifically to a receptor

for IL-21.

Embodiment E43. The multi-chain chimeric polypeptide of any one of

embodiments E1-E38, wherein the first target-binding domain binds specifically to a

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receptor for IL-21, and the second target-binding domain bind specifically to a ligand of

TGFBRII.

Embodiment E44. The multi-chain chimeric polypeptide of embodiment E43,

wherein the first target-binding domain comprises a soluble IL-21.

Embodiment E45. The multi-chain chimeric polypeptide of embodiment E44,

wherein the soluble IL-21 is a soluble human IL-21.

Embodiment E46. The multi-chain chimeric polypeptide of embodiment E45,

wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID

NO: 83.

Embodiment E47. The multi-chain chimeric polypeptide of embodiment E46,

wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID

NO: 83.

Embodiment E48. The multi-chain chimeric polypeptide of embodiment E47,

wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID

NO: 83.

Embodiment E49. The multi-chain chimeric polypeptide of embodiment E48,

wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 83.

Embodiment E50. The multi-chain chimeric polypeptide of any one of

embodiments E43-E49, wherein the second target-binding domain comprises a soluble

TGFßRII.

Embodiment E51. The multi-chain chimeric polypeptide of embodiment E50,

wherein the soluble TGFBRII is a soluble human TGFßRII.

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Embodiment E52. The multi-chain chimeric polypeptide of embodiment E51,

wherein the soluble human TGFßRII comprises a first sequence of soluble human

TGFßRII and a second sequence of soluble human TGFBRII.

Embodiment E53. The multi-chain chimeric polypeptide of embodiment E52,

wherein the soluble human TGFßRII further comprises a linker sequence between the

first sequence of soluble human TGFßRII and the second sequence of soluble human

TGFBRII.

Embodiment E54. The multi-chain chimeric polypeptide of embodiment E53,

wherein the linker sequence comprises SEQ ID NO: 102.

Embodiment E55. The multi-chain chimeric polypeptide of any one of

embodiments E52-E54, wherein the first sequence of soluble human TGFßRII comprises

a sequence that is at least 80% identical to SEQ ID NO: 183.

Embodiment E56. The multi-chain chimeric polypeptide of embodiment E55,

wherein the first sequence of soluble human TGFßRII comprises a sequence that is at

least 90% identical to SEQ ID NO: 183.

Embodiment E57. The multi-chain chimeric polypeptide of embodiment E56,

wherein the first sequence of soluble human TGFßRII comprises a sequence that is at

least 95% identical to SEQ ID NO: 183.

Embodiment E58. The multi-chain chimeric polypeptide of embodiment E57,

wherein the first sequence of soluble human TGFßRII comprises SEQ ID NO: 183.

Embodiment E59. The multi-chain chimeric polypeptide of any one of

embodiments E52-E58, wherein the second sequence of soluble human TGFßRII

comprises a sequence that is at least 80% identical to SEQ ID NO: 184.

Embodiment E60. The multi-chain chimeric polypeptide of embodiment E59,

wherein the second sequence of soluble human TGFßRII comprises a sequence that is at

least 90% identical to SEQ ID NO: 184.

Embodiment E61. The mule-chain chimeric polypeptide of embodiment E60,

wherein the second sequence of soluble human TGFßRII comprises a sequence that is at

least 95% identical to SEQ ID NO: 184.

Embodiment E62. The multi-chain chimeric polypeptide of embodiment E61,

wherein the second sequence of soluble human TGFßRII comprises SEQ ID NO: 184.

Embodiment E63. The multi-chain chimeric polypeptide of embodiment E1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 189.

Embodiment E64. The multi-chain chimeric polypeptide of embodiment E63,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 189.

Embodiment E65. The multi-chain chimeric polypeptide of embodiment E64,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 189.

Embodiment E66. The multi-chain chimeric polypeptide of embodiment E65,

wherein the first chimeric polypeptide comprises SEQ ID NO: 189.

Embodiment E67. The multi-chain chimeric polypeptide of embodiment E66,

wherein the first chimeric polypeptide comprises SEQ ID NO: 191.

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Embodiment E68. The multi-chain chimeric polypeptide of any one of

embodiments E1 and E63-E67, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 193.

Embodiment E69. The multi-chain chimeric polypeptide of embodiment E68,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 193.

Embodiment E70. The multi-chain chimeric polypeptide of embodiment E69,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 193.

Embodiment E71. The multi-chain chimeric polypeptide of embodiment E70,

wherein the second chimeric polypeptide comprises SEQ ID NO: 193.

Embodiment E72. The multi-chain chimeric polypeptide of embodiment E71,

wherein the second chimeric polypeptide comprises SEQ ID NO: 195.

Embodiment E73. The multi-chain chimeric polypeptide of any one of

embodiments E1-E62, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domain(s), where at least one of the one or more

additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains.

Embodiment E74. The multi-chain chimeric polypeptide of embodiment E73,

wherein the first chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the at least one of the one or more additional antigen-

binding domain(s), and/or a linker sequence between the at least one of the one or more

additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

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Embodiment E75. The multi-chain chimeric polypeptide of any one of

embodiments E1-E62, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal and/or C-terminal end of the

first chimeric polypeptide.

Embodiment E76. The multi-chain chimeric polypeptide of embodiment E75,

wherein at least one of the one or more additional target-binding domains directly abuts

the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment E77. The multi-chain chimeric polypeptide of embodiment E75,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first domain of the

pair of affinity domains.

Embodiment E78. The multi-chain chimeric polypeptide of embodiment E75,

wherein the at least one of the one or more additional target-binding domains directly

abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment E79. The multi-chain chimeric polypeptide of embodiment E75,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first target-binding

domain.

Embodiment E80. The multi-chain chimeric polypeptide of embodiment E75,

wherein at least one of the one or more additional target-binding domains is disposed at

the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment E81. The multi-chain chimeric polypeptide of embodiment E80,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the N-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment E82. The multi-chain chimeric polypeptide of embodiment E80,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment E83. The multi-chain chimeric polypeptide of embodiment E80,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the C-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment E84. The multi-chain chimeric polypeptide of embodiment E80,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment E85. The multi-chain chimeric polypeptide of embodiment E80,

wherein the at least one of the one or more additional target-binding domains positioned

between the soluble tissue factor domain and the first domain of the pair of affinity

domains, directly abuts the soluble tissue factor domain and/or the first domain of the

pair of affinity domains.

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Embodiment E86. The multi-chain chimeric polypeptide of embodiment E80,

wherein the first chimeric polypeptide further comprises a linker sequence disposed (i)

between the soluble tissue factor domain and the at least one of the one or more

additional target-binding domains positioned between the soluble tissue factor domain

and the first domain of the pair of affinity domains, and/or (ii) between the first domain

of the pair of affinity domains and the at least one of the one or more additional target-

binding domains positioned between the soluble tissue factor domain and the first domain

of the pair of affinity domains.

Embodiment E87. The multi-chain chimeric polypeptide of any one of

embodiments E1-E62 and E73-E86, wherein the second chimeric polypeptide further

comprises one or more additional target-binding domains at the N-terminal end or the C-

terminal end of the second chimeric polypeptide.

Embodiment E88. The multi-chain chimeric polypeptide of embodiment E87,

wherein at least one of the one or more additional target-binding domains directly abuts

the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment E89. The multi-chain chimeric polypeptide of embodiment E87,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second domain of

the pair of affinity domains in the second chimeric polypeptide.

Embodiment E90. The multi-chain chimeric polypeptide of embodiment E87,

wherein at least one of the one or more additional target-binding domains directly abuts

the second target-binding domain in the second chimeric polypeptide.

Embodiment E91. The multi-chain chimeric polypeptide of embodiment E87,

wherein the second chimeric polypeptide further comprises a linker sequence between at

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least one of the one or more additional target-binding domains and the second target-

binding domain in the second chimeric polypeptide.

Embodiment E92. The multi-chain chimeric polypeptide of any one of

embodiments E73-E91, wherein two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

bind specifically to the same antigen.

Embodiment E93. The multi-chain chimeric polypeptide of embodiment E92,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope.

Embodiment E94. The multi-chain chimeric polypeptide of embodiment E93,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains comprise the same amino

acid sequence.

Embodiment E95. The multi-chain chimeric polypeptide of any one of

embodiments E73-E91, wherein the first target-binding domain, the second target-

binding domain, and the one or more additional target-binding domains bind specifically

to different antigens.

Embodiment E96. The multi-chain chimeric polypeptide of any one of

embodiments E73-E95, wherein the one or more additional antigen-binding domains bind

specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33,

CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT,

PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30,

CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3,

PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein,

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HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-D, a ligand of TGF-B

receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46,

a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a

ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a

receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor

for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for

IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem

cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a

receptor for a ULP16-binding protein, a receptor for CD155, and a receptor for CD28.

Embodiment E97. The multi-chain chimeric polypeptide of any one of

embodiments E73-E95, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine protein.

Embodiment E98 The multi-chain chimeric polypeptide of embodiment E97,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment E99. The multi-chain chimeric polypeptide of any one of

embodiments E73-E95, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine receptor.

Embodiment E100. The multi-chain chimeric polypeptide of embodiment E99,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-B RII) a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAM1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, , or a soluble CD28.

Embodiment E101. A composition comprising any of the multi-chain chimeric

polypeptides of embodiments E1-E100.

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Embodiment E102. The composition of embodiment E101, wherein the

composition is a pharmaceutical composition.

Embodiment E103. A kit comprising at least one dose of the composition of

embodiment E101 or E102.

Embodiment E104. Nucleic acid encoding any of the multi-chain chimeric

polypeptides of any one of embodiments E1-E100.

Embodiment E105. A vector comprising the nucleic acid of embodiment E 104.

Embodiment E106. The vector of embodiment E105, wherein the vector is an

expression vector.

Embodiment E107. A cell comprising the nucleic acid of embodiment C104 or

the vector of embodiment E105 or E106.

Embodiment E108. A method of producing a multi-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment E107 in a culture medium under conditions

sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment E109. A multi-chain chimeric polypeptide produced by the method

of embodiment E108.

Embodiment E110. The multi-chain chimeric polypeptide of embodiment E12,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

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Embodiment E111. The multi-chain chimeric polypeptide of embodiment E110,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment E112. The multi-chain chimeric polypeptide of embodiment E111,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment E113. The multi-chain chimeric polypeptide of embodiment E112,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

Embodiment E114. The multi-chain chimeric polypeptide of embodiment E12,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment E115. The multi-chain chimeric polypeptide of embodiment E114,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment E116. The multi-chain chimeric polypeptide of embodiment E115,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment E117. The multi-chain chimeric polypeptide of embodiment E116,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

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Embodiment F1. A multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate

through the binding of the first domain and the second domain of the pair of affinity

domains;

the first target-binding domain and the second targeting-binding domain each

independently bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment F2. The multi-chain chimeric polypeptide of embodiment F1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other in the first chimeric polypeptide.

Embodiment F3. The multi-chain chimeric polypeptide of embodiment F1,

wherein the first chimeric polypeptide further comprises a linker sequence between the

first target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

Embodiment F4. The multi-chain chimeric polypeptide of any one of

embodiments F1-F3, wherein the soluble tissue factor domain and the first domain of the

pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment F5. The multi-chain chimeric polypeptide of any one of

embodiments F1-F3, wherein the first chimeric polypeptide further comprises a linker

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sequence between the soluble tissue factor domain and the first domain of the pair of

affinity domains in the first chimeric polypeptide.

Embodiment F6. The multi-chain chimeric polypeptide of any one of

embodiments F1-F5, wherein the second domain of the pair of affinity domains and the

second target-binding domain directly abut each other in the second chimeric

polypeptide.

Embodiment F7. The multi-chain chimeric polypeptide of any one of

embodiments F1-F5, wherein second chimeric polypeptide further comprises a linker

sequence between the second domain of the pair of affinity domains and the second

target-binding domain in the second chimeric polypeptide.

Embodiment F8. The multi-chain chimeric polypeptide of any one of

embodiments F1-F7, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment F9. The multi-chain chimeric polypeptide of embodiment F8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment F10. The multi-chain chimeric polypeptide of embodiment F9,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment F11. The multi-chain chimeric polypeptide of embodiment F10,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

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Embodiment F12. The multi-chain chimeric polypeptide of embodiment F11,

wherein the soluble human tissue factor domain comprises SEQ ID NO: 93.

Embodiment F13. The multi-chain chimeric polypeptide of embodiment F8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

Embodiment F14. The multi-chain chimeric polypeptide of embodiment F13,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment F15. The multi-chain chimeric polypeptide of embodiment F14,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment F16. The multi-chain chimeric polypeptide of embodiment F15,

wherein the soluble human tissue factor domain comprises SEQ ID NO: 97.

Embodiment F17. The multi-chain chimeric polypeptide of embodiment F8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment F18. The multi-chain chimeric polypeptide of embodiment F17,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment F19. The multi-chain chimeric polypeptide of embodiment F18,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

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Embodiment F20. The multi-chain chimeric polypeptide of embodiment F19,

wherein the soluble human tissue factor domain comprises SEQ ID NO: 98.

Embodiment F21. The multi-chain chimeric polypeptide of any one of

embodiments F8-F11, F13-F15, and F17-F19, wherein the soluble human tissue factor

domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment F22. The multi-chain chimeric polypeptide of embodiment F21,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

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an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment F23. The multi-chain chimeric polypeptide of any one of

embodiments F1-F22, wherein the soluble tissue factor domain is not capable of binding

to Factor VIIa.

Embodiment F24. The multi-chain chimeric polypeptide of any one of

embodiments F1-F23, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment F25. The multi-chain chimeric polypeptide of any one of

embodiments F1-F24, wherein the multi-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment F26. The multi-chain chimeric polypeptide of any one of

embodiments F1-F25, wherein the first chimeric polypeptide further comprises a peptide

tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment F27. The multi-chain chimeric polypeptide of any one of

embodiments F1-F26, wherein the second chimeric polypeptide further comprises a

peptide tag at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

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Embodiment F28. The multi-chain chimeric polypeptide of any one of

embodiments F1-F27, wherein the first chimeric polypeptide and/or the second chimeric

polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment F29. The multi-chain chimeric polypeptide of embodiment F28,

wherein the signal sequence comprises SEQ ID NO: 117.

Embodiment F30. The multi-chain chimeric polypeptide of embodiment F28,

wherein the signal sequence is SEQ ID NO: 328.

Embodiment F31. The multi-chain chimeric polypeptide of any one of

embodiments F1-F30, wherein the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra) and a soluble IL-15.

Embodiment F32. The multi-chain chimeric polypeptide of embodiment F31,

wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment F33. The multi-chain chimeric polypeptide of embodiment F31,

wherein the soluble IL-15 comprises a sequence that is at least 80% identical to SEQ ID

NO: 82.

Embodiment F34. The multi-chain chimeric polypeptide of embodiment F33,

wherein the soluble IL-15 comprises a sequence that is at least 90% identical to SEQ ID

NO: 82.

Embodiment F35. The multi-chain chimeric polypeptide of embodiment F34,

wherein the soluble IL-15 comprises a sequence that is at least 95% identical to SEQ ID

NO: 82.

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Embodiment F36. The multi-chain chimeric polypeptide of embodiment F35,

wherein the soluble IL-15 comprises SEQ ID NO: 82.

Embodiment F37. The multi-chain chimeric polypeptide of any one of

embodiments F31-F36, wherein the sushi domain of IL-15Ra comprises a sushi domain

from human IL-15Ra.

Embodiment F38. The multi-chain chimeric polypeptide of embodiment F37,

wherein the sushi domain from human IL-15Ra comprises a sequence that is at least 80%

identical to SEQ ID NO: 113.

Embodiment F39. The multi-chain chimeric polypeptide of embodiment F38,

wherein the sushi domain from human IL-15Ra comprises a sequence that is at least 90%

identical to SEQ ID NO: 113.

Embodiment F40. The multi-chain chimeric polypeptide of embodiment F39,

wherein the sushi domain from human IL-15Ra comprises a sequence that is at least 95%

identical to SEQ ID NO: 113.

Embodiment F41. The multi-chain chimeric polypeptide of embodiment F40,

wherein the sushi domain from human IL-15Ra comprises SEQ ID NO: 113.

Embodiment F42. The multi-chain chimeric polypeptide of embodiment F37,

wherein the sushi domain from human IL-15Ra is a mature full-length IL-15Ra.

Embodiment F43. The multi-chain chimeric polypeptide of any one of

embodiments F1-F30, wherein the pair of affinity domains is selected from the group

consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules

based on mutated RNase I fragments, and SNARE modules based on interactions of the

proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

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Embodiment F44. The multi-chain chimeric polypeptide of any one of

embodiments F1-F43, wherein one or both of the first target-binding domain and the

second target-binding domain is an agonistic antigen-binding domain.

Embodiment F45. The multi-chain chimeric polypeptide of embodiment F44,

wherein the first target-binding domain and the second target-binding domain are each

agonistic antigen-binding domains.

Embodiment F46. The multi-chain chimeric polypeptide of embodiment F44 or

F45, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment F47. The multi-chain chimeric polypeptide of any one of

embodiments F1-F43, wherein one or both of the first target-binding domain and the

second target-binding domain is a soluble IL-21 or a soluble IL-7.

Embodiment F48. The multi-chain chimeric polypeptide of embodiment F47,

wherein the first target-binding domain and the second target-binding domain are each

independently a soluble IL-21 or a soluble IL-7.

Embodiment F49. The multi-chain chimeric polypeptide of any one of

embodiments F1-F48, wherein the first target-binding domain and the second target-

binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment F50. The multi-chain chimeric polypeptide of embodiment F49,

wherein the first target-binding domain and the second target-binding domain bind

specifically to the same epitope.

Embodiment F51. The multi-chain chimeric polypeptide of embodiment F50,

wherein the first target-binding domain and the second target-binding domain comprise

the same amino acid sequence.

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Embodiment F52. The multi-chain chimeric polypeptide of any one of

embodiments F1-F48, wherein the first target-binding domain binds specifically to a

receptor for IL-21, and the second target-binding domain binds specifically to a receptor

for IL-7.

Embodiment F53. The multi-chain chimeric polypeptide of any one of

embodiments F1-F48, wherein the first target-binding domain binds specifically to a

receptor for IL-7, and the second target-binding domain bind specifically to a receptor for

IL-21.

Embodiment F54. The multi-chain chimeric polypeptide of embodiment F53,

wherein the first target-binding domain comprises a soluble IL-21.

Embodiment F55. The multi-chain chimeric polypeptide of embodiment F54,

wherein the soluble IL-21 is a soluble human IL-21.

Embodiment F56. The multi-chain chimeric polypeptide of embodiment F55,

wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID

NO: 83.

Embodiment F57. The multi-chain chimeric polypeptide of embodiment F56,

wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID

NO: 83.

Embodiment F58. The multi-chain chimeric polypeptide of embodiment F57,

wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID

NO: 83.

Embodiment F59. The multi-chain chimeric polypeptide of embodiment F58,

wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 83.

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Embodiment F60. The multi-chain chimeric polypeptide of any one of

embodiments F53-F59, wherein the second target-binding domain comprises a soluble

IL-7.

Embodiment F61. The multi-chain chimeric polypeptide of embodiment D60,

wherein the soluble IL-7 is a soluble human IL-7.

Embodiment F62. The multi-chain chimeric polypeptide of embodiment F61,

wherein the soluble human IL-7 comprises a sequence at least 80% identical to SEQ ID

NO: 79.

Embodiment F63. The multi-chain chimeric polypeptide of embodiment F62,

wherein the soluble human IL-7 comprises a sequence at least 90% identical to SEQ ID

NO: 79.

Embodiment F64. The multi-chain chimeric polypeptide of embodiment F63,

wherein the soluble human IL-7 comprises a sequence at least 95% identical to SEQ ID

NO: 79.

Embodiment F65. The multi-chain chimeric polypeptide of embodiment F64,

wherein the soluble human IL-7 comprises a sequence of SEQ ID NO: 79.

Embodiment F66. The multi-chain chimeric polypeptide of embodiment F1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment F67. The multi-chain chimeric polypeptide of embodiment F66,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

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Embodiment F68. The multi-chain chimeric polypeptide of embodiment F67,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment F69. The multi-chain chimeric polypeptide of embodiment F68,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment F70. The multi-chain chimeric polypeptide of embodiment F69,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment F71. The multi-chain chimeric polypeptide of any one of

embodiments F1 and F66-F70, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 211.

Embodiment F72. The multi-chain chimeric polypeptide of embodiment F71,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 211.

Embodiment F73. The multi-chain chimeric polypeptide of embodiment F72,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 211.

Embodiment F74. The multi-chain chimeric polypeptide of embodiment F73,

wherein the second chimeric polypeptide comprises SEQ ID NO: 211.

Embodiment F75. The multi-chain chimeric polypeptide of embodiment F74,

wherein the second chimeric polypeptide comprises SEQ ID NO: 213.

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Embodiment F76. The multi-chain chimeric polypeptide of embodiment F1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 199.

Embodiment F77. The multi-chain chimeric polypeptide of embodiment F76,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 199.

Embodiment F78. The multi-chain chimeric polypeptide of embodiment F77,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 199.

Embodiment F79. The multi-chain chimeric polypeptide of embodiment F68,

wherein the first chimeric polypeptide comprises SEQ ID NO: 199.

Embodiment F80. The multi-chain chimeric polypeptide of embodiment F69,

wherein the first chimeric polypeptide comprises SEQ ID NO: 201.

Embodiment F81. The multi-chain chimeric polypeptide of any one of

embodiments F1 and F76-F80, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 203.

Embodiment F82. The multi-chain chimeric polypeptide of embodiment F81,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 203.

Embodiment F83. The multi-chain chimeric polypeptide of embodiment F82,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 203.

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Embodiment F84. The multi-chain chimeric polypeptide of embodiment F83,

wherein the second chimeric polypeptide comprises SEQ ID NO: 203.

Embodiment F85. The multi-chain chimeric polypeptide of embodiment F84,

wherein the second chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment F86. The multi-chain chimeric polypeptide of any one of

embodiments F1-F65, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domain(s), where at least one of the one or more

additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains.

Embodiment F87. The multi-chain chimeric polypeptide of embodiment F86,

wherein the first chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the at least one of the one or more additional antigen-

binding domain(s), and/or a linker sequence between the at least one of the one or more

additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment F88. The multi-chain chimeric polypeptide of any one of

embodiments F1-F65, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal and/or C-terminal end of the

first chimeric polypeptide.

Embodiment F89. The multi-chain chimeric polypeptide of embodiment F88,

wherein at least one of the one or more additional target-binding domains directly abuts

the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment F90. The multi-chain chimeric polypeptide of embodiment F88,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

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least one of the one or more additional target-binding domains and the first domain of the

pair of affinity domains.

Embodiment F91. The multi-chain chimeric polypeptide of embodiment F88,

wherein the at least one of the one or more additional target-binding domains directly

abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment F92. The multi-chain chimeric polypeptide of embodiment F88,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first target-binding

domain.

Embodiment F93. The multi-chain chimeric polypeptide of embodiment F88,

wherein at least one of the one or more additional target-binding domains is disposed at

the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment F94. The multi-chain chimeric polypeptide of embodiment F93,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the N-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment F95. The multi-chain chimeric polypeptide of embodiment F93,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

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Embodiment F96. The multi-chain chimeric polypeptide of embodiment F93,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the C-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment F97. The multi-chain chimeric polypeptide of embodiment F93,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment F98. The multi-chain chimeric polypeptide of embodiment F93,

wherein the at least one of the one or more additional target-binding domains positioned

between the soluble tissue factor domain and the first domain of the pair of affinity

domains, directly abuts the soluble tissue factor domain and/or the first domain of the

pair of affinity domains.

Embodiment F99. The multi-chain chimeric polypeptide of embodiment F93,

wherein the first chimeric polypeptide further comprises a linker sequence disposed (i)

between the soluble tissue factor domain and the at least one of the one or more

additional target-binding domains positioned between the soluble tissue factor domain

and the first domain of the pair of affinity domains, and/or (ii) between the first domain

of the pair of affinity domains and the at least one of the one or more additional target-

binding domains positioned between the soluble tissue factor domain and the first domain

of the pair of affinity domains.

Embodiment F100. The multi-chain chimeric polypeptide of any one of

embodiments F1-F65 and F86-F99, wherein the second chimeric polypeptide further

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comprises one or more additional target-binding domains at the N-terminal end or the C-

terminal end of the second chimeric polypeptide.

Embodiment F101. The multi-chain chimeric polypeptide of embodiment F100,

wherein at least one of the one or more additional target-binding domains directly abuts

the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment F102. The multi-chain chimeric polypeptide of embodiment F100,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second domain of

the pair of affinity domains in the second chimeric polypeptide.

Embodiment F103. The multi-chain chimeric polypeptide of embodiment F100,

wherein at least one of the one or more additional target-binding domains directly abuts

the second target-binding domain in the second chimeric polypeptide.

Embodiment F104. The multi-chain chimeric polypeptide of embodiment F100,

wherein the second chimeric polypeptide further comprises a linker sequence between at

least one of the one or more additional target-binding domains and the second target-

binding domain in the second chimeric polypeptide.

Embodiment F105. The multi-chain chimeric polypeptide of any one of

embodiments F86-F104, wherein two or more of the first target-binding domain, the

second target-binding domain, and the one or more additional target-binding domains

bind specifically to the same antigen.

Embodiment F106. The multi-chain chimeric polypeptide of embodiment F105,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains bind specifically to the

same epitope.

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Embodiment F107. The multi-chain chimeric polypeptide of embodiment F106,

wherein two or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains comprise the same amino

acid sequence.

Embodiment F108. The multi-chain chimeric polypeptide of any one of

embodiments F86-F104, wherein the first target-binding domain, the second target-

binding domain, and the one or more additional target-binding domains bind specifically

to different antigens.

Embodiment F109. The multi-chain chimeric polypeptide of any one of

embodiments F86-F108, wherein the one or more additional antigen-binding domains

bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3,

CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1,

TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2,

CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2,

HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding

protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of

TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of

NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for

IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a

receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a

receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a

receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a

receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, and a

receptor for CD28.

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Embodiment F110. The multi-chain chimeric polypeptide of any one of

embodiments F86-F108, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine protein.

Embodiment F111. The multi-chain chimeric polypeptide of embodiment F110,

wherein the soluble interleukin, cytokine, or ligand protein is selected from the group

consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,

PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment F112. The multi-chain chimeric polypeptide of any one of

embodiments F86-F108, wherein the one or more additional target-binding domains is a

soluble interleukin or cytokine receptor.

Embodiment F113. The multi-chain chimeric polypeptide of embodiment F112,

wherein the soluble receptor is a soluble TGF-B receptor II (TGF-BRII), a soluble TGF-

BRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble

DNAMI, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a

soluble CD28.

Embodiment F114. A composition comprising any of the multi-chain chimeric

polypeptides of embodiments F1-F113.

Embodiment F115. The composition of embodiment F114, wherein the

composition is a pharmaceutical composition.

Embodiment F116. A kit comprising at least one dose of the composition of

embodiment F114 or F115.

Embodiment F117. Nucleic acid encoding any of the multi-chain chimeric

polypeptides of any one of embodiments F1-F113.

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Embodiment F118. A vector comprising the nucleic acid of embodiment F 17.

Embodiment F119. The vector of embodiment F118, wherein the vector is an

expression vector.

Embodiment F120. A cell comprising the nucleic acid of embodiment F 117 or the

vector of embodiment F118 or F119.

Embodiment F121. A method of producing a multi-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment F120 in a culture medium under conditions

sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment F122. A multi-chain chimeric polypeptide produced by the method

of embodiment F121.

Embodiment G1. A multi-chain chimeric polypeptide comprising:

(e) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(f) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate

through the binding of the first domain and the second domain of the pair of affinity

domains; and

the first target-binding domain and the second targeting-binding domain each

independently bind specifically to: a receptor for IL-7, CD16, a receptor for IL-21, TGF-

B, or a receptor for CD137L.

Embodiment G2. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first target-binding domain and the soluble tissue factor domain directly abut

each other in the first chimeric polypeptide.

Embodiment G3. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide further comprises a linker sequence between the

first target-binding domain and the soluble tissue factor domain in the first chimeric

polypeptide.

Embodiment G4. The multi-chain chimeric polypeptide of any one of

embodiments G1-G3, wherein the soluble tissue factor domain and the first domain of the

pair of affinity domains directly abut each other in the first chimeric polypeptide.

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Embodiment G5. The multi-chain chimeric polypeptide of any one of

embodiments G1-G3, wherein the first chimeric polypeptide further comprises a linker

sequence between the soluble tissue factor domain and the first domain of the pair of

affinity domains in the first chimeric polypeptide.

Embodiment G6. The multi-chain chimeric polypeptide of any one of

embodiments G1-G5, wherein the second domain of the pair of affinity domains and the

second target-binding domain directly abut each other in the second chimeric

polypeptide.

Embodiment G7. The multi-chain chimeric polypeptide of any one of

embodiments G1-G5, wherein second chimeric polypeptide further comprises a linker

sequence between the second domain of the pair of affinity domains and the second

target-binding domain in the second chimeric polypeptide.

Embodiment G8. The multi-chain chimeric polypeptide of any one of

embodiments G1-G7, wherein the soluble tissue factor domain is a soluble human tissue

factor domain.

Embodiment G9. The multi-chain chimeric polypeptide of embodiment G8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 93.

Embodiment G10. The multi-chain chimeric polypeptide of embodiment G9,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 93.

Embodiment G11. The multi-chain chimeric polypeptide of embodiment G10,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 93.

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Embodiment G12. The multi-chain chimeric polypeptide of any one of

embodiments G8-G11, wherein the soluble human tissue factor domain does not

comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment G13. The multi-chain chimeric polypeptide of embodiment G12,

wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

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an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment G14. The multi-chain chimeric polypeptide of any one of

embodiments G1-G13, wherein the soluble tissue factor domain is not capable of binding

to Factor VIIa.

Embodiment G15. The multi-chain chimeric polypeptide of any one of

embodiments G1-G14, wherein the soluble tissue factor domain does not convert inactive

Factor X into Factor Xa.

Embodiment G16. The multi-chain chimeric polypeptide of any one of

embodiments G1-G15, wherein the multi-chain chimeric polypeptide does not stimulate

blood coagulation in a mammal.

Embodiment G17. The multi-chain chimeric polypeptide of any one of

embodiments G1-G16, wherein the first chimeric polypeptide further comprises a peptide

tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment G18. The multi-chain chimeric polypeptide of any one of

embodiments G1-G17, wherein the second chimeric polypeptide further comprises a

peptide tag at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

Embodiment G19. The multi-chain chimeric polypeptide of any one of

embodiments G1-G18, wherein the first chimeric polypeptide and/or the second chimeric

polypeptide further comprises a signal sequence at its N-terminal end.

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Embodiment G20, The multi-chain chimeric polypeptide of embodiment G19,

wherein the signal sequence comprises SEQ ID NO: 117.

Embodiment G21. The multi-chain chimeric polypeptide of embodiment G20,

wherein the signal sequence is SEQ ID NO: 117.

Embodiment G22. The multi-chain chimeric polypeptide of any one of

embodiments G1-G21, wherein the pair of affinity domains is a sushi domain from an

alpha chain of human IL-15 receptor (IL-15Ra) and a soluble IL-15.

Embodiment G23. The multi-chain chimeric polypeptide of embodiment G22,

wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment G24. The multi-chain chimeric polypeptide of embodiment G22,

wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 82.

Embodiment G25. The multi-chain chimeric polypeptide of embodiment G24,

wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 82.

Embodiment G26. The multi-chain chimeric polypeptide of embodiment G25,

wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 82.

Embodiment G27. The multi-chain chimeric polypeptide of embodiment G26,

wherein the soluble IL-15 comprises SEQ ID NO: 82.

Embodiment G28. The multi-chain chimeric polypeptide of any one of

embodiments G22-G27, wherein the sushi domain of IL-15Ra comprises a sushi domain

from human IL-15Ra.

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Embodiment G29. The multi-chain chimeric polypeptide of embodiment G28,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 80%

identical to SEQ ID NO: 113.

Embodiment G30. The multi-chain chimeric polypeptide of embodiment G29,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 90%

identical to SEQ ID NO: 113.

Embodiment G31. The multi-chain chimeric polypeptide of embodiment G30,

wherein the sushi domain from human IL-15Ra comprises a sequence that is 95%

identical to SEQ ID NO: 113.

Embodiment G32. The multi-chain chimeric polypeptide of embodiment G31,

wherein the sushi domain from human IL-15Ra comprises SEQ ID NO: 113.

Embodiment G33. The multi-chain chimeric polypeptide of embodiment G28,

wherein the sushi domain from human IL-15Ra is a mature full-length IL-15Ra.

Embodiment G34. The multi-chain chimeric polypeptide of any one of

embodiments G1-G21, wherein the pair of affinity domains is selected from the group

consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules

based on mutated RNase I fragments, and SNARE modules based on interactions of the

proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment G35. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to a receptor for IL-7, CD16, or a

receptor for IL-21.

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Embodiment G36. The multi-chain chimeric polypeptide of embodiment G35,

wherein the first target-binding domain binds specifically to a receptor IL-7 and the

second target-binding domain binds specifically to CD16 or a receptor for IL-21.

Embodiment G37. The multi-chain chimeric polypeptide of embodiment G36,

wherein the first target-binding domain comprises a soluble IL-7 protein.

Embodiment G38. The multi-chain chimeric polypeptide of embodiment G37,

wherein the soluble IL-7 protein is a soluble human IL-7.

Embodiment G39. The multi-chain chimeric polypeptide of any one of

embodiments G36-G38, wherein the second antigen-binding domain comprises an

antigen-binding domain that binds specifically to CD16.

Embodiment G40. The multi-chain chimeric polypeptide of embodiment G39,

wherein the second antigen-binding domain comprises an scFv that binds specifically to

CD16.

Embodiment G41. The multi-chain chimeric polypeptide of any one of

embodiments G36-G38, wherein the second antigen-binding domain bind specifically to

a receptor for IL-21.

Embodiment G42. The multi-chain chimeric polypeptide of embodiment G41,

wherein the second antigen-binding domain comprises a soluble IL-21.

Embodiment G43. The multi-chain chimeric polypeptide of embodiment G42,

wherein the soluble IL-21 is a soluble human IL-21.

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Embodiment G44. The multi-chain chimeric polypeptide of any one of

embodiments G36-G40, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to a receptor for IL-21.

Embodiment G45. The multi-chain chimeric polypeptide of embodiment G44,

wherein the additional target-binding domain comprises a soluble IL-21.

Embodiment G46. The multi-chain chimeric polypeptide of embodiment G45,

wherein the soluble IL-21 is a soluble human IL-12.

Embodiment G47. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to TGF-B, CD16, or a receptor for

IL-21.

Embodiment G48. The multi-chain chimeric polypeptide of embodiment G47,

wherein the first target-binding domain binds specifically to a TGF-B and the second

target-binding domain binds specifically to CD16 or a receptor of IL-21.

Embodiment G49. The multi-specific chimeric polypeptide of embodiment G48,

wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G50. The multi-specific chimeric polypeptide of embodiment G49,

wherein soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G51. The multi-specific chimeric polypeptide of any one of

embodiments G48-G50, wherein the second target-binding domain binds specifically to

CD16.

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Embodiment G52. The multi-specific chimeric polypeptide of embodiment G51,

wherein the second antigen-binding domain comprises an antigen-binding domain that

binds specifically to CD16.

Embodiment G53. The multi-chain chimeric polypeptide of embodiment G52,

wherein the second antigen-binding domain comprises an scFv that binds specifically to

CD16.

Embodiment G54. The multi-chain chimeric polypeptide of any one of

embodiments G48-G50, wherein the second target-binding domain binds specifically to a

receptor for IL-21.

Embodiment G55. The multi-chain chimeric polypeptide of embodiment G54,

wherein the second target-binding domain comprises a soluble IL-21.

Embodiment G56. The multi-chain chimeric polypeptide of embodiment G55,

wherein the second target-binding domain comprises a soluble human IL-21.

Embodiment G57. The multi-chain chimeric polypeptide of any one of

embodiments G48-G53, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to a receptor for IL-21.

Embodiment G58. The multi-chain chimeric polypeptide of embodiment G57,

wherein the additional target-binding domain comprises a soluble IL-21.

Embodiment G59. The multi-chain chimeric polypeptide of embodiment G58,

wherein the soluble IL-21 is a soluble human IL-21.

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Embodiment G60. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second target-

binding domain each independently bind specifically to a receptor for IL-7.

Embodiment G61. The multi-chain chimeric polypeptide of embodiment G60,

wherein the first target-binding domain and the second target-binding domain include a

soluble IL-7.

Embodiment G62. The multi-chain chimeric polypeptide of embodiment G61,

wherein the soluble IL-7 is a soluble human IL-7.

Embodiment G63. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second target-

binding domain each independently bind specifically to TGF-B.

Embodiment G64. The multi-specific chimeric polypeptide of embodiment G63,

wherein the first target-binding domain and the second target-binding domain is a soluble

TGF-B receptor.

Embodiment G65. The multi-specific chimeric polypeptide of embodiment G64,

wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G66. The multi-specific chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to a receptor for IL-7, a receptor for

IL-21, or a receptor for CD137L.

Embodiment G67. The multi-chain chimeric polypeptide of embodiment G66,

wherein the first target-binding domain binds specifically to a receptor for IL-7 and the

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second target-binding domain binds specifically to a receptor for IL-21 or a receptor for

CD137L.

Embodiment G68. The multi-specific chimeric polypeptide of embodiment G67,

wherein the first target-binding domain is a soluble IL-7.

Embodiment G69. The multi-specific chimeric polypeptide of embodiment G68,

wherein the soluble IL-7 is a soluble human IL-7.

Embodiment G70. The multi-chain chimeric polypeptide of any one of

embodiments G67-G69, wherein the second target-binding domain binds specifically to a

receptor for IL-21.

Embodiment G71. The multi-chain chimeric polypeptide of embodiment G70,

wherein the second target-binding domain is a soluble IL-21.

Embodiment G72. The multi-chain chimeric polypeptide of embodiment G71,

wherein the soluble IL-21 is a soluble human IL-21.

Embodiment G73. The multi-chain chimeric polypeptide of any one of

embodiments G67-G69, wherein the second antigen-binding domain binds specifically to

a receptor for CD137L.

Embodiment G74. The multi-chain chimeric polypeptide of embodiment G73,

wherein the second antigen-binding domain is a soluble CD137L.

Embodiment G75. The multi-chain chimeric polypeptide of embodiment G74,

wherein the soluble CD137L is a soluble human CD137L.

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Embodiment G76. The multi-chain chimeric polypeptide of any one of

embodiments G67-G72, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to a receptor for CD137L.

Embodiment G77. The multi-chain chimeric polypeptide of embodiment G76,

wherein the additional target-binding domain comprises a soluble CD137L.

Embodiment G78. The multi-chain chimeric polypeptide of embodiment G77,

wherein the soluble CD137L is a soluble human CD137L.

Embodiment G79. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to a receptor for IL-7 or TGF-B.

Embodiment G80. The multi-chain chimeric polypeptide of embodiment G79,

wherein the first target-binding domain binds specifically to a receptor IL-7 and the

second target-binding domain binds specifically to TGF-B.

Embodiment G81. The multi-chain chimeric polypeptide of embodiment G80,

wherein the first target-binding domain comprises a soluble IL-7 protein.

Embodiment G82. The multi-chain chimeric polypeptide of embodiment G81,

wherein the soluble IL-7 protein is a soluble human IL-7.

Embodiment G83. The multi-chain chimeric polypeptide of any one of

embodiments G80-G82, wherein the second antigen-binding domain comprises an

antigen-binding domain that binds specifically to TGF-B.

Embodiment G84. The multi-specific chimeric polypeptide of embodiment G83,

wherein the second target-binding domain is a soluble TGF-B receptor.

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Embodiment G85. The multi-specific chimeric polypeptide of embodiment G84,

wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G86. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to TGF-B, a receptor for IL-21, or a

receptor for CD137L.

Embodiment G87. The multi-chain chimeric polypeptide of embodiment G86,

wherein the first target-binding domain binds specifically to a TGF-B and the second

target-binding domain binds specifically to a receptor for IL-21 or a receptor for

CD137L.

Embodiment G88. The multi-specific chimeric polypeptide of embodiment G87,

wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G89. The multi-specific chimeric polypeptide of embodiment G88,

wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G90. The multi-specific chimeric polypeptide of any one of

embodiments G87-G89, wherein the second target-binding domain binds specifically to a

receptor for IL-21.

Embodiment G91. The multi-chain chimeric polypeptide of embodiment G90,

wherein the second target-binding domain comprises a soluble IL-21.

Embodiment G92. The multi-chain chimeric polypeptide of embodiment G91,

wherein the second target-binding domain comprises a soluble human IL-21.

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Embodiment G93. The multi-specific chimeric polypeptide of any one of

embodiments G87-G89, wherein the second target-binding domain binds specifically to a

receptor for CD137L.

Embodiment G94. The multi-chain chimeric polypeptide of embodiment G93,

wherein the second target-binding domain comprises a soluble CD137L.

Embodiment G95. The multi-chain chimeric polypeptide of embodiment G94,

wherein the second target-binding domain comprises a soluble human CD137L.

Embodiment G96. The multi-chain chimeric polypeptide of any one of

embodiments G87-G92, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to a receptor for CD137L.

Embodiment G97. The multi-chain chimeric polypeptide of embodiment G96,

wherein the additional target-binding domain comprises a soluble CD137L.

Embodiment G98. The multi-chain chimeric polypeptide of embodiment G97,

wherein the soluble CD137L is a soluble human CD137L.

Embodiment G99. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to TGF-B or a receptor for IL-21.

Embodiment G100. The multi-chain chimeric polypeptide of embodiment G99,

wherein the first target-binding domain binds specifically to a TGF-B and the second

target-binding domain binds specifically to TGF-B or a receptor for IL-21.

Embodiment G101. The multi-specific chimeric polypeptide of embodiment

G100, wherein the first target-binding domain is a soluble TGF-B receptor.

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Embodiment G102. The multi-specific chimeric polypeptide of embodiment

G101, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G103. The multi-specific chimeric polypeptide of any one of

embodiments G100-G102, wherein the second target-binding domain binds specifically

to a receptor for IL-21.

Embodiment G104. The multi-chain chimeric polypeptide of embodiment G103,

wherein the second target-binding domain comprises a soluble IL-21.

Embodiment G105. The multi-chain chimeric polypeptide of embodiment G104,

wherein the second target-binding domain comprises a soluble human IL-21.

Embodiment G106. The multi-specific chimeric polypeptide of any one of

embodiments G100-G102, wherein the second target-binding domain binds specifically

to TGF-B.

Embodiment G107. The multi-specific chimeric polypeptide of embodiment

G106, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G108. The multi-specific chimeric polypeptide of embodiment

G107, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G109. The multi-specific chimeric polypeptide of any one of

embodiments G100-G105, wherein the second polypeptide further comprises an

additional target-binding domain that binds specifically to TGF-B.

Embodiment G110. The multi-specific chimeric polypeptide of embodiment

G109, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G111. The multi-specific chimeric polypeptide of embodiment

G110, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G112. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to TGF-B or IL-16.

Embodiment G113. The multi-chain chimeric polypeptide of embodiment G112,

wherein the first target-binding domain binds specifically to a TGF-B and the second

target-binding domain binds specifically to TGF-B or IL-16.

Embodiment G114. The multi-specific chimeric polypeptide of embodiment

G113, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G115. The multi-specific chimeric polypeptide of embodiment

G114, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G116. The multi-specific chimeric polypeptide of any one of

embodiments G113-G115, wherein the second target-binding domain binds specifically

to IL-16.

Embodiment G117. The multi-specific chimeric polypeptide of embodiment

G116, wherein the second antigen-binding domain comprises an antigen-binding domain

that binds specifically to CD16.

Embodiment G118. The multi-chain chimeric polypeptide of embodiment G117,

wherein the second antigen-binding domain comprises an scFv that binds specifically to

CD16.

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Embodiment G119. The multi-specific chimeric polypeptide of any one of

embodiments G113-G115, wherein the second target-binding domain binds specifically

to TGF-B.

Embodiment G120. The multi-specific chimeric polypeptide of embodiment

G119, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G121. The multi-specific chimeric polypeptide of embodiment

G120, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G122. The multi-specific chimeric polypeptide of any one of

embodiments G113-G118, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to TGF-B.

Embodiment G123. The multi-specific chimeric polypeptide of embodiment

G122, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G124. The multi-specific chimeric polypeptide of embodiment

G123, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G125. The multi-chain chimeric polypeptide of any one of

embodiments G1-G34, wherein the first target-binding domain and the second targeting-

binding domain each independently bind specifically to a TGF-B or a receptor for

CD137L.

Embodiment G126. The multi-chain chimeric polypeptide of embodiment G125,

wherein the first target-binding domain binds specifically to TGF-B and the second

target-binding domain binds specifically to a receptor for CD137L.

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Embodiment G127. The multi-specific chimeric polypeptide of embodiment

G126, wherein the first target-binding domain is a soluble TGF-B receptor.

Embodiment G128. The multi-specific chimeric polypeptide of embodiment

G127, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G129. The multi-chain chimeric polypeptide of embodiment G128,

wherein the second target-binding domain comprises a soluble CD137L protein.

Embodiment G130. The multi-chain chimeric polypeptide of embodiment G129,

wherein the soluble CD137L protein is a soluble human CD137L.

Embodiment G131. The multi-chain chimeric polypeptide of any one of

embodiments G126-G130, wherein the second chimeric polypeptide further comprises an

additional target-binding domain that binds specifically to TGF-B.

Embodiment G132. The multi-specific chimeric polypeptide of embodiment

G131, wherein the additional target-binding domain is a soluble TGF-B receptor.

Embodiment G133. The multi-specific chimeric polypeptide of embodiment

G132, wherein the soluble TGF-B receptor is a soluble TGFßRII receptor.

Embodiment G134. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment G135. The multi-chain chimeric polypeptide of embodiment G134,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

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Embodiment G136. The multi-chain chimeric polypeptide of embodiment G135,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment G137. The multi-chain chimeric polypeptide of embodiment G136,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment G138. The multi-chain chimeric polypeptide of embodiment G137,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment G139. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G134-G138, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 232.

Embodiment G140. The multi-chain chimeric polypeptide of embodiment G139,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 232.

Embodiment G141. The multi-chain chimeric polypeptide of embodiment G140,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 232.

Embodiment G142. The multi-chain chimeric polypeptide of embodiment G141,

wherein the second chimeric polypeptide comprises SEQ ID NO: 232.

Embodiment G143. The multi-chain chimeric polypeptide of embodiment G142,

wherein the second chimeric polypeptide comprises SEQ ID NO: 234.

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Embodiment G144. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G145. The multi-chain chimeric polypeptide of embodiment G144,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

Embodiment G146. The multi-chain chimeric polypeptide of embodiment G145,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G147. The multi-chain chimeric polypeptide of embodiment G146,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

Embodiment G148. The multi-chain chimeric polypeptide of embodiment G147,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G149. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G144-G148, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 232.

Embodiment G150. The multi-chain chimeric polypeptide of embodiment G149,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 232.

Embodiment G151. The multi-chain chimeric polypeptide of embodiment G150,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 232.

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Embodiment G152. The multi-chain chimeric polypeptide of embodiment G151,

wherein the second chimeric polypeptide comprises SEQ ID NO: 232.

Embodiment G153. The multi-chain chimeric polypeptide of embodiment G152,

wherein the second chimeric polypeptide comprises SEQ ID NO: 234.

Embodiment G154. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment G155. The multi-chain chimeric polypeptide of embodiment G154,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

Embodiment G156. The multi-chain chimeric polypeptide of embodiment G155,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment G157. The multi-chain chimeric polypeptide of embodiment G156,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment G158. The multi-chain chimeric polypeptide of embodiment G157,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment G159. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G154-G158, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 203.

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Embodiment G160. The multi-chain chimeric polypeptide of embodiment G159,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 203.

Embodiment G161. The multi-chain chimeric polypeptide of embodiment G160,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 203.

Embodiment G162. The multi-chain chimeric polypeptide of embodiment G161,

wherein the second chimeric polypeptide comprises SEQ ID NO: 203.

Embodiment G163. The multi-chain chimeric polypeptide of embodiment G162,

wherein the second chimeric polypeptide comprises SEQ ID NO: 250.

Embodiment G164. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G165. The multi-chain chimeric polypeptide of embodiment G164,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

Embodiment G166. The multi-chain chimeric polypeptide of embodiment G165,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G167. The multi-chain chimeric polypeptide of embodiment G166,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

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Embodiment G168. The multi-chain chimeric polypeptide of embodiment G167,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G169. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G164-G168, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 193.

Embodiment G170. The multi-chain chimeric polypeptide of embodiment G169,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 193.

Embodiment G171. The multi-chain chimeric polypeptide of embodiment G170,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 193.

Embodiment G172. The multi-chain chimeric polypeptide of embodiment G171,

wherein the second chimeric polypeptide comprises SEQ ID NO: 193.

Embodiment G173. The multi-chain chimeric polypeptide of embodiment G172,

wherein the second chimeric polypeptide comprises SEQ ID NO: 195.

Embodiment G174. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment G175. The multi-chain chimeric polypeptide of embodiment G174,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

Embodiment G176. The multi-chain chimeric polypeptide of embodiment G175,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment G177. The multi-chain chimeric polypeptide of embodiment G176,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment G178. The multi-chain chimeric polypeptide of embodiment G177,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment G179. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G174-G178, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 268.

Embodiment G180. The multi-chain chimeric polypeptide of embodiment G179,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 268.

Embodiment G181. The multi-chain chimeric polypeptide of embodiment G180,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 268.

Embodiment G182. The multi-chain chimeric polypeptide of embodiment G181,

wherein the second chimeric polypeptide comprises SEQ ID NO: 268.

Embodiment G183. The multi-chain chimeric polypeptide of embodiment G182,

wherein the second chimeric polypeptide comprises SEQ ID NO: 270.

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Embodiment G184. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment G185. The multi-chain chimeric polypeptide of embodiment G184,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

Embodiment G186. The multi-chain chimeric polypeptide of embodiment G185,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment G187. The multi-chain chimeric polypeptide of embodiment G186,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment G188. The multi-chain chimeric polypeptide of embodiment G187,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment G189. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G184-G188, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 272.

Embodiment G190. The multi-chain chimeric polypeptide of embodiment G189,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 272.

Embodiment G191. The multi-chain chimeric polypeptide of embodiment G190,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 272.

Embodiment G192. The multi-chain chimeric polypeptide of embodiment G191,

wherein the second chimeric polypeptide comprises SEQ ID NO: 272.

Embodiment G193. The multi-chain chimeric polypeptide of embodiment G192,

wherein the second chimeric polypeptide comprises SEQ ID NO: 272.

Embodiment G194. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 207.

Embodiment G195. The multi-chain chimeric polypeptide of embodiment G194,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 207.

Embodiment G196. The multi-chain chimeric polypeptide of embodiment G195,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 207.

Embodiment G197. The multi-chain chimeric polypeptide of embodiment G196,

wherein the first chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment G198. The multi-chain chimeric polypeptide of embodiment G197,

wherein the first chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment G199. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G194-G198, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 193.

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Embodiment G200, The multi-chain chimeric polypeptide of embodiment G199,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 193.

Embodiment G201. The multi-chain chimeric polypeptide of embodiment G200,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 193.

Embodiment G202. The multi-chain chimeric polypeptide of embodiment G201,

wherein the second chimeric polypeptide comprises SEQ ID NO: 193.

Embodiment G203. The multi-chain chimeric polypeptide of embodiment G202,

wherein the second chimeric polypeptide comprises SEQ ID NO: 195.

Embodiment G204. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G205. The multi-chain chimeric polypeptide of embodiment G204,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

Embodiment G206, The multi-chain chimeric polypeptide of embodiment G205,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G207. The multi-chain chimeric polypeptide of embodiment G206,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

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Embodiment G208. The multi-chain chimeric polypeptide of embodiment G207,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G209. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G204-G208, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 268.

Embodiment G210. The multi-chain chimeric polypeptide of embodiment G209,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 268.

Embodiment G211. The multi-chain chimeric polypeptide of embodiment G210,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 268.

Embodiment G212. The multi-chain chimeric polypeptide of embodiment G211,

wherein the second chimeric polypeptide comprises SEQ ID NO: 268.

Embodiment G213. The multi-chain chimeric polypeptide of embodiment G212,

wherein the second chimeric polypeptide comprises SEQ ID NO: 270.

Embodiment G214. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G215. The multi-chain chimeric polypeptide of embodiment G214,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

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Embodiment G216. The multi-chain chimeric polypeptide of embodiment G215,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G217. The multi-chain chimeric polypeptide of embodiment G216,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

Embodiment G218. The multi-chain chimeric polypeptide of embodiment G217,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G219. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G214-G218, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 300.

Embodiment G220. The multi-chain chimeric polypeptide of embodiment G219,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 300.

Embodiment G221. The multi-chain chimeric polypeptide of embodiment G220,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 300.

Embodiment G222. The multi-chain chimeric polypeptide of embodiment G221,

wherein the second chimeric polypeptide comprises SEQ ID NO: 300.

Embodiment G223. The multi-chain chimeric polypeptide of embodiment G222,

wherein the second chimeric polypeptide comprises SEQ ID NO: 302.

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Embodiment G224. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G225. The multi-chain chimeric polypeptide of embodiment G224,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

Embodiment G226. The multi-chain chimeric polypeptide of embodiment G225,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G227. The multi-chain chimeric polypeptide of embodiment G226,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

Embodiment G228. The multi-chain chimeric polypeptide of embodiment G227,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G229. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G224-G228, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 308.

Embodiment G230. The multi-chain chimeric polypeptide of embodiment G229,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 308.

Embodiment G231. The multi-chain chimeric polypeptide of embodiment G230,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 308.

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Embodiment G232. The multi-chain chimeric polypeptide of embodiment G231,

wherein the second chimeric polypeptide comprises SEQ ID NO: 308.

Embodiment G233. The multi-chain chimeric polypeptide of embodiment G232,

wherein the second chimeric polypeptide comprises SEQ ID NO: 310.

Embodiment G234. The multi-chain chimeric polypeptide of embodiment G1,

wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical

to SEQ ID NO: 236.

Embodiment G235. The multi-chain chimeric polypeptide of embodiment G234,

wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical

to SEQ ID NO: 236.

Embodiment G236. The multi-chain chimeric polypeptide of embodiment G235,

wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical

to SEQ ID NO: 236.

Embodiment G237. The multi-chain chimeric polypeptide of embodiment G236,

wherein the first chimeric polypeptide comprises SEQ ID NO: 236.

Embodiment G238. The multi-chain chimeric polypeptide of embodiment G237,

wherein the first chimeric polypeptide comprises SEQ ID NO: 238.

Embodiment G239. The multi-chain chimeric polypeptide of any one of

embodiments G1 and G234-G238, wherein the second chimeric polypeptide comprises a

sequence that is at least 80% identical to SEQ ID NO: 316.

910

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Embodiment G240. The multi-chain chimeric polypeptide of embodiment G239,

wherein the second chimeric polypeptide comprises a sequence that is at least 90%

identical to SEQ ID NO: 316.

Embodiment G241. The multi-chain chimeric polypeptide of embodiment G240,

wherein the second chimeric polypeptide comprises a sequence that is at least 95%

identical to SEQ ID NO: 316.

Embodiment G242. The multi-chain chimeric polypeptide of embodiment G241,

wherein the second chimeric polypeptide comprises SEQ ID NO: 316.

Embodiment G243. The multi-chain chimeric polypeptide of embodiment G242,

wherein the second chimeric polypeptide comprises SEQ ID NO: 318.

Embodiment G244. The multi-chain chimeric polypeptide of any one of

embodiments G1-G133, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domain(s), where at least one of the one or more

additional antigen-binding domain(s) is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains.

Embodiment G245. The multi-chain chimeric polypeptide of embodiment G244,

wherein the first chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the at least one of the one or more additional antigen-

binding domain(s), and/or a linker sequence between the at least one of the one or more

additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment G246. The multi-chain chimeric polypeptide of any one of

embodiments G1-G133, wherein the first chimeric polypeptide further comprises one or

more additional target-binding domains at the N-terminal and/or C-terminal end of the

first chimeric polypeptide.

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Embodiment G247. The multi-chain chimeric polypeptide of embodiment G246,

wherein at least one of the one or more additional target-binding domains directly abuts

the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment G248. The multi-chain chimeric polypeptide of embodiment G246,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first domain of the

pair of affinity domains.

Embodiment G249. The multi-chain chimeric polypeptide of embodiment G246,

wherein the at least one of the one or more additional target-binding domains directly

abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment G250. The multi-chain chimeric polypeptide of embodiment G246,

wherein the first chimeric polypeptide further comprises a linker sequence between the at

least one of the one or more additional target-binding domains and the first target-binding

domain.

Embodiment G251. The multi-chain chimeric polypeptide of embodiment G246,

wherein at least one of the one or more additional target-binding domains is disposed at

the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or

more additional target-binding domains is positioned between the soluble tissue factor

domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment G252. The multi-chain chimeric polypeptide of embodiment G251,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the N-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

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Embodiment G253. The multi-chain chimeric polypeptide of embodiment G251,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment G254. The multi-chain chimeric polypeptide of embodiment G251,

wherein the at least one additional target-binding domain of the one or more additional

target-binding domains disposed at the C-terminus directly abuts the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment G255. The multi-chain chimeric polypeptide of embodiment G251,

wherein the first chimeric polypeptide further comprises a linker sequence disposed

between the at least one additional target-binding domain and the first target-binding

domain or the first domain of the pair of affinity domains in the first chimeric

polypeptide.

Embodiment G256. The multi-chain chimeric polypeptide of embodiment G251,

wherein the at least one of the one or more additional target-binding domains positioned

between the soluble tissue factor domain and the first domain of the pair of affinity

domains, directly abuts the soluble tissue factor domain and/or the first domain of the

pair of affinity domains.

Embodiment G257. The multi-chain chimeric polypeptide of embodiment G251,

wherein the first chimeric polypeptide further comprises a linker sequence disposed (i)

between the soluble tissue factor domain and the at least one of the one or more

additional target-binding domains positioned between the soluble tissue factor domain

and the first domain of the pair of affinity domains, and/or (ii) between the first domain

of the pair of affinity domains and the at least one of the one or more additional target-

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binding domains positioned between the soluble tissue factor domain and the first domain

of the pair of affinity domains.

Embodiment G258. The multi-chain chimeric polypeptide of any one of

embodiments G44-G46, G57-G59, G76-G78, G96-G98, G109-G111, G122-G124, and

G131-G133, wherein the second chimeric polypeptide further comprises the additional

target-binding domain at the N-terminal end or the C-terminal end of the second chimeric

polypeptide.

Embodiment G259. The multi-chain chimeric polypeptide of embodiment G258,

wherein the additional target-binding domain directly abuts the second domain of the pair

of affinity domains in the second chimeric polypeptide.

Embodiment G260. The multi-chain chimeric polypeptide of embodiment G258,

wherein the second chimeric polypeptide further comprises a linker sequence between the

additional target-binding domain and the second domain of the pair of affinity domains in

the second chimeric polypeptide.

Embodiment G261. The multi-chain chimeric polypeptide of embodiment G258,

wherein the additional target-binding domain directly abuts the second target-binding

domain in the second chimeric polypeptide.

Embodiment G262. The multi-chain chimeric polypeptide of embodiment G258,

wherein the second chimeric polypeptide further comprises a linker sequence between the

additional target-binding domain and the second target-binding domain in the second

chimeric polypeptide.

Embodiment G263. A composition comprising any of the multi-chain chimeric

polypeptides of embodiments G1-G262.

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Embodiment G264. The composition of embodiment G263, wherein the

composition is a pharmaceutical composition.

Embodiment G265. A kit comprising at least one dose of the composition of

embodiment G263 or G264.

Embodiment G266. Nucleic acid encoding any of the multi-chain chimeric

polypeptides of any one of embodiments G1-G262.

Embodiment G267. A vector comprising the nucleic acid of embodiment G266.

Embodiment G268. The vector of embodiment G267, wherein the vector is an

expression vector.

Embodiment G269. A cell comprising the nucleic acid of embodiment G323 or

the vector of embodiment G267 or G268.

Embodiment G270. A method of producing a multi-chain chimeric polypeptide,

the method comprising:

culturing the cell of embodiment G269 in a culture medium under conditions

sufficient to result in the production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or the culture

medium.

Embodiment G271. A multi-chain chimeric polypeptide produced by the method

of embodiment G270.

Embodiment G272. The multi-chain chimeric polypeptide of embodiment G8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 97.

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Embodiment G273. The multi-chain chimeric polypeptide of embodiment G272,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 97.

Embodiment G274. The multi-chain chimeric polypeptide of embodiment G273,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 97.

Embodiment G275. The multi-chain chimeric polypeptide of embodiment G274,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 97.

Embodiment G276. The multi-chain chimeric polypeptide of embodiment G8,

wherein the soluble human tissue factor domain comprises a sequence that is at least 80%

identical to SEQ ID NO: 98.

Embodiment G277. The multi-chain chimeric polypeptide of embodiment G276,

wherein the soluble human tissue factor domain comprises a sequence that is at least 90%

identical to SEQ ID NO: 98.

Embodiment G278. The multi-chain chimeric polypeptide of embodiment G277,

wherein the soluble human tissue factor domain comprises a sequence that is at least 95%

identical to SEQ ID NO: 98.

Embodiment G279. The multi-chain chimeric polypeptide of embodiment G278,

wherein the soluble human tissue factor domain comprises a sequence that is 100%

identical to SEQ ID NO: 98.

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Embodiment H1. A method of treating an aging-related disease or condition in a

subject in need thereof, the method comprising administering to a subject identified as

having an aging-related disease or condition a therapeutically effective amount of one or

more natural killer (NK) cell activating agent(s).

Embodiment H2. A method of killing or reducing the number of senescent cells in

a subject in need thereof, the method comprising administering to the subject a

therapeutically effective amount of one or more NK cell activating agent(s).

Embodiment H3. The method of embodiment H2, wherein the senescent cells are

senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent

astrocytes, senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent

dermal fibroblasts, senescent keratinocytes, or other differentiated tissue-specific dividing

functional cells.

Embodiment H4. The method of embodiment H3, wherein the senescent cancer

cells are chemotherapy-induced senescent cells or radiation-induced senescent cells.

Embodiment H5. The method of embodiment H2, wherein the subject has been

identified or diagnosed as having an aging-related disease or condition.

Embodiment H6. The method of embodiment H1 or H5, wherein the aging-

related disease or condition is selected from the group consisting of: a cancer, an

autoimmune disease, a metabolic disease, a neurodegenerative disease, a cardiovascular

disease, a skin disease, a progeria disease, and a fragility disease.

Embodiment H7. The method of embodiment H6, wherein the cancer is selected

from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,

glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,

osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-

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Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL),

acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic

leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,

retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma,

gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer,

colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and

neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular

carcinoma.

Embodiment H8. The method of embodiment H6, wherein the autoimmune

disease is type-1 diabetes.

Embodiment H9. The method of embodiment H6, wherein the metabolic disease

is selected from the group consisting of: obesity, a lipodystrophy, and type 2 diabetes

mellitus.

Embodiment H10. The method of embodiment H6, wherein the

neurodegenerative disease is selected from the group consisting of: Alzheimer's disease,

Parkinson's disease, and dementia.

Embodiment H11. The method of embodiment H6, wherein the cardiovascular

disease is selected from the group consisting of: coronary artery disease, atherosclerosis,

and pulmonary arterial hypertension.

Embodiment H12. The method of embodiment H6, wherein the skin disease is

selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo,

skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

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Embodiment H13. The method of embodiment H6, wherein the progeria disease

is selected from the group consisting of: progeria and Hutchinson-Gilford Progeria

Syndrome.

Embodiment H14. The method of embodiment H6, wherein the fragility disease is

selected from the group consisting of: frailty, responsiveness to vaccination, osteoporosis,

and sarcopenia.

Embodiment H15. The method of embodiment H1 or H5, wherein the aging-

related disease or condition is selected from the group of: age-related macular

degeneration osteoarthritis, adipose atrophy, chronic obstructive pulmonary disease,

idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,

sarcopenia, age-associated loss of lung tissue elasticity, osteoporosis, age-associated renal

dysfunction, and chemical-induced renal dysfunction.

Embodiment H16. The method of embodiment H1 or H5, wherein the aging-

related disease or condition is type 2 diabetes or atherosclerosis.

Embodiment H17. The method of any one of embodiments H1-H16, wherein the

administering results in a decrease in the number of senescent cells in a target tissue in

the subject.

Embodiment H18. The method of embodiment H17, wherein the target tissue is

selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung

tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin

tissue, muscle tissue, and secondary lympho-organ tissue.

Embodiment H19. The method of any one of embodiments H1-H18, wherein the

administering results in an increase in the expression levels of CD25, CD69, MTOR-C1,

SREBP1, IFN-y, and granzyme B in activated NK cells.

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Embodiment H20. A method of treating an aging-related disease or condition in a

subject in need thereof, the method comprising administering to a subject identified as

having an aging-related disease or condition a therapeutically effective number of

activated NK cells.

Embodiment H21. A method of killing or reducing the number of senescent cells

in a subject in need thereof, the method comprising administering to the subject a

therapeutically effective number of activated NK cells.

Embodiment H22. The method of embodiment H21, wherein the senescent cells

are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent

astrocytes, senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent

dermal fibroblasts, senescent keratinocytes, or other differentiated tissue-specific dividing

functional cells.

Embodiment H23. The method of embodiment H22, wherein the senescent cancer

cells are chemotherapy-induced senescent cells or radiation-induced senescent cells.

Embodiment H24. The method of embodiment H21, wherein the subject has been

identified or diagnosed as having an aging-related disease or condition.

Embodiment H25. The method of embodiment H20 or H24, wherein the aging-

related disease or condition is selected from the group consisting of: a cancer, an

autoimmune disease, a metabolic disease, a neurodegenerative disease, a cardiovascular

disease, a skin disease, a progeria disease, and a fragility disease.

Embodiment H26. The method of embodiment H25, wherein the cancer is

selected from the group consisting of: solid tumor, hematological tumor, sarcoma,

osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing

sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H27. The method of embodiment H25, wherein the autoimmune

disease is type-1 diabetes.

Embodiment H28. The method of embodiment H25, wherein the metabolic

disease is selected from the group consisting of: obesity, a lipodystrophy, and type 2

diabetes mellitus.

Embodiment H29. The method of embodiment H25, wherein the

neurodegenerative disease is selected from the group consisting of: Alzheimer's disease,

Parkinson's disease, and dementia.

Embodiment H30. The method of embodiment H25, wherein the cardiovascular

disease is selected from the group consisting of: coronary artery disease, atherosclerosis,

and pulmonary arterial hypertension.

Embodiment H31. The method of embodiment H25, wherein the skin disease is

selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo,

skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

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Embodiment H32. The method of embodiment H25, wherein the progeria disease

is selected from the group consisting of: progeria and Hutchinson-Gilford Progeria

Syndrome.

Embodiment H33. The method of embodiment H25, wherein the fragility disease

is selected from the group consisting of: frailty, responsiveness to vaccination,

osteoporosis, and sarcopenia.

Embodiment H34. The method of embodiment H20 or H24, wherein the aging-

related disease or condition is selected from the group consisting of: age-related macular

degeneration, osteoarthritis, adipose atrophy, chronic obstructive pulmonary disease,

idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,

sarcopenia, age-associated loss of lung tissue elasticity, osteoporosis, age-associated

renal dysfunction, and chemical-induced renal dysfunction.

Embodiment H35. The method of any one of embodiments H20-H34, wherein the

method further comprises:

obtaining a resting NK cell; and

contacting the resting NK cell in vitro in a liquid culture medium comprising one

or more NK cell activating agent(s), wherein the contacting results in the generation of

the activated NK cells that are subsequently administered to the subject.

Embodiment H36 The method of embodiment H35, wherein the resting NK cell

is an autologous NK cell obtained from the subject.

Embodiment H37. The method of embodiment H35, wherein the resting NK cell

is an allogeneic resting NK cell.

Embodiment H38. The method of embodiment H35, wherein the resting NK cell

is an artificial NK cell.

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Embodiment H39. The method of embodiment H35, wherein the resting NK cell

is a haploidentical resting NK cell.

Embodiment H40. The method of any one of embodiments H35-H39, wherein the

resting NK cell is a genetically-engineered NK cell carrying a chimeric antigen receptor

or recombinant T cell receptor.

Embodiment H41. The method of any one of embodiments H35-H40, wherein the

method further comprises isolating the activated NK cells before the activated NK cells

are administered to the subject.

Embodiment H42. A method of improving the texture and/or appearance of skin

and/or hair in a subject in need thereof over a period of time, the method comprising

administering to the subject a therapeutically effective amount of one or more natural

killer (NK) cell activating agent(s).

Embodiment H43. A method of improving the texture and/or appearance of skin

and/or hair in a subject in need thereof over a period of time, the method comprising

administering to the subject a therapeutically effective number of activated NK cells.

Embodiment H44. The method of embodiment H43, wherein the method further

comprises:

obtaining a resting NK cell; and

contacting the resting NK cell in vitro in a liquid culture medium comprising one

or more NK cell activating agent(s), wherein the contacting results in the generation of

the activated NK cells that are subsequently administered to the subject.

Embodiment H45. The method of embodiment H44, wherein the resting NK cell

is an autologous NK cell obtained from the subject.

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Embodiment H46. The method of embodiment H44, wherein the resting NK cell

is an allogeneic resting NK cell.

Embodiment H47 The method of embodiment H44, wherein the resting NK cell

is an artificial NK cell.

Embodiment H48. The method of embodiment H44, wherein the resting NK cell

is a haploidentical resting NK cell.

Embodiment H49. The method of any one of embodiments H44-H48, wherein the

resting NK cell is a genetically-engineered NK cell carrying a chimeric antigen receptor

or recombinant T cell receptor.

Embodiment H50. The method of any one of embodiments H44-H49, wherein the

method further comprises isolating the activated NK cells before the activated NK cells

are administered to the subject.

Embodiment H51. The method of any one of embodiments H42-H50, wherein the

method provides for an improvement in the texture and/or appearance of skin of the

subject over the period of time.

Embodiment H52. The method of embodiment H51, wherein the method results

in a decrease in the rate of formation of wrinkles in the skin of the subject over the period

of time.

Embodiment H53. The method of embodiment H51 or H52, wherein the method

results in an improvement in the coloration of skin of the subject over the period of time.

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Embodiment H54. The method of any one of embodiments H51-H53, wherein the

method results in an improvement in the texture of skin of the subject over the period of

time.

Embodiment H55. The method of any one of embodiments H42-H50, wherein the

method provides for an improvement in the texture and/or appearance of hair of the

subject over the period of time.

Embodiment H56. The method of embodiment H55, wherein the method results

in a decrease in the rate of formation of gray hair in the subject over the period of time.

Embodiment H57. The method of embodiment H55 or H56, wherein the method

results in a decrease in the number of gray hairs of the subject over the period of time.

Embodiment H58. The method of any one of embodiments H55-H57, wherein the

method results in a decrease in the rate of hair loss in the subject over time.

Embodiment H59. The method of any one of embodiments H55-H58, wherein the

method results in an improvement in the texture of hair of the subject over the period of

time.

Embodiment H60. The method of any one of embodiments H42-H59, wherein the

period of time is between about one month and about 10 years.

Embodiment H61. The method of any one of embodiments H42-H60, wherein the

method results in a decrease in the number of senescent dermal fibroblasts in the skin of

the subject over the period of time.

Embodiment H62. A method of assisting in the treatment of obesity in a subject in

need thereof over a period of time, the method comprising administering to the subject a

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therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s).

Embodiment H63. A method of assisting in the treatment of obesity in a subject in

need thereof over a period of time, the method comprising administering to the subject a

therapeutically effective number of activated NK cells.

Embodiment H64. The method of embodiment H63, wherein the method further

comprises:

obtaining a resting NK cell; and

contacting the resting NK cell in vitro in a liquid culture medium comprising one

or more NK cell activating agent(s), wherein the contacting results in the generation of

the activated NK cells that are subsequently administered to the subject.

Embodiment H65. The method of embodiment H64, wherein the resting NK cell

is an autologous NK cell obtained from the subject.

Embodiment H66. The method of embodiment H64, wherein the resting NK cell

is an allogeneic resting NK cell.

Embodiment H67. The method of embodiment H64, wherein the resting NK cell

is an artificial NK cell.

Embodiment H68. The method of embodiment H64, wherein the resting NK cell

is a haploidentical resting NK cell.

Embodiment H69. The method of any one of embodiments H64-H68, wherein the

resting NK cell is a genetically-engineered NK cell carrying a chimeric antigen receptor

or recombinant T cell receptor.

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Embodiment H70. The method of any one of embodiments H64-H69, wherein the

method further comprises isolating the activated NK cells before the activated NK cells

are administered to the subject.

Embodiment H71. The method of any one of embodiments H62-H70, wherein the

method results in a decrease in the mass of the subject over the period of time.

Embodiment H72. The method of any one of embodiments H62-H71, wherein the

method results in a decrease in the body mass index (BMI) of the subject over the period

of time.

Embodiment H73. The method of any one of embodiments H62-H70, wherein the

method results in a decrease in the rate of progression from pre-diabetes to type 2

diabetes in the subject.

Embodiment H74. The method of any one of embodiments H62-H70, wherein the

method results in a decrease in fasting serum glucose level in the subject.

Embodiment H75. The method of any one of embodiments H62-H70, wherein the

method results in an increase in insulin sensitivity in the subject.

Embodiment H76. The method of any one of embodiments H62-H70, wherein the

method results in a decrease in the severity of atherosclerosis in the subject.

Embodiment H77. The method of any one of embodiments H62-H76, wherein the

period of time is between about two weeks and about 10 years.

Embodiment H78 The method of any one of embodiments H1-H19, H35-H42,

H44-H62, and H64-H77, wherein at least one of the one or more NK cell activating

agent(s) results in activation of one or more of: a receptor for IL-2, a receptor for IL-7, a

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receptor for IL-12, a receptor for IL-15, a receptor for IL-18, a receptor for IL-21, a

receptor for IL-33, CD16, CD69, CD25, CD36, CD59, CD352, NKp80, DNAM-1, 2B4,

NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1.

Embodiment H79. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-2

is a soluble IL-2 or an agonistic antibody that binds specifically to an IL-2 receptor.

Embodiment H80. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-7

is a soluble IL-7 or an agonistic antibody that binds specifically to an IL-7 receptor.

Embodiment H81. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

12 is a soluble IL-12 or an agonistic antibody that binds specifically to an IL-12 receptor.

Embodiment H82. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

15 is a soluble IL-15 or an agonistic antibody that binds specifically to an IL-15 receptor.

Embodiment H83. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

21 is a soluble IL-21 or an agonistic antibody that binds specifically to an IL-21 receptor.

Embodiment H84. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for IL-

33 is a soluble IL-33 or an agonistic antibody that binds specifically to an IL-33 receptor.

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Embodiment H85. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD16 is an agonistic antibody that binds specifically to a CD16.

Embodiment H86. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD69 is an agonistic antibody that binds specifically to a CD69.

Embodiment H87. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD25, CD36, CD59 is an agonistic antibody that binds specifically to a CD25, CD6,

CD59.

Embodiment H88. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

CD352 is an agonistic antibody that binds specifically to a CD352.

Embodiment H89. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp80 is an agonistic antibody that binds specifically to an NKp80.

Embodiment H90. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

DNAM-1 is an agonistic antibody that binds specifically to a DNAM-1.

Embodiment H91. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for 2B4

is an agonistic antibody that binds specifically to a 2B4.

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Embodiment H92. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp30 is an agonistic antibody that binds specifically to an NKp30.

Embodiment H93. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp44 is an agonistic antibody that binds specifically to an NKp44.

Embodiment H94. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKp46 is an agonistic antibody that binds specifically to an NKp46.

Embodiment H95. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

NKG2D is an agonistic antibody that binds specifically to an NKG2D.

Embodiment H96. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS1 is an agonistic antibody that binds specifically to a KIR2DS1.

Embodiment H97. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS2/3 is an agonistic antibody that binds specifically to a KIR2DS2/3.

Embodiment H98. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DL4 is an agonistic antibody that binds specifically to a KIR2DL4.

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Embodiment H99. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS4 is an agonistic antibody that binds specifically to a KIR2DS4.

Embodiment H100. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR2DS5 is an agonistic antibody that binds specifically to a KIR2DS5.

Embodiment H101. The method of embodiment H78, wherein the at least one of

the one or more NK cell activating agent(s) that results in activation of a receptor for

KIR3DS1 is an agonistic antibody that binds specifically to a KIR3DS1.

Embodiment H102. The method of any one of embodiments H1-H19, H35-H42,

H44-H62, and H64-H101, wherein at least one of the one or more NK cell activating

agent(s) results in a decrease in the activation of one or more of: PD-1, a TGF-B receptor,

TIGIT, CD1, TIM-3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/KLRD1, KIR2DL1,

KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2.

Embodiment H103. The method of embodiment H102, wherein the at least one of

the one or more NK cell activating agent(s) that results in a decrease in the activation of

PD-1 is an antagonistic antibody that binds specifically to PD-1, a soluble PD-1, a soluble

PD-L1, or an antibody that binds specifically to PD-L1.

Embodiment H104. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of a

TGF-B receptor is a soluble TGF-B receptor, an antibody that binds specifically to TGF-B,

or an antagonistic antibody that binds specifically to a TGF-B receptor.

Embodiment H105. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

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TIGIT is an antagonistic antibody that binds specifically to TIGIT, a soluble TIGIT, or an

antibody that binds specifically to a ligand of TIGIT.

Embodiment H106. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of CD1

is an antagonistic antibody that binds specifically to CD1, a soluble CD1, or an antibody

that binds specifically to a ligand of CD1.

Embodiment H107. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

TIM-3 is an antagonistic antibody that binds specifically to TIM-3, a soluble TIM-3, or

an antibody that binds specifically to a ligand of TIM-3.

Embodiment H108. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

Siglec-7 is an antagonistic antibody that binds specifically to Siglec-7 or an antibody that

binds specifically to a ligand of Siglec-7.

Embodiment H109. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

IRP60 is an antagonistic antibody that binds specifically to IRP60 or an antibody that

binds specifically to a ligand of IRP60.

Embodiment H110. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

Tactile is an antagonistic antibody that binds specifically to Tactile or an antibody that

binds specifically to a ligand of Tactile.

Embodiment H111. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

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IL1R8 is an antagonistic antibody that binds specifically to IL1R8 or an antibody that

binds specifically to a ligand of IL 1R8

Embodiment H112. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

NKG2A/KLRD1 is an antagonistic antibody that binds specifically to NKG2A/KLRD1

or an antibody that binds specifically to a ligand of NKG2A/KLRD1.

Embodiment H113. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR2DL1 is an antagonistic antibody that binds specifically to KIR2DL1 or an antibody

that binds specifically to a ligand of KIR2DL1.

Embodiment H114. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR2DL2/3 is an antagonistic antibody that binds specifically to KIR2DL2/3 or an

antibody that binds specifically to a ligand of KIR2DL2/3.

Embodiment H115. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR2DL5 is an antagonistic antibody that binds specifically to KIR2DL5 or an antibody

that binds specifically to a ligand of KIR2DL5.

Embodiment H116. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

KIR3DL1 is an antagonistic antibody that binds specifically to KIR3DL1 or an antibody

that binds specifically to a ligand of KIR3DL1.

Embodiment H117. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

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KIR3DL2 is an antagonistic antibody that binds specifically to KIR3DL2 or an antibody

that binds specifically to a ligand of KIR3DL2.

Embodiment H118. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

ILT2/LIR-1 is an antagonistic antibody that binds specifically to ILT2/LIR-1 or an

antibody that binds specifically to a ligand of ILT2/LIR-1.

Embodiment H119. The method of embodiment H102, wherein at least one of the

one or more NK cell activating agent(s) that results in a decrease in the activation of

LAG-2is an antagonistic antibody that binds specifically to LAG-2 or an antibody that

binds specifically to a ligand of LAG-2.

Embodiment H120. The method of any one of embodiments H1-H19, H35-H42,

H44-H62, and H64-H77, wherein at least one of the one or more NK cell activating

agent(s) is a single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain.

Embodiment H121. The method of embodiment H120, wherein the first target-

binding domain and the soluble tissue factor domain directly abut each other.

Embodiment H122. The method of embodiment H120, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between the first target-binding

domain and the soluble tissue factor domain.

Embodiment H123. The method of any one of embodiments H120-H122, wherein

the soluble tissue factor domain and the second target-binding domain directly abut each

other.

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Embodiment H124. The method of any one of embodiments H120-H122, wherein

the single-chain chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the second target-binding domain.

Embodiment H125. The method of embodiment H120, wherein the first target-

binding domain and the second target-binding domain directly abut each other

Embodiment H126. The method of embodiment H120, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between the first target-binding

domain and the second target-binding domain.

Embodiment H127. The method of embodiment H125 or H126, wherein the

second target-binding domain and the soluble tissue factor domain directly abut each

other.

Embodiment H128. The method of embodiment H125 or H126, wherein the

single-chain chimeric polypeptide further comprises a linker sequence between the

second target-binding domain and the soluble tissue factor domain.

Embodiment H129. The method of any one of embodiments H120-H128, wherein

the first target-binding domain and the second target-binding domain bind specifically to

the same antigen.

Embodiment H130. The method of embodiment H129, wherein the first target-

binding domain and the second target-binding domain bind specifically to the same

epitope.

Embodiment H131. The method of embodiment H130, wherein the first target-

binding domain and the second target-binding domain comprise the same amino acid

sequence.

Embodiment H132. The method of any one of embodiments H120-H128, wherein

the first target-binding domain and the second target-binding domain bind specifically to

different antigens.

Embodiment H133. The method of any one of embodiments H120-H132, wherein

one or both of the first target-binding domain and the second target-binding domain is an

antigen-binding domain.

Embodiment H134. The method of embodiment H133, wherein the first target-

binding domain and the second target-binding domain are each an antigen-binding

domain.

Embodiment H135. The method of embodiment H134, wherein antigen-binding

domain comprises a scFv or a single domain antibody.

Embodiment H136. The method of any one of embodiments H120-H135, wherein

one or both of the first target-binding domain and the second target-binding domain bind

to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22,

CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26,

CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR,

HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-

DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a ligand of

DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of

NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor

for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine

kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a

ULP16-binding protein, a receptor for CD155, and a receptor for CD122.

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Embodiment H137. The method of any one of embodiments H120-H128,

wherein one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine protein.

Embodiment H138. The method of embodiment H137, wherein the soluble

interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3,

IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment H139. The method of any one of embodiments H120-H128, wherein

one or both of the first target-binding domain and the second target-binding domain is a

soluble interleukin or cytokine receptor.

Embodiment H140. The method of embodiment H139, wherein the soluble

interleukin or cytokine receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble

TGF-BRIII, a soluble receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor

for IL-10

Embodiment H141. The method of any one of embodiments H120-H140, wherein

the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H142. The method of embodiment H141, wherein the soluble

human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID

NO: 93.

Embodiment H143. The method of embodiment H142, wherein the soluble

human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID

NO: 93.

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Embodiment H144. The method of embodiment H143, wherein the soluble

human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID

NO: 93.

Embodiment H145. The method of any one of embodiments H141-H144, wherein

the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment H146. The method of embodiment H145, wherein the soluble

human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

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an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment H147. The method of any one of embodiments H120-H146, wherein

the soluble tissue factor domain is not capable of binding Factor VIIa.

Embodiment H148. The method of any one of embodiments H120-H147, wherein

the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H149. The method of any one of embodiments H120-H148, wherein

the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H150. The method of any one of embodiments H120-H149, wherein

the single-chain chimeric polypeptide further comprises one or more additional target-

binding domains at its N- and/or C-terminus.

Embodiment H151. The method of embodiment H150, wherein the single-chain

chimeric polypeptide comprises one or more additional target-binding domains at its N-

terminus.

Embodiment H152. The method of embodiment H151, wherein one or more

additional target-binding domains directly abuts the first target-binding domain, the

second target-binding domain, or the soluble tissue factor domain.

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Embodiment H153. The method of embodiment H152, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between one of the at least one

additional target-binding domains and the first target-binding domain, the second target-

binding domain, or the soluble tissue factor domain.

Embodiment H154. The method of embodiment H150, wherein the single-chain

chimeric polypeptide comprises one or more additional target-binding domains at its C-

terminus.

Embodiment H155. The method of embodiment H154, wherein one of the one or

more additional target-binding domains directly abuts the first target-binding domain, the

second target-binding domain, or the soluble tissue factor domain.

Embodiment H156. The method of embodiment H154, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between one of the at least one

additional target-binding domains and the first target-binding domain, the second target-

binding domain, or the soluble tissue factor domain.

Embodiment H157. The method of embodiment H150, wherein the single-chain

chimeric polypeptide comprises one or more additional target binding domains at its N-

terminus and the C-terminus.

Embodiment H158. The method of embodiment H157, wherein one of the one or

more additional antigen binding domains at the N-terminus directly abuts the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment H159. The method of embodiment H157, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between one of the one or more

additional antigen-binding domains at the N-terminus and the first target-binding domain,

the second target-binding domain, or the soluble tissue factor domain.

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Embodiment H160. The method of embodiment H157, wherein one of the one or

more additional antigen binding domains at the C-terminus directly abuts the first target-

binding domain, the second target-binding domain, or the soluble tissue factor domain.

Embodiment H161. The method of embodiment H157, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between one of the one or more

additional antigen-binding domains at the C-terminus and the first target-binding domain,

the second target-binding domain, or the soluble tissue factor domain.

Embodiment H162. The method of any one of embodiments H150-H161, wherein

two or more of the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains bind specifically to the same antigen.

Embodiment H163. The method of embodiment H162, wherein two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same epitope.

Embodiment H164. The method of embodiment H163, wherein two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains comprise the same amino acid sequence.

Embodiment H165. The method of embodiment H162, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each bind specifically to the same antigen.

Embodiment H166. The method of embodiment H165, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each bind specifically to the same epitope.

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Embodiment H167. The method of embodiment H166, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each comprise the same amino acid sequence.

Embodiment H168. The method of any one of embodiments H150-H161, wherein

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to different antigens.

Embodiment H169. The method of any one of embodiments H150-H168, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more target-binding domains is an antigen-binding domain.

Embodiment H170. The method of embodiment H169, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains are each an antigen-binding domain.

Embodiment H171. The method of embodiment H170, wherein antigen-binding

domain comprises a scFv or a single domain antibody.

Embodiment H172. The method of any one of embodiments H150-H171, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more target-binding domains bind specifically to a target selected from the group

consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3,

CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-

1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR,

DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II

(TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of

NKp44, a ligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand for a

scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a receptor for stem cell factor

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(SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for

MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for

CD155, and a receptor for CD122.

Embodiment H173. The method of any one of embodiments H150-H161, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment H174. The method of embodiment H173, wherein the soluble

interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3,

IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment H175. The method of any one of embodiments H150-H161, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains is a soluble interleukin or cytokine

receptor.

Embodiment H176. The method of embodiment H175, wherein the soluble

receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble

receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment H177. The method of any one of embodiments H1-H19, H35-H42,

H44-H62, and H64-H77, wherein at least one of the one or more NK cell activating

agent(s) is a multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide

associate through the binding of the first domain and the second domain of the pair of

affinity domains.

Embodiment H178. The method of embodiment H177, wherein the first target-

binding domain and the soluble tissue factor domain directly abut each other in the first

chimeric polypeptide.

Embodiment H179. The method of embodiment H177, wherein the first chimeric

polypeptide further comprises a linker sequence between the first target-binding domain

and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment H180. The method of any one of embodiments H177-H179, wherein

the soluble tissue factor domain and the first domain of the pair of affinity domains

directly abut each other in the first chimeric polypeptide.

Embodiment H181. The method of any one of embodiments H177-H179, wherein

the first chimeric polypeptide further comprises a linker sequence between the soluble

tissue factor domain and the first domain of the pair of affinity domains in the first

chimeric polypeptide.

Embodiment H182. The method of any one of embodiments H177-H181, wherein

the second domain of the pair of affinity domains and the second target-binding domain

directly abut each other in the second chimeric polypeptide.

Embodiment H183. The method of any one of embodiments H177-H181, wherein

second chimeric polypeptide further comprises a linker sequence between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

Embodiment H184. The method of any one of embodiments H177-H183, wherein

the first target-binding domain and the second target-binding domain bind specifically to

the same antigen.

Embodiment H185. The method of embodiment H184, wherein the first target-

binding domain and the second target-binding domain bind specifically to the same

epitope.

Embodiment H186. The method of embodiment H185, wherein the first target-

binding domain and the second target-binding domain comprise the same amino acid

sequence.

Embodiment H187. The method of any one of embodiments H177-H183, wherein

the first target-binding domain and the second target-binding domain bind specifically to

different antigens.

Embodiment H188. The method of any one of embodiments H177-H187, wherein

one or both of the first target-binding domain and the second target-binding domain is an

antigen-binding domain.

Embodiment H189. The method of embodiment H188, wherein the first target-

binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment H190. The method of embodiment H188 or H189, wherein antigen-

binding domain comprises a scFv or a single domain antibody.

Embodiment H191. The method of any one of embodiments H177-H190, wherein

one or both of the first target-binding domain and the second target-binding domain bind

specifically to a target selected from the group consisting of: CD16a, CD33, CD20,

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CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,

TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,

CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,

CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a

ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand

of NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a

receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like

tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor

for a ULP16-binding protein, a receptor for CD155, and a receptor for CD122.

Embodiment H192. The method of any one of embodiments H177-H183,

wherein one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine protein.

Embodiment H193. The method of embodiment H192, wherein the soluble

interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3,

IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment H194. The method of any one of embodiments H177-H183, wherein

one or both of the first target-binding domain and the second target-binding domain is a

soluble interleukin or cytokine receptor.

Embodiment H195. The method of embodiment H194, wherein the soluble

receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble

receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment H196. The method of any one of embodiments H177-H195, wherein

the first chimeric polypeptide further comprises one or more additional target-binding

domain(s), where at least one of the one or more additional antigen-binding domain(s) is

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positioned between the soluble tissue factor domain and the first domain of the pair of

affinity domains.

Embodiment H197. The method of embodiment H196, wherein the first chimeric

polypeptide further comprises a linker sequence between the soluble tissue factor domain

and the at least one of the one or more additional antigen-binding domain(s), and/or a

linker sequence between the at least one of the one or more additional antigen-binding

domain(s) and the first domain of the pair of affinity domains.

Embodiment H198. The method of any one of embodiments H177-H195, wherein

the first chimeric polypeptide further comprises one or more additional target-binding

domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment H199. The method of embodiment H198, wherein at least one of the

one or more additional target-binding domains directly abuts the first domain of the pair

of affinity domains in the first chimeric polypeptide.

Embodiment H200. The method of embodiment H198, wherein the first chimeric

polypeptide further comprises a linker sequence between the at least one of the one or

more additional target-binding domains and the first domain of the pair of affinity

domains.

Embodiment H201. The method of embodiment H198, wherein the at least one of

the one or more additional target-binding domains directly abuts the first target-binding

domain in the first chimeric polypeptide.

Embodiment H202. The method of embodiment H198, wherein the first chimeric

polypeptide further comprises a linker sequence between the at least one of the one or

more additional target-binding domains and the first target-binding domain.

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Embodiment H203. The method of embodiment H198, wherein at least one of the

one or more additional target-binding domains is disposed at the N- and/or C-terminus of

the first chimeric polypeptide, and at least one of the one or more additional target-

binding domains is positioned between the soluble tissue factor domain and the first

domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H204. The method of embodiment H203, wherein the at least one

additional target-binding domain of the one or more additional target-binding domains

disposed at the N-terminus directly abuts the first target-binding domain or the first

domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H205. The method of embodiment H203, wherein the first chimeric

polypeptide further comprises a linker sequence disposed between the at least one

additional target-binding domain and the first target-binding domain or the first domain

of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H206. The method of embodiment H203, wherein the at least one

additional target-binding domain of the one or more additional target-binding domains

disposed at the C-terminus directly abuts the first target-binding domain or the first

domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H207. The method of embodiment H203, wherein the first chimeric

polypeptide further comprises a linker sequence disposed between the at least one

additional target-binding domain and the first target-binding domain or the first domain

of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H208. The method of embodiment H203, wherein the at least one of

the one or more additional target-binding domains positioned between the soluble tissue

factor domain and the first domain of the pair of affinity domains, directly abuts the

soluble tissue factor domain and/or the first domain of the pair of affinity domains.

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Embodiment H209. The method of embodiment H203, wherein the first chimeric

polypeptide further comprises a linker sequence disposed (i) between the soluble tissue

factor domain and the at least one of the one or more additional target-binding domains

positioned between the soluble tissue factor domain and the first domain of the pair of

affinity domains, and/or (ii) between the first domain of the pair of affinity domains and

the at least one of the one or more additional target-binding domains positioned between

the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment H210. The method of any one of embodiments H177-H209, wherein

the second chimeric polypeptide further comprises one or more additional target-binding

domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment H211. The method of embodiment H210, wherein at least one of the

one or more additional target-binding domains directly abuts the second domain of the

pair of affinity domains in the second chimeric polypeptide.

Embodiment H212. The method of embodiment H210, wherein the second

chimeric polypeptide further comprises a linker sequence between at least one of the one

or more additional target-binding domains and the second domain of the pair of affinity

domains in the second chimeric polypeptide.

Embodiment H213. The method of embodiment H210, wherein at least one of the

one or more additional target-binding domains directly abuts the second target-binding

domain in the second chimeric polypeptide.

Embodiment H214. The method of embodiment H210, wherein the second

chimeric polypeptide further comprises a linker sequence between at least one of the one

or more additional target-binding domains and the second target-binding domain in the

second chimeric polypeptide.

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Embodiment H215. The method of any one of embodiments H196-H214, wherein

two or more of the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains bind specifically to the same antigen.

Embodiment H216. The method of embodiment H215, wherein two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to the same epitope.

Embodiment H217. The method of embodiment H216, wherein two or more of

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains comprise the same amino acid sequence.

Embodiment H218. The method of embodiment H215, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each bind specifically to the same antigen.

Embodiment H219. The method of embodiment H218, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each bind specifically to the same epitope.

Embodiment H220. The method of embodiment H219, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains each comprise the same amino acid sequence.

Embodiment H221. The method of any one of embodiments H196-H214, wherein

the first target-binding domain, the second target-binding domain, and the one or more

additional target-binding domains bind specifically to different antigens.

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Embodiment H222. The method of any one of embodiments H196-H221, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more target-binding domains is an antigen-binding domain.

Embodiment H223. The method of embodiment H222, wherein the first target-

binding domain, the second target-binding domain, and the one or more additional target-

binding domains are each an antigen-binding domain.

Embodiment H224. The method of embodiment H223, wherein antigen-binding

domain comprises a scFv.

Embodiment H225. The method of any one of embodiments H196-H224, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more target-binding domains bind specifically to a target selected from the group

consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3,

CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-

1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR,

DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-B receptor II

(TGF-BRII), a ligand of TGF-BRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of

NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a

scMHCII, a ligand for a scTCR, a receptor for PDGF-DD, a receptor for stem cell factor

(SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for

MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for

CD155, and a receptor for CD122.

Embodiment H226. The method of any one of embodiments H196-H214,

wherein one or more of the first target-binding domain, the second target-binding

domain, and the one or more additional target-binding domains is a soluble interleukin or

cytokine protein.

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Embodiment H227. The method of embodiment H226, wherein the soluble

interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3,

IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment H228. The method of any one of embodiments H196-H214, wherein

one or more of the first target-binding domain, the second target-binding domain, and the

one or more additional target-binding domains is a soluble interleukin or cytokine

receptor.

Embodiment H229. The method of embodiment H228, wherein the soluble

receptor a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor

for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment H230, The method of any one of embodiments H196-H229, wherein

the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H231. The method of embodiment H230, wherein the soluble

human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID

NO: 93.

Embodiment H232. The method of embodiment H231, wherein the soluble

human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID

NO: 93.

Embodiment H233. The method of embodiment H232, wherein the soluble

human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID

NO: 93.

Embodiment H234. The method of any one of embodiments H230-H233, wherein

the soluble human tissue factor domain does not comprise one or more of:

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a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment H235. The method of embodiment H234, wherein the soluble

human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of

mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22

of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45

of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position

58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of

mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135

of mature wildtype human tissue factor protein; and

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a phenylalanine at an amino acid position that corresponds to amino acid position

140 of mature wildtype human tissue factor protein.

Embodiment H236. The method of any one of embodiments H196-H235, wherein

the soluble tissue factor domain is not capable of binding to Factor VIIa.

Embodiment H237. The method of any one of embodiments H196-H236, wherein

the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment H238. The method of any one of embodiments H196-H237, wherein

the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment H239. The method of any one of embodiments H196-H238, wherein

the pair of affinity domains is a sushi domain from an alpha chain of human IL-15

receptor (IL-15Ra) and a soluble IL-15.

Embodiment H240. The method of embodiment H239, wherein the soluble IL-15

has a D8N or D8A amino acid substitution.

Embodiment H241. The method of embodiment H239 or H240, wherein the

human IL-15Ra is a mature full-length IL-15Ra.

Embodiment H242. The method of any one of embodiments H196-H238,

wherein the pair of affinity domains is selected from the group consisting of: barnase and

barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I

fragments, and SNARE modules based on interactions of the proteins syntaxin,

synaptotagmin, synaptobrevin, and SNAP25.

954

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Embodiment H243. The method of any one of embodiments H1-H19, H35-H42,

H44-H62, and H64-H77, wherein at least one of the one or more NK cell activating

agent(s) is a multi-chain chimeric polypeptide comprising:

(a) a first and second chimeric polypeptides, wherein each comprises:

(i) a first target-binding domain;

(ii) a Fc domain; and

(iii) a first domain of a pair of affinity domains;

(b) a third and fourth chimeric polypeptide, wherein each comprises:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first and second chimeric polypeptides and the third and fourth

chimeric polypeptides associate through the binding of the first domain and the second

domain of the pair of affinity domains, and the first and second chimeric polypeptides

associate through their Fc domains.

Embodiment H244. The method of embodiment H243, wherein the first target-

binding domain and the Fc domain directly abut each other in the first and second

chimeric polypeptides.

Embodiment H245. The method of embodiment H243, wherein the first and

second chimeric polypeptides further comprise a linker sequence between the first target-

binding domain and the Fc domain in the first and second chimeric polypeptides.

Embodiment H246. The method of any one of embodiments H243-H245, wherein

the Fc domain and the first domain of the pair of affinity domains directly abut each other

in the first and second chimeric polypeptides.

Embodiment H247. The method of any one of embodiments H243-H245, wherein

the first chimeric polypeptide further comprises a linker sequence between the Fc domain and the first domain of the pair of affinity domains in the first and second chimeric polypeptides.

Embodiment H248. The method of any one of embodiments H243-H247, wherein

the second domain of the pair of affinity domains and the second target-binding domain

directly abut each other in the third and fourth chimeric polypeptides.

Embodiment H249. The method of any one of embodiments H243-H247, wherein

third and fourth chimeric polypeptides further comprise a linker sequence between the

second domain of the pair of affinity domains and the second target-binding domain in

the third and fourth chimeric polypeptides.

Embodiment H250. The method of any one of embodiments H243-H249, wherein

the first target-binding domain and the second target-binding domain bind specifically to

the same antigen.

Embodiment H251. The method of embodiment H250, wherein the first target-

binding domain and the second target-binding domain bind specifically to the same

epitope.

Embodiment H252. The method of embodiment H251, wherein the first target-

binding domain and the second target-binding domain comprise the same amino acid

sequence.

Embodiment H253. The method of any one of embodiments H243-H249, wherein

the first target-binding domain and the second target-binding domain bind specifically to

different antigens.

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Embodiment H254. The method of any one of embodiments H243-H253, wherein

one or both of the first target-binding domain and the second target-binding domain is an

antigen-binding domain.

Embodiment H255. The method of embodiment H254, wherein the first target-

binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment H256. The method of embodiment H254 or H255, wherein antigen-

binding domain comprises a scFv or a single domain antibody.

Embodiment H257. The method of any one of embodiments H243-H256, wherein

one or both of the first target-binding domain and the second target-binding domain bind

specifically to a target selected from the group consisting of: CD16a, CD33, CD20,

CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,

TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,

CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,

CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a

ligand of DNAMI, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand

of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a

receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like

tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor

for a ULP16-binding protein, a receptor for CD155, and a receptor for CD122.

Embodiment H258. The method of any one of embodiments H243-H256,

wherein one or both of the first target-binding domain and the second target-binding

domain is a soluble interleukin or cytokine protein.

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Embodiment H259. The method of embodiment H258, wherein the soluble

interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3,

IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment H260. The method of any one of embodiments H243-H256, wherein

one or both of the first target-binding domain and the second target-binding domain is a

soluble interleukin or cytokine receptor.

Embodiment H261. The method of embodiment H260, wherein the soluble

receptor is a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble

receptor for TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

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Embodiment Il. A method of treating an aging-related disease or condition in a

subject in need thereof, the method comprising administering to a subject identified as

having an aging-related disease or condition a therapeutically effective amount of one or

more natural killer (NK) cell activating agent(s).

Embodiment 12. A method of killing or reducing the number of senescent cells in

a subject in need thereof, the method comprising administering to the subject a

therapeutically effective amount of one or more NK cell activating agent(s).

Embodiment I3. The method of embodiment Il or I2, wherein the administering

results in a decrease in the number of senescent cells in a target tissue in the subject.

Embodiment I4. The method of embodiment I3, wherein the target tissue is

selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung

tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin

tissue, muscle tissue, and secondary lympho-organ tissue.

Embodiment I5. A method of treating an aging-related disease or condition in a

subject in need thereof, the method comprising administering to a subject identified as

having an aging-related disease or condition a therapeutically effective number of

activated NK cells.

Embodiment I6. A method of killing or reducing the number of senescent cells in

a subject in need thereof, the method comprising administering to the subject a

therapeutically effective number of activated NK cells.

Embodiment I7. The method of any one of embodiments I1-16, wherein the

subject has been identified or diagnosed as having an aging-related disease or condition.

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Embodiment I8. The method of embodiment I7, wherein the aging-related disease

or condition is selected from the group consisting of: a cancer, an autoimmune disease, a

metabolic disease, a neurodegenerative disease, a cardiovascular disease, a skin disease, a

progeria disease, and a fragility disease.

Embodiment I9. The method of embodiment I8, wherein the cancer is selected

from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,

glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,

osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-

Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL),

acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic

leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,

retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma,

gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal

cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck

carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular

carcinoma.

Embodiment I10. The method of embodiment I8, wherein the autoimmune

disease is type-1 diabetes.

Embodiment I11. The method of embodiment I8, wherein the metabolic disease is

selected from the group consisting of: obesity, a lipodystrophy, and type 2 diabetes

mellitus.

Embodiment I12. The method of embodiment I8, wherein the neurodegenerative

disease is selected from the group consisting of: Alzheimer's disease, Parkinson's

disease, amyotrophic lateral sclerosis, and dementia.

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Embodiment I13. The method of embodiment 8, wherein the cardiovascular

disease is selected from the group consisting of: coronary artery disease, atherosclerosis,

and pulmonary arterial hypertension.

Embodiment I14. The method of embodiment I8, wherein the skin disease is

selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo,

skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Embodiment I15. The method of embodiment I8, wherein the progeria disease is

selected from the group consisting of: progeria and Hutchinson-Gilford Progeria

Syndrome.

Embodiment I16. The method of embodiment I8, wherein the fragility disease is

selected from the group consisting of: frailty, responsiveness to vaccination, osteoporosis,

and sarcopenia.

Embodiment I17. The method of any one of embodiments I1-16, wherein the

aging-related disease or condition is selected from the group consisting of: osteoarthritis,

adipose atrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis,

kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-associated loss

of lung tissue elasticity, osteoporosis, age-associated renal dysfunction, and chemical-

induced renal dysfunction.

Embodiments I18. The method of any one of embodiments I1-I6, wherein the

aging-related disease or condition is type 2 diabetes or atherosclerosis.

Embodiments I19. A method of improving the texture and/or appearance of skin

and/or hair in a subject in need thereof over a period of time, the method comprising

administering to the subject a therapeutically effective amount of one or more natural

killer (NK) cell activating agent(s).

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Embodiment I20. A method of improving the texture and/or appearance of skin

and/or hair in a subject in need thereof over a period of time, the method comprising

administering to the subject a therapeutically effective number of activated NK cells.

Embodiment I21. The method of embodiment I19 or I20, wherein the method

provides for an improvement in the texture and/or appearance of skin of the subject over

the period of time.

Embodiment I22. The method of embodiment I21, wherein the method results in a

decrease in the rate of formation of wrinkles in the skin of the subject over the period of

time. time.

Embodiment I23. The method of embodiment I21 or I22, wherein the method

results in an improvement in the coloration of skin of the subject over the period of time.

Embodiment I24. The method of any one of embodiments I21-I23, wherein the

method results in an improvement in the texture of skin of the subject over the period of

time.

Embodiment I25. The method of any one of embodiments I20-124, wherein the

method provides for an improvement in the texture and/or appearance of hair of the

subject over the period of time.

Embodiment I26. The method of embodiment I25, wherein the method results in a

decrease in the rate of formation of gray hair in the subject over the period of time.

Embodiment I27. The method of embodiment I25 or I26, wherein the method

results in a decrease in the number of gray hairs of the subject over the period of time.

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Embodiment I28. The method of any one of embodiments I25-127, wherein the

method results in a decrease in the rate of hair loss in the subject over time.

Embodiment I29. The method of any one of embodiments I25-128, wherein the

method results in an improvement in the texture of hair of the subject over the period of

time.

Embodiment I30. The method of any one of embodiments I19-129, wherein the

method results in a decrease in the number of senescent dermal fibroblasts in the skin of

the subject over the period of time.

Embodiment I31. A method of assisting in the treatment of obesity in a subject in

need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of one or more natural killer (NK) cell activating

agent(s).

Embodiment I32. A method of assisting in the treatment of obesity in a subject in

need thereof over a period of time, the method comprising administering to the subject a

therapeutically effective number of activated NK cells.

Embodiment I33. The method of any one of embodiments I1-I32, wherein the

method further comprises:

obtaining a resting NK cell; and

contacting the resting NK cell in vitro in a liquid culture medium comprising one

or more NK cell activating agent(s), wherein the contacting results in the generation of

the activated NK cells that are subsequently administered to the subject.

Embodiment I34. The method of embodiment I33, wherein the resting NK cell is

a genetically-engineered NK cell carrying a chimeric antigen receptor or recombinant T

cell receptor.

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Embodiment I35, The method of embodiment I33, wherein the method further

comprises introducing a nucleic acid that encodes a chimeric antigen receptor or a

recombinant T cell receptor into the resting NK cell or the activated NK cell prior to

administration to the subject.

Embodiment I36. The method of any one of embodiments I31-I35, wherein the

method results in a decrease in the mass of the subject over the period of time.

Embodiment I37. The method of any one of embodiments I31-I36, wherein the

method results in a decrease in the body mass index (BMI) of the subject over the period

of time.

Embodiment 138. The method of any one of embodiments I31-I35, wherein the

method results in a decrease in the rate of progression from pre-diabetes to type 2

diabetes in the subject.

Embodiment I39. The method of any one of embodiments I31-I35, wherein the

method results in a decrease in fasting serum glucose level in the subject.

Embodiment I40. The method of any one of embodiments I31-I35, wherein the

method results in an increase in insulin sensitivity in the subject.

Embodiment I41. The method of any one of embodiments I31-I35, wherein the

method results in a decrease in the severity of atherosclerosis in the subject.

Embodiment I42. The method of any one of embodiments I1-141, wherein at least

one of the one or more NK cell activating agent(s) results in activation of one or more of:

a receptor for IL-2, a receptor for IL-7, a receptor for IL-12, a receptor for IL-15, a

receptor for IL-18, a receptor for IL-21, a receptor for IL-33, CD16, CD69, CD25, CD36,

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CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1.

Embodiment I43. The method of any one of embodiments I1-142, wherein at least

one of the one or more NK cell activating agent(s) results in a decrease in the activation

of one or more of: PD-1, a TGF-B receptor, TIGIT, CD1, TIM-3, Siglec-7, IRP60,

Tactile, IL1R8, NKG2A/KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1,

KIR3DL2, ILT2/LIR-1, and LAG-2.

Embodiment I44. The method of any one of embodiments I1-141, wherein at least

one of the one or more NK cell activating agent(s) is a single-chain chimeric polypeptide

comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain.

Embodiment I45. The method of embodiment I44, wherein the first target-binding

domain and the soluble tissue factor domain directly abut each other.

Embodiment I46. The method of embodiment I44, wherein the single-chain

chimeric polypeptide further comprises a linker sequence between the first target-binding

domain and the soluble tissue factor domain.

Embodiment I47. The method of any one of embodiments I44-146, wherein the

soluble tissue factor domain and the second target-binding domain directly abut each

other.

Embodiment I48. The method of any one of embodiments I44-146, wherein the

single-chain chimeric polypeptide further comprises a linker sequence between the

soluble tissue factor domain and the second target-binding domain.

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Embodiment I49. The method of any one of embodiments I1-141, wherein at least

one of the one or more NK cell activating agent(s) is a multi-chain chimeric polypeptide

comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide

associate through the binding of the first domain and the second domain of the pair of

affinity domains.

Embodiment I50. The method of embodiment I49, wherein the first target-binding

domain and the soluble tissue factor domain directly abut each other in the first chimeric

polypeptide.

Embodiment I51. The method of embodiment I49, wherein the first chimeric

polypeptide further comprises a linker sequence between the first target-binding domain

and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment I52. The method of any one of embodiments I49-I51, wherein the

soluble tissue factor domain and the first domain of the pair of affinity domains directly

abut each other in the first chimeric polypeptide.

Embodiment I53. The method of any one of embodiments I49-I51, wherein the

first chimeric polypeptide further comprises a linker sequence between the soluble tissue

factor domain and the first domain of the pair of affinity domains in the first chimeric

polypeptide.

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Embodiment I54. The method of any one of embodiments I49-153, wherein the

second domain of the pair of affinity domains and the second target-binding domain

directly abut each other in the second chimeric polypeptide.

Embodiment I55. The method of any one of embodiments 149-I53, wherein

second chimeric polypeptide further comprises a linker sequence between the second

domain of the pair of affinity domains and the second target-binding domain in the

second chimeric polypeptide.

Embodiment I56. The method of any one of embodiments I1-141, wherein at least

one of the one or more NK cell activating agent(s) is a multi-chain chimeric polypeptide

comprising:

(a) a first and second chimeric polypeptides, wherein each comprises:

(i) a first target-binding domain;

(ii) a Fc domain; and

(iii) a first domain of a pair of affinity domains;

(b) a third and fourth chimeric polypeptide, wherein each comprises:

(i) a second domain of a pair of affinity domains; and

(ii) a second target-binding domain,

wherein the first and second chimeric polypeptides and the third and fourth

chimeric polypeptides associate through the binding of the first domain and the second

domain of the pair of affinity domains, and the first and second chimeric polypeptides

associate through their Fc domains.

Embodiment I57. The method of any one of embodiments I44-156, wherein one or

both of the first target-binding domain and the second target-binding domain bind

specifically to a target selected from the group consisting of: CD16a, CD33, CD20,

CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,

TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,

CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,

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CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,

CD155, PDGF-DD, a ligand of TGF-B receptor II (TGF-BRII), a ligand of TGF-BRIII, a

ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand

of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a

receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like

tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor

for a ULP16-binding protein, a receptor for CD155, and a receptor for CD122.

Embodiment I58. The method of any one of embodiments I44-156, wherein one or

both of the first target-binding domain and the second target-binding domain is a soluble

interleukin or cytokine protein.

Embodiment I59. The method of embodiment I58, wherein the soluble interleukin

or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8,

IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment I60. The method of any one of embodiments I44-I56, wherein one or

both of the first target-binding domain and the second target-binding domain is a soluble

interleukin or cytokine receptor.

Embodiment I61. The method of embodiment I60, wherein the soluble receptor is

a soluble TGF-B receptor II (TGF-BRII) a soluble TGF-BRIII, a soluble receptor for

TNFa, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment I62. The method of any one of embodiments I44-I55, wherein the

soluble tissue factor domain is a soluble human tissue factor domain that does not

stimulate blood coagulation.

Embodiment I63. The method of any one of embodiments I43-155, wherein the

soluble tissue factor domain comprises or consists of a sequence from a wild-type soluble

human tissue factor.

Claims (100)

WHAT IS CLAIMED IS: 24 Feb 2026

1. A method of killing or reducing the number of naturally occurring senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agent(s) that specifically bind(s) TGF-β and thereby result(s) in a decrease in the plasma levels of TGF-β, wherein the agent is a single-chain or multi-chain fusion protein comprised of at least one target-binding domain that is at least 90% identical to SEQ ID NO: 183. 2021283199

2. A method of decreasing the accumulation of naturally occurring senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agent(s) that specifically bind(s) TGF-β and thereby result(s) in a decrease in the plasma levels of TGF-β, wherein the agent is a single-chain or multi-chain fusion protein comprised of at least one target-binding domain that is at least 90% identical to SEQ ID NO: 183.

3. A method of decreasing a level of a marker of naturally occurring senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agent(s) that specifically bind(s) TGF-β and thereby result(s) in a decrease in the plasma levels of TGF-β, wherein the agent is a single-chain or multi-chain fusion protein comprised of at least one target-binding domain that is at least 90% identical to SEQ ID NO: 183.

4. A method of reducing the activity of naturally occurring senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agent(s) that specifically bind(s) TGF-β and thereby result(s) in a decrease in the plasma levels of TGF-β, wherein the agent is a single-chain or multi-chain fusion protein comprised of at least one target-binding domain that is at least 90% identical to SEQ ID NO: 183.

5. A method of decreasing levels and/or activity of one or more SASP factor(s) derived from naturally occurring senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agent(s) that specifically bind(s) TGF-β and thereby result(s) in a decrease in the plasma levels of TGF-β, wherein the agent is a single-chain or multi-chain fusion protein comprised of at least one 24 Feb 2026 target-binding domain that is at least 90% identical to SEQ ID NO: 183.

6. The method of any one of claims 1-5, wherein the subject has been previously diagnosed or identified as having an aging-related disease or an inflammatory disease.

7. The method of claim 5, wherein the aging-related disease is selected from the group 2021283199

consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, intervertebral disc degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, Parkinson’s disease, age-associated loss of lung tissue elasticity, age-related macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

8. The method of claim 5, wherein the aging-related disease is a cancer selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

9. The method of claim 5, wherein the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Crohn’s disease, multiple sclerosis, Guillain-Barre syndrome, 24 Feb 2026 psoriasis, Grave’s disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders and cancer treatment-related cognitive impairment.

10. The method of any one of claims 1-9, wherein the administration of the one or more agent(s) that specifically bind(s) TGF-β results in a decrease in the number or activity of naturally occurring senescent cells in a target tissue in the subject. 2021283199

11. The method of claim 10, wherein the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lympho-organ tissue.

12. The method of any one of claims 1-11, wherein the one or more agent(s) that specifically bind(s) TGF-β is a TGF-β receptor II (TGF-βRII).

13. The method of any one of claims 1-11, wherein the TGF-β receptor is a TGF- βRIII.

14. The method of any one of claims 1-13, wherein at least one of the one or more agent(s) that specifically bind(s) TGF-β is a soluble TGF-β receptor, an extracellular domain of TGF-β receptor, an antibody that binds specifically to TGF-β, an antagonistic antibody that binds to a TGF-β receptor, an agent that binds to a LAP, or an agent that binds to a TGF- β/LAP complex.

15. The method of claim 14, wherein the one or more agent(s) that specifically bind(s) TGF-β decrease(s) the activation of a TGF-β receptor through binding to a LAP, or to a TGF- β/LAP complex.

16. The method of any one of claims 1-13, wherein at least one of the one or more agent(s) that specifically bind(s) TGF-β is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain;

(ii) a soluble tissue factor domain; and 24 Feb 2026

(iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein one or both of the first target-binding domain and the second target-binding domain binds specifically to a ligand of a TGF-β receptor; or 2021283199

one or both of the first target-binding domain and the second target-binding domain is an antagonistic antigen-binding domain that binds specifically to a TGF-β receptor.

17. The method of claim 16, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

18. The method of claim 16, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

19. The method of any one of claims 16-18, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

20. The method of any one of claims 16-18, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

21. The method of any one of claims 16-20, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

22. The method of any one of claims 16-20, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

23. The method of any one of claims 16-22, wherein the first target-binding domain 24 Feb 2026

and the second target-binding domain bind specifically to the same antigen.

24. The method of any one of claims 16-22, wherein the first target-binding domain and the second target-binding domain bind specifically to different antigens.

25. The method of any one of claims 16-24, wherein the first chimeric polypeptide 2021283199

further comprises one or more additional target-binding domain(s).

26. The method of any one of claims 16-25, wherein the second chimeric polypeptide further comprises one or more additional target-binding domain(s).

27. The method of any one of claims 16-26, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

28. The method of claim 27, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 93.

29. The method of any one of claims 16-28, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

30. The method of claim 29, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

31. The method of any one of claims 29-30, wherein the soluble IL-15 comprises a mutation to reduce or eliminate IL-15 activity.

32. The method of any one of claims 16-28, wherein the pair of affinity domains is selected from the group consisting of: barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

33. The method of any one of claims 16-28, wherein the first domain or the second 24 Feb 2026

domain of a pair of affinity domains is a soluble common gamma-chain family cytokine or an antigen-binding domain that binds specifically to a common gamma-chain family cytokine receptor.

34. The method of any one of claims 16-33, wherein the first target-binding domain and/or the second target-binding domain comprise a soluble TGF-β receptor. 2021283199

35. The method of claim 34, wherein the soluble TGF-β receptor is a soluble TGF- βRII.

36. The method of claim 35, wherein the soluble TGF-βRII comprises a first sequence that is at least 80% identical to SEQ ID NO: 183, and a second sequence that is at least 80% identical to SEQ ID NO: 183, wherein the first and second sequence are separated by a linker.

37. The method of claim 36, wherein the soluble TGF-βRII comprises a first sequence that is at least 90% identical to SEQ ID NO: 183, and a second sequence that is at least 90% identical to SEQ ID NO: 183.

38. The method of claim 37, wherein the soluble TGF-βRII comprises a first sequence of SEQ ID NO: 183, and a second sequence of SEQ ID NO: 183.

39. The method of claim 36, wherein the linker comprises a sequence of SEQ ID NO: 102.

40. The method of claim 35, wherein the soluble TGF-βRII comprises a sequence that is at least 80% identical to SEQ ID NO: 188.

41. The method of claim 40, wherein the soluble TGF-βRII comprises a sequence that is at least 90% identical to SEQ ID NO: 188.

42. The method of claim 41, wherein the soluble TGF-βRII comprises a sequence of SEQ ID NO: 188.

43. The method of claim 16, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 236.

44. The method of claim 43, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 236. 2021283199

45. The method of claim 44, wherein the first chimeric polypeptide comprises a sequence of SEQ ID NO: 236.

46. The method of claim 16, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 193.

47. The method of claim 46, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 236.

48. The method of claim 47, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 193.

49. The method of claim 48, wherein the second chimeric polypeptide comprises a sequence of SEQ ID NO: 193.

50. The method of claim 49, wherein the first chimeric polypeptide comprises a sequence of SEQ ID NO: 236.

51. The method of any one of claims 1-13, wherein at least one of the one or more agent(s) that specifically bind(s) TGF-β is a single-chain chimeric polypeptide comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a second target-binding domain, wherein one or both of the first target-binding domain and the second target-binding domain binds specifically to TGF-β.

52. The method of claim 51, wherein the first target-binding domain and the soluble 24 Feb 2026

tissue factor domain directly abut each other.

53. The method of claim 51, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain. 2021283199

54. The method of any one of claims 51-53, wherein the soluble tissue factor domain and the second target-binding domain directly abut each other.

55. The method of any one of claims 51-53, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target-binding domain.

56. The method of any one of claims 51-55, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

57. The method of claim 56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 93.

58. The method of any one of claims 51-57, wherein the single-chain chimeric polypeptide further comprises one or more additional target-binding domains at its N- and/or C-terminus.

59. The method of any one of claims 51-58, wherein the first target-binding domain and/or the second target-binding domain comprise a soluble TGF-β receptor.

60. The method of claim 59, wherein the soluble TGF-β receptor is a soluble TGF- βRII.

61. The method of claim 60, wherein the soluble TGF-βRII comprises a first sequence that is at least 80% identical to SEQ ID NO: 183, and a second sequence that is at least 80% identical to SEQ ID NO: 183, wherein the first and second sequence are separated by a linker.

62. The method of claim 61, wherein the soluble TGF-βRII comprises a first sequence that is at least 90% identical to SEQ ID NO: 183, and a second sequence that is at least 90% identical to SEQ ID NO: 183.

63. The method of claim 62, wherein the soluble TGF-βRII comprises a first sequence of SEQ ID NO: 183, and a second sequence of SEQ ID NO: 183. 2021283199

64. The method of claim 61, wherein the linker comprises a sequence of SEQ ID NO: 102.

65. The method of claim 64, wherein the soluble TGF-βRII comprises a sequence that is at least 80% identical to SEQ ID NO: 188.

66. The method of claim 65, wherein the soluble TGF-βRII comprises a sequence that is at least 90% identical to SEQ ID NO: 188.

67. The method of claim 66, wherein the soluble TGF-βRII comprises a sequence of SEQ ID NO: 188.

68. The method of any one of claims 1-67, wherein the method comprises administering two or more doses of the one or more agent(s) that result(s) in a decrease in the plasma levels of TGF-β in the subject.

69. The method of claim 68, wherein any two consecutive doses of the two or more doses are administered about 1 week to about one year apart.

70. The method of claim 69, wherein any two consecutive doses of the two or more doses are administered about 1 week to about 6 months apart.

71. The method of claim 70, wherein any two consecutive doses of the two or more doses are administered about 1 week to about 2 months apart.

72. The method of claim 71, wherein any two consecutive doses of the two or more 24 Feb 2026

doses are administered about 1 week to about 1 month apart.

73. The method of any one of claims 68-72, wherein the two or more doses are administered by subcutaneous administration.

74. The method of any one of claims 68-72, wherein the two or more doses are 2021283199

administered by intramuscular administration.

75. The method of any one of claims 68-74, wherein the two or more doses are administered over a period of time of about 1 year to about 60 years.

76. The method of claim 75, wherein the two or more doses are administered over a period of time of about 1 year to about 50 years.

77. The method of claim 76, wherein the two or more doses are administered over a period of time of about 1 year to about 40 years.

78. The method of claim 77, wherein the two or more doses are administered over a period of time of about 1 year to about 30 years.

79. The method of claim 78, wherein the two or more doses are administered over a period of time of about 1 year to about 20 years.

80. The method of claim 79, wherein the two or more doses are administered over a period of time of about 1 year to about 10 years.

81. The method of any one of claims 1-80, wherein a first dose of the one or more agent(s) that result(s) in a decrease in the activation of a TGF-β receptor begins when the subject reaches an age of at least 30 years.

82. The method of claim 81, wherein a first dose of the one or more agent(s) that 24 Feb 2026

result(s) in a decrease in the plasma levels of TGF-β begins when the subject reaches an age of at least 40 years.

83. The method of claim 82, wherein a first dose of the one or more agent(s) that result(s) in a decrease in the plasma levels of TGF-β begins when the subject reaches an age of at least 50 years. 2021283199

84. The method of claim 83, wherein a first dose of the one or more agent(s) that result(s) in a decrease in the plasma levels of TGF-β begins when the subject reaches an age of at least 60 years.

85. The method of any one of claims 1-84, wherein each of the two or more doses are administered at a dosage of about 0.01-10 mg/kg of each agent that results in a decrease in the plasma levels of TGF-β.

86. The method of claim 85, wherein each of the two or more doses are administered at a dosage of about 0.02-5 mg/kg of each agent that results in a decrease in the plasma levels of TGF-β.

87. The method of any one of claims 1-3 and 8-86, wherein the subject is not diagnosed or identified as having an aging-related disease or an inflammatory disease.

88. The method of any one of claims 1-3 and 8-87, wherein the subject has not been previously treated with a therapeutic agent that induces cellular senescence.

89. The method of claim1, wherein the method further comprises administering to the subject at least one or more agent(s) that result(s) in a decrease in the activation of a TGF-β receptor.

90. The method of claim 89, wherein the one or more agent(s) that result(s) in a decrease in the activation of a TGF-β receptor is a soluble TGF-β receptor, an extracellular domain of TGF-β receptor, an antibody that binds specifically to TGF-β, an antagonistic antibody that binds to a TGF-β receptor, an agent that binds to a LAP, or an agent that binds 24 Feb 2026 to a TGF-β/LAP complex.

91. The method of claim 90, wherein the one or more agent(s) that result(s) in a decrease in the activation of a TGF-β decrease(s) the activation of a TGF-β receptor through binding to a LAP, or to a TGF-β/LAP complex. 2021283199

92. The method of any one of claims 27, 28, 56 and 57, wherein the soluble human tissue factor domain does not initiate blood coagulation.

93. The method of any one of claims 1-92, wherein the method further comprises administering an additional therapeutic agent selected from the group consisting of: checkpoint inhibitors, chemotherapy drugs, and therapeutic antibodies.

94. The method of any one of claims 51-67, wherein the single-chain chimeric polypeptide is stable in human serum for at least 10 days at 37 °C.

95. The method of any one of claims 16-50, wherein the multi-chain chimeric polypeptide is stable in human serum for at least 10 days at 37 °C.

96. The method of any one of claims 1-95, wherein the method results in rejuvenation of aged immune cells in the subject

97. The method of claim 96, wherein the rejuvenation of the aged immune cells results in a reduction of number of diseased cells or infectious agents in the subject.

98. The method of claim 96 or 97, wherein the aged immune cells include one or more of aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, aged Kupffer cells, and aged microgial cells.

99. The methods of claim 98, wherein the diseased cells include cancer cells, virally infected cells, and intracellularly-bacterially-infected cells.

100. The methods of claim 97, wherein the infectious agents include viruses bacterium, fungus, and parasites. 2021283199