JP7664175B2 - Trispecific binding proteins, methods and uses thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62/831,572, filed April 9, 2019, and EP Application No. EP19306311.2, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.

Submission of a Sequence Listing in an ASCII Text File The contents of the following submission in an ASCII text file are incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 183952032040SEQLIST.TXT, Date of Accession: April 6, 2020, Size: 526KB).

FIELD The present disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein has dual variable domains with a crossover orientation. The disclosure also relates to methods of making trispecific and/or trivalent binding proteins and uses of such binding proteins.

Monoclonal antibody-based biopharmaceuticals have become an important tool for new drug development. Monoclonal antibody technology provides specific targeting, precise signaling and/or payload to unique cell populations, and long-lasting biological effects through its Fc function. Efforts in antibody engineering have enabled the development of bispecific antibodies that combine the specificities of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development. Newly discovered neutralizing antibodies with improved breadth and potency may provide more options for developing biopharmaceuticals to treat complex diseases, such as cancer, arthritis, and/or inflammatory disorders.

Immuno-oncology is a promising and emerging therapeutic approach for disease management in cancer. The immune system is the first line of defense against cancer development and progression. There is now growing evidence that T cells can control tumor growth and prolong the survival of cancer patients in both early and late stages of the disease. However, tumor-specific T cells are restricted in a number of ways that prevent them from controlling the disease.

As part of human adaptive immunity, T cell immunity plays a crucial role in controlling viral infections and cancer, potentially eliminating infected and malignant cells, resulting in the clearance of viral infections or the cure of cancer. In chronic infectious diseases, such as herpesvirus infections (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans by various mechanisms, including immunosuppression, T cell exhaustion, and establishment of latency. Nevertheless, viral infections generally induce viral antigen-specific immunity, including antigen-specific CD8 T cells that can readily recognize infected cells for control or killing through cytokine release or cytotoxic T cell (CTL)-mediated killing processes.

Therefore, in vivo and/or ex vivo activation and/or expansion of viral antigen-specific T cells may provide a therapeutic strategy for chronic viral infections.

All references referred to herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

To meet these and other needs, trispecific binding proteins (e.g., antibodies) that form three antigen binding sites are provided herein. These binding proteins can specifically bind one, two, or three antigen targets or target proteins, such as CD28, CD3, and a tumor target protein. Some tumors express specific antigens. For example, amplification and overexpression of HER2 can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung, and prostate cancer. Optimal activation of T cells requires two factors: 1. antigen recognition and 2. costimulation. Using the trispecific HER2/CD28×CD3 trispecific binding proteins described herein, signal 1 is provided by the agonist anti-CD3 binding site and signal 2 is provided by the agonist anti-CD28 binding site. The trispecific binding proteins recruit T cells to the tumor via binding sites that recognize HER2, CD38, or another tumor target protein, and activate the engaged T cells via anti-CD3 and CD28. The resulting activation induces the killing potential of immune cells against nearby tumor cells. Additionally, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and with removed potential manufacturing inconveniences (e.g., deamidation sites).

Further provided herein are anti-CD38/CD28xCD3 trispecific antibodies that have been developed and evaluated for their potential in T cell activation and subsequent proliferation and/or expansion of antigen-specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations in vitro, including antigen-specific CD8 T central memory and effector memory cells. In particular, in vitro expansion of CMV, EBV, HIV-1, and influenza-specific CD8 central and effector memory cells has been demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibit novel properties by engaging CD3/CD28/CD38 and providing a signaling pathway to stimulate and expand T cells, which may provide an effective strategy to treat chronic infectious diseases, such as HSV, CMV, EBV, HIV-1, and HBV infections.

To meet these and other needs, binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus CD38 polypeptides) are provided herein, including monospecific, bispecific, or trispecific binding proteins having at least one antigen-binding site that binds to a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells and subsequently activate the T cells to promote activated T cell killing of adjacent cancer cells through a granzyme/perforin mechanism, providing a different mode of action for anti-tumor activity than anti-CD38 antibodies, such as DARZALEX® (daratumumab). Furthermore, the ability to bind to both human and cynomolgus CD38 polypeptides allows the binding proteins to be readily tested in preclinical toxicology studies, for example, to assess their safety profile for subsequent clinical use.

In some embodiments, a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain has the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
_VH1 and _VL1 form a first antigen-binding site;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and the V _L2 domain is QSLVHX ₁ NX _{2 X 3 TY} , where X ₁ is E or Q, X ₂ is A _or L, _and X ₃ is Q, R, or F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and V _H3 and V _L3 form a third antigen-binding site.
Said binding proteins are provided herein.

In some embodiments, the first binding site binds to a CD28 polypeptide. In some embodiments, the V _H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the V _L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54). In some embodiments, the _VH1 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and/or The _L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92).

In some embodiments, the CDR-L1 sequence of the V _L2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, the _VH2 domain comprises the amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); and/or the _VL2 domain comprises the amino acid sequence of:
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS In some embodiments, the amino acid sequence of V is selected from the group consisting of: VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). The _H2 domain is QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQL and/or the _VL2 Domain comprises the amino acid sequence of:
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, a binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, a binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, a binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.

In some embodiments, the third antigen-binding site binds to a tumor targeting protein. In some embodiments, the tumor targeting protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor targeting protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, tumor targeting proteins of the present disclosure include A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCCL5 (also known as CCCL6), CCCL7 (also known as CCCL8), CCCL9 (also known as CCCL10), CCCL11 (also known as CCCL12), CCCL12 (also known as CCCL13), CCCL14 (also known as CCCL15), CCCL15 (also known as CCCL16), CCCL16 (also known as CCCL17), CCCL18 (also known as CCCL19), CCCL20 (also known as CCCL21), CCCL22 (also known as CCCL22), CCCL23 (also known as CCCL24), CCCL25 (also known as CCCL26), CCCL26 (also known as CCCL27), CCCL28 (also known as CCCL29), CCCL30 (also known as CCCL31), CCCL32 (also known as CCCL32), CCCL33 (also known as CCCL34), CCCL35 (also known as CCCL35), CCCL36 (also known as CCCL37), CCCL37 (also known as CCCL38), CCCL L5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2) , CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 ( CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, C SF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF F, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25) known as the receptor for IL33), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTP Dase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33 ), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

In some embodiments, the third antigen-binding site binds a human CD38 polypeptide. In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).

In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:79), and/or The _L3 domain comprises the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81), and/or the _VL3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87), and/or The _L3 domain comprises the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO:88). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89), and/or The _L3 domain comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:90). In some embodiments, the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85), and/or The _L3 domain comprises the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86).

In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:159. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:163. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:167. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:171. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:175. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:179. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:184. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:185 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:186 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:187 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:188 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:188.

In some embodiments, the third antigen-binding site binds to a human HER2 polypeptide. In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR- _L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWV RQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADT SKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWG and/or the _VL3 Domain comprises the amino acid sequence of:
It comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78).

In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:100; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:101; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:102; and the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:107. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:115. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:123. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:127. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:131. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:135. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:139. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:143. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:147. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:151. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:155. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:298 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:299 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:300 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:301 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:301.

_In some embodiments that may be combined with any other embodiments described herein, at least one of _L1 , L2, _L3 , or _L4 is independently 0 amino acids in length. In some embodiments, _L1 , _L2 , _L3 , and _L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S,RT,TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71). In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67) and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L ₁ comprises the sequence GQPKAAP (SEQ ID NO: 67), L ₂ comprises the sequence TKGPS (SEQ ID NO: 68), L ₃ comprises the sequence S and L ₄ comprises the sequence RT. In some embodiments, at least one of L ₁ , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO: 66). In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ comprise the sequence DKTHT (SEQ ID NO: 66).

In some embodiments that may be combined with any other embodiment described herein, the hinge-C _H2 -C _H3 Domains of the second and third polypeptide chains are human IgG4 hinge-C _H2 -C _H3 Domains, which comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, and the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C _H2 -C _H3 Domains of the second and third polypeptide chains are human IgG4 hinge- _{C H2} -C _H3 Domains _, which comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the _EU index, respectively, _and the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C _H2 -C _H3 Domains of the second and third polypeptide chains are human IgG4 hinge-C _{H2 -C H3} Domains, which comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, respectively, and the amino acid substitutions are S228P and R409K. In some embodiments, the hinge- _{C H2} -C _H3 Domains of the second and third polypeptide chains are human IgG1 hinge- _{C H2 -C H3} Domains, which comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, respectively, _and the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG1 hinge-C _H2 -C _H3 domains, wherein the hinge-C _H2 -C _H3 domains comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-C _H2 -C _H3 Domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and the hinge-C _H2 -C _H3 Domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C _H2 -C _H3 Domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and the hinge-C _H2 -C _H3 Domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

In some embodiments, provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding protein of any one of the above embodiments. In some embodiments, provided herein is an expression vector comprising a nucleic acid molecule of any one of the above embodiments. In some embodiments, provided herein is an isolated host cell comprising a nucleic acid molecule of any one of the above embodiments or an expression vector of any one of the above embodiments. In some embodiments, the host cell is a mammalian or insect cell.

In some embodiments, provided herein is a pharmaceutical composition comprising a binding protein of any one of the above embodiments and a pharma- ceutically acceptable carrier.

In some embodiments, provided herein is a method of preventing and/or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of at least one of the binding proteins or pharmaceutical compositions of any one of the above embodiments. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in a method of preventing and/or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in the manufacture of a medicament for preventing and/or treating cancer in a patient.

In some embodiments, at least one binding protein is administered in combination with a chemotherapeutic agent. In some embodiments, the patient is a human.

In some embodiments, the third antigen-binding site binds to a human CD38 polypeptide, and the cancer cells from the individual or patient express CD38. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, prior to administration of the binding protein, the patient has been treated with daratumumab without a washout period.

In some embodiments, the third antigen-binding site binds to a human HER2 polypeptide and the cancer cells from the individual or patient express HER2. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

In some embodiments, a method of expanding virus-specific memory T cells comprises contacting a virus-specific memory T cell with a binding protein, the binding protein comprising four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide;
Such methods are provided herein.

In some embodiments, a binding protein for use in the expansion of virus-specific memory T cells comprises four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. Provided herein is a binding protein for said use, comprising a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein _VH3 and _VL3 form a third antigen-binding site that binds to a CD38 polypeptide.

In some embodiments, the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cells with the binding protein causes activation and/or proliferation of the virus-specific memory T cells.

In some embodiments, a method of expanding T cells comprises contacting a T cell in vitro or ex vivo with a binding protein, the binding protein comprising four polypeptide chains that form three antigen-binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide;
Such methods are provided herein.

In some embodiments, a binding protein for use in a method of expanding T cells comprises four polypeptide chains that form three antigen-binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide;
Binding proteins for said uses are provided herein.

In some embodiments, the T cell is a memory T cell or an effector T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

In some embodiments, a method of treating a chronic viral infection comprises administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide;
Such methods are provided herein.

In some embodiments, a binding protein for use in a method of treating a chronic viral infection comprises four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence that comprises the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence that comprises the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence that comprises the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain is QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is _A or L, and X ₃ is Q, _R , or _F. a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide;
The method comprises administering to an individual or patient in need thereof an effective amount of a binding protein.
Binding proteins for said uses are provided herein.

In some embodiments, the individual or patient is a human. In some embodiments, the binding protein is administered to the individual or patient in a pharmaceutical formulation comprising the binding protein and a pharma- ceutically acceptable carrier. In some embodiments, administration of the binding protein results in activation and/or expansion of virus-specific memory T cells in the individual or patient.

In some embodiments that may be combined with any other embodiments described herein, the memory T cells are CD8+ or CD4+ memory T cells. In some embodiments, the memory T cells are central memory T cells ( _TCM ) or effector memory T cells ( _TEM ).

In some embodiments that may be combined with any other embodiment described herein, the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2).

In some embodiments that may be combined with any other embodiment described herein, the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide.

In some embodiments, a vector system is provided herein that includes one or more vectors encoding the first, second, third, and fourth polypeptide chains of the binding protein of any one of the above embodiments. In some embodiments, the vector system includes a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.

In some embodiments, provided herein is a kit comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above embodiments. In some embodiments, the kit further comprises instructions for using the polypeptide chains or binding protein according to any of the methods or uses described herein, e.g., above.

In some embodiments, a kit is provided herein, the kit comprising one, two, three, or four polynucleotides according to any one of the above embodiments. In some embodiments, a kit of polynucleotides comprising: (a) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 198, a polynucleotide of SEQ ID NO: 199. (d) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:210, (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224. (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (l) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, (m) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:240; (n) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:244. Nucleotides; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256. (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, and a polynucleotide of SEQ ID NO:267. (u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:272; or (v) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:276. The kit includes one, two, three, or four of the following polynucleotides:

It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

1 provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and HER2. The first pair of polypeptides has dual variable domains with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes HER2. The trispecific binding protein shown in FIG. 1A uses a constant region with a "knob-into-hole" mutation, where the knob is in the second pair of polypeptides with the single variable domain. FIG. 1B provides the fold change in binding affinity (relative to parent) of anti-CD28/CD3/HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. Mutations 3233QQ-QEQ (top to bottom) refer to mutations introduced at residues 32-35 of the VL domain of the anti-CD3 binding site (indicated by ^* ); the remaining mutations were introduced in the VH or VL domains of the trastuzumab anti-HER2 binding site (indicated by #; numbering follows Kabat). For mutations in the anti-HER2 binding site, mutation 30Q was introduced in the VL domain and the remaining mutations were introduced in the VH domain. Binding affinity was measured by ELISA and the values provided are relative to the parent trispecific antibody. FIG. 1C provides binding curves of the trispecific antibodies that bind to the indicated human HER2, human CD28, and CD3 as determined by ELISA. FIG. 1D provides a proposed mechanism of action of HER2/CD28×CD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing. Figure 2A provides a schematic diagram of a trispecific binding protein that includes four polypeptide chains that form three antigen-binding sites that bind three target proteins: CD28, CD3, and CD38. The first pair of polypeptides has dual variable domains with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes CD38. The trispecific binding protein shown in Figure 2A uses an IgG4 constant region with a "knob-into-hole" mutation, where the knob is in the second pair of polypeptides with the single variable domain. Figures 2B-2E show the binding affinity of CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains to the target antigens human CD38 (Figure 2B), cynomolgus monkey CD38 (Figure 2C), human CD3 (Figure 2D), and human CD28 (Figure 2E) as measured by ELISA. Continued from Figure 2-1. Continued from Figure 2-2. Figure 3 shows an SPR competition assay for binding to CD38 by daratumumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains. If the antibody recognized an epitope on CD38 different from that of daratumumab, injection of the antibody resulted in an increase in the SPR signal. If the antibody recognized an overlapping epitope as daratumumab, injection of the antibody did not increase the SPR signal. 4A-4B show the in vitro cell killing activity of CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human multiple myeloma NCI-H929 cells (CD38+/CD28+). Assays were performed in the presence of 5 nM isotype control antibody (FIG. 4A) or daratumumab (FIG. 4B). In the presence of daratumumab, the trispecific antibody continued to exhibit cell killing activity. 5A-5B show the in vitro cell killing activity of CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human lymphoma OCI-Ly19 cells (CD38+/CD28-). Assays were performed in the presence of 5 nM isotype control antibody (FIG. 5A) or daratumumab (FIG. 5B). Daratumumab caused a decrease in the cell killing activity of the anti-CD38/CD28xCD3 trispecific antibody. 6A-6J show characterization of in vitro T cell subset expansion in PBMCs collected from CMV-infected donor D in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate CMV-specific CD8+ T cells (Figs. 6A-6B), CMV-specific T _cm CD8+ cells (Figs. 6C-6D), and CMV-specific T _em CD8+ cells (Figs. 6E-6F). In addition, the percentages of CMV-specific T _cm (Figs. 6G-6H) and T _em (Figs. 6I-6J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of CMV-specific memory CD8+ T cells in a dose-responsive manner with different potencies and kinetics. Continued from Figure 6-1. Continued from Figure 6-2. Continued from Figure 6-3. Continued from Figure 6-4. 7A-7J show characterization of in vitro T cell subset expansion in PBMCs collected from CMV-infected donor E in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). Antibodies shown from top to bottom in the legend are shown from left to right in the graph. T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM. Flow cytometry was used to quantitate CMV-specific CD8+ T cells (Figs. 7A-7B), CMV-specific T _cm CD8+ cells (Figs. 7C-7D), and CMV-specific T _em CD8+ cells (Figs. 7E-7F). In addition, the percentages of CMV-specific T _cm (Figs. 7G-7H) and T _em (Figs. 7I-7J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. Continued from Figure 7-1. Continued from Figure 7-2. Continued from Figure 7-3. Continued from Figure 7-4. 8A-8J show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected donor C in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate EBV-specific CD8+ T cells (Figs. 8A-8B), CMV-specific T _cm CD8+ cells (Figs. 8C-8D), and CMV-specific T _em CD8+ cells (Figs. 8E-8F). In addition, the percentages of EBV-specific T _cm (Figs. 8G-8H) and T _em (Figs. 8I-8J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. Continued from Figure 8-1. Continued from Figure 8-2. Continued from Figure 8-3. Continued from Figure 8-4. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected donor D in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate EBV-specific CD8+ T cells (Figs. 9A-9B), EBV-specific T _cm CD8+ cells (Figs. 9C-9D), and EBV-specific T _em CD8+ cells (Figs. 9E-9F). In addition, the percentages of EBV-specific T _cm (Figs. 9G-10) and T _em (Figs. 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. Continued from Figure 9-1. Continued from Figure 9-2. Continued from Figure 9-3. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected donor D in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate EBV-specific CD8+ T cells (Figs. 9A-9B), EBV-specific T _cm CD8+ cells (Figs. 9C-9D), and EBV-specific T _em CD8+ cells (Figs. 9E-9F). In addition, the percentages of EBV-specific T _cm (Figs. 9G-10) and T _em (Figs. 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected donor D in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate EBV-specific CD8+ T cells (Figs. 9A-9B), EBV-specific T _cm CD8+ cells (Figs. 9C-9D), and EBV-specific T _em CD8+ cells (Figs. 9E-9F). In addition, the percentages of EBV-specific T _cm (Figs. 9G-10) and T _em (Figs. 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected donor D in response to CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1/ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate EBV-specific CD8+ T cells (Figs. 9A-9B), EBV-specific T _cm CD8+ cells (Figs. 9C-9D), and EBV-specific T _em CD8+ cells (Figs. 9E-9F). In addition, the percentages of EBV-specific T _cm (Figs. 9G-10) and T _em (Figs. 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. Figures 13A-13D show the change (in days) in tumor volume (Figure 13A) and body weight (Figure 13B) over time in NSG mice bearing ZR-75-1 tumors engrafted with in vitro expanded human CD3+ T cells. Groups of 10 mice were treated with either vehicle or Her2/CD28xCD3 trispecific antibody at the indicated dosages. Arrowheads indicate the day of administration. Tumor volumes are depicted as mean ± SEM, ^mm3 . Body weight change is depicted as % change, mean ± SEM. The X-axis shows days after ZR-75-1 cell implantation. Figure 13C shows tumor volume ( ^mm3 ) over time per individual mouse in each treatment group. Figure 13D shows tumor weight (mg) for each treatment group. ^** = p<0.001; ^*** = p<0.0003 (two-way ANOVA, control vs. 100 and 10 ug/kg). Continued from Figure 13-1. Continued from Figure 13-2. Continued from Figure 13-3. Figures 14A-14C show the effect of Her2/CD28xCD3 trispecific antibody treatment on T cells from whole blood. Figure 14A shows analysis of hCD45+, CD8+, CD4+, and mCD45+ cells by flow cytometry. Figure 14B shows the effect of control or Her2/CD28xCD3 trispecific antibody treatment (at the doses shown) on hCD45+, CD8+, CD4+, and mCD45+ cell counts. Figure 14C shows the effect of control or Her2/CD28xCD3 trispecific antibody treatment (at the doses shown) on human cell ratios (CD4+/CD45+ and CD8+/CD45+). For each x-axis parameter shown in Figures 14B and 14C, the conditions are (from left to right): control, 100ug/kg trispecific antibody, 10ug/kg trispecific antibody, 1ug/kg trispecific antibody, and 0.1ug/kg trispecific antibody. The percentages shown in Figures 14B and 14C are based on the control sample versus 100ug/kg. Continued from Figure 14-1. Figures 15A-15C show the effect of Her2/CD28xCD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TILs) as examined by immunohistochemistry (IHC). Arrows indicate tumor infiltrating T cells identified in ZR-75-1 breast tumors. Top image is 1x magnification; bottom image is 20x magnification. In both sets of images, staining for human CD45, human CD4, and human CD8 is shown from left to right. Tumors from mice treated with vehicle control (Figure 15A), 100ug/kg trispecific antibody (Figure 15B), or 0.1ug/kg trispecific antibody (Figure 15C) are shown. Continued from Figure 15-1. Continued from Figure 15-2. Figures 16A-16C show quantification of the effect of Her2/CD28xCD3 trispecific antibody treatment on TILs as measured by IHC. Each dot represents one tumor from an individual mouse; rectangles represent group means; error bars show standard deviation. ^* = p<0.05 compared to vehicle control group (ANOVA). Numbers of CD45+ (Figure 16A), CD4+ (Figure 16B), or CD8+ (Figure 16C) cells are shown. In Figure 16C, an area quantification approach was used for CD8+ cells instead of a cell counting algorithm due to the excess nonspecific signal in the CD8 IHC slide. Continued from Figure 16-1. Continued from Figure 16-2. Figures 17A-17F show in vitro lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by a Her2/CD28xCD3 trispecific antibody with wild-type trastuzumab and anti-CD3 antigen binding domains and no 32/35QQ mutation in the VL domain ("ctl") compared to a Her2/CD28xCD3 trispecific antibody with mutations in the VL domains of the anti-HER2 and anti-CD3 arms (numbering as indicated in Table 1). Cell killing activity against cell lines showing different expression of HER2 is depicted: HCC1954 with high HER2 expression (Figure 17A), BT20 with intermediate HER2 expression (Figure 17C), and MDA-MD-231 with low HER2 expression (Figure 17E). Graphs are shown depicting cell killing as a function of antibody concentration against target cells HCC1954 (FIG. 17B), BT20 (FIG. 17D), and MDA-MD-231 (FIG. 17F), comparing binding protein #2 to a control or binding proteins #1 and #5 to a control. Continued from Figure 17-1. Continued from Figure 17-2. Continued from Figure 17-3. Continued from Figure 17-4. Continued from Figure 17-5. Figures 18A and 18B summarize the mean EC50 (pM) of in vitro cell killing by experimental or control Her2/CD28xCD3 trispecific antibodies against the indicated breast (Figure 18A) or gastric cancer cell lines (Figure 18B). The amino acid sequences of the indicated trispecific antibodies are provided in Table 1. Continued from Figure 18-1.

The present disclosure provides a trispecific and/or trivalent binding protein comprising four polypeptide chains forming three antigen-binding sites that specifically bind to one or more target proteins, the first pair of polypeptides forming the binding protein having dual variable domains with crossover orientation.

General Definitions As utilized in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "comprise," "consist," and "consist essentially of."

The term "polynucleotide" as used herein refers to a single- or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Such modifications can include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates. The term "polynucleotide" specifically includes single- and double-stranded forms of DNA.

An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, where (1) the isolated polynucleotide is not associated with all or a portion of a polynucleotide with which it is found in nature, (2) it is linked to a polynucleotide with which it is not linked in nature, or (3) it does not occur in nature as part of a larger sequence.

An "isolated polypeptide" is a polypeptide that is (1) free from at least some other polypeptides with which it is normally found, (2) essentially free from other polypeptides from the same source, e.g., the same species, (3) expressed by cells from a different species, (4) separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) not associated (by covalent or non-covalent interactions) with portions of polypeptides with which it is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) not naturally occurring. Such isolated polypeptides can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination of these. Preferably, an isolated polypeptide is substantially free from polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, with each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain", as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids that are typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain ( _VH ) and three constant domains (C _H1 , C _H2 , and C _H3 ), with the _VH domain being at the amino-terminus of the polypeptide and the C _H3 domain being at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide comprises a variable domain ( _VL ) and a constant domain (C _L ), with the _VL domain being at the amino-terminus of the polypeptide and the C _L domain being at the carboxyl-terminus.

Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly further divided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 additional amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair typically form the antigen-binding site. Variable domains of naturally occurring antibodies typically exhibit the same overall structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 199:111-112; 342:877-83) found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. These subportions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described in Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Still other CDR boundary definitions may not strictly follow one of the systems herein, but may nevertheless overlap with the Kabat CDRs, but may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using amino acid sequences is described in Martin, A. C., "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2. Kontermann R. , Dubel S., ed., Springer-Verlag, Berlin, pp. 33-51 (2010), are well known in the art. The amino acid sequences of the heavy and/or light chain variable domains can be inspected to identify the sequences of the CDRs by other conventional methods, for example, by determining regions of sequence hypervariability in comparison with known amino acid sequences of other heavy and light chain variable regions. The numbered sequences can be aligned by eye or by using an alignment program, for example, one of the programs in the CLUSTAL suite, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are conventionally used to accurately delineate the framework and CDR regions and therefore the accurate sequence-based assignments.

The term "Fc" as used herein refers to a molecule, whether in monomeric or multimeric form, that contains a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or produced by other means, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins, with IgG1 and IgG2 being preferred. Fc molecules are composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent associations into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of an Fc is the disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is inclusive of monomeric, dimeric, and multimeric forms.

An F(ab) fragment typically contains one light chain and one heavy chain _VH and C _H1 domains, and the V _H -C _H1 heavy chain portion of an F(ab) fragment cannot form disulfide bonds with another heavy chain polypeptide. As used herein, an F(ab) fragment can also contain one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains and a C _H1 domain separated by an amino acid linker.

An F(ab') fragment typically contains one light chain and a portion of one heavy chain containing an additional constant region (between the C _H1 and C _H2 domains) such that an interchain disulfide bond can form between the two heavy chains to form an F(ab') ₂ molecule.

The term "binding protein," as used herein, refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. Trispecific binding proteins of the present disclosure, unless otherwise specified, typically comprise four polypeptide chains that form at least three antigen-binding sites, with the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
having a structure represented by
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
having a structure represented by
The third polypeptide chain has the formula:
V _H3 -C _H1 [III]
having a structure represented by
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
having a structure represented by
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

A "recombinant" molecule is one that is produced, expressed, created, or isolated by recombinant means.

One embodiment of the disclosure provides a binding protein having biological and immunological specificity for one to three target antigens. Another embodiment of the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Yet another embodiment of the disclosure provides a host cell that expresses such a binding protein (i.e., comprises a nucleic acid molecule or vector encoding a polypeptide chain forming such a binding protein).

The term "swappability" as used herein refers to the interchangeability of variable domains within a binding protein format and with retention of folding and ultimate binding affinity. "Total swappability" refers to the ability to swap (i.e., reverse) the order of both the _VH1 and _VH2 domains, and thus the VL1 and _VL2 domains, in a polypeptide chain of formula I or a polypeptide chain of formula II while maintaining full functionality of the binding protein as evidenced _by retention of binding affinity. Furthermore, it should be noted that the designation _VH and _VL only refers to the location of the domains on a particular protein chain in the final format. For example, _VH1 and _VH2 can be derived from the _VL1 and _VL2 domains in a parent antibody and placed in the _VH1 and _VH2 positions in the binding protein. Similarly, _VL1 and _VL2 can be derived from _{the VH1} and _VH2 domains in a parent antibody and placed in the _VH1 and _VH2 positions in the binding protein. Thus, the designation _VH and _VL refers to their current positions, and not to their original positions in the parent antibody. _{The VH} and _VL domains are thus "swappable".

The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or portion of a molecule to which a binding protein can bind and which can additionally be used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with intact antibodies that recognize the target antigen.

The term "Her2" refers to human epidermal growth factor receptor 2, a member of the epidermal growth factor receptor family.

"CD3" is cluster of differentiation factor 3 polypeptide, a T cell surface protein that is typically part of the T cell receptor (TCR) complex.

"CD28" is cluster of differentiation 28 polypeptide, a T cell surface protein that provides a costimulatory signal for T cell activation and survival.

"CD38" is cluster of differentiation 38 polypeptide, a glycoprotein found on the surface of many immune cells.

The term "T cell engager" refers to a binding protein that is directed to a tumor targeting protein in addition to being directed to the host's immune system, and more specifically, the cytotoxic activity of T cells.

The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

The term "bivalent binding protein" refers to a binding protein that has two binding sites.

The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

The term "trivalent binding protein" refers to a binding protein that has three binding sites. In certain embodiments, a trivalent binding protein can bind to one antigen target. In other embodiments, a trivalent binding protein can bind to two antigen targets. In other embodiments, a trivalent binding protein can bind to three antigen targets.

An "isolated" binding protein is a binding protein that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the binding protein and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding protein includes the binding protein in situ in a recombinant cell, since at least one component of the binding protein's natural environment is not present.

The terms "substantially pure" or "substantially purified," as used herein, refer to a compound or species that is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis). In some embodiments, a substantially purified fraction is a composition in which the species constitutes at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition is a composition in which all macromolecular species present in the composition constitute greater than about 80%, 85%, 90%, 95%, or 99%. In still other embodiments, the species is purified to an essential homogeneity state in which the composition consists essentially of a single macromolecular species (contaminant species cannot be detected in the composition by conventional detection methods).

The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have distinctive three-dimensional structural features and/or distinctive charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is ≦10 ⁻⁸ M, more preferably when the equilibrium dissociation constant is ≦10 ⁻⁹ M, and most preferably when the dissociation constant is ≦10 ⁻¹⁰ M.

The dissociation constant (K _D ) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "K _D " as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

The term "specifically binds," as used herein, refers to the ability of a binding protein or antigen-binding fragment thereof to bind to an antigen containing the epitope with a Kd of about 1× ^{10 −6} M, 1×10 ⁻⁷ M, 1×10 ⁻⁸ M, 1×10 ⁻⁹ M, 1×10 ⁻¹⁰ M, 1×10 ⁻¹¹ M, 1×10 −12 M, or less, and/or to bind to an epitope with an affinity that is at least two-fold higher than its affinity for a nonspecific antigen.

In some embodiments, the antigen-binding domains and/or binding proteins of the present disclosure are "cross-reactive" with human and cynomolgus CD38 polypeptides, e.g., CD38 extracellular domain, human CD38 isoform A, human CD38 isoform E, and cynomolgus CD38. Binding of a binding protein to antigen 1 (Ag1) is "cross-reactive" to antigen 2 (Ag2) if _{the EC50} is in a similar range for both antigens. In the present application, binding of a binding protein to Ag1 is cross-reactive to Ag2 if the ratio of the affinity of Ag2 to the affinity of Ag1 is equal or less than 20, where affinity is measured using the same method for both antigens.

The term "linker" as used herein refers to one or more amino acid residues that are inserted between immunoglobulin domains to provide the light and heavy chain domains with sufficient mobility to fold into a crossover dual variable region immunoglobulin. The linker is inserted at the transition between the variable domains or between the variable and constant domains at the sequence level. The approximate sizes of immunoglobulin domains are well understood, allowing the transition between domains to be identified. The precise location of the domain transition can be determined by locating secondary structure elements, such as peptide stretches that do not form beta sheets or alpha-helices, as demonstrated by experimental data or as can be predicted by modeling or secondary structure prediction techniques. The linkers described herein are referred to _{as L1} , located on the light chain between the C-terminus of the V _L2 domain and the N-terminus of the V _L1 domain; and _L2 , located on the light chain between the C-terminus of the V _L1 domain and the N-terminus of the _CL domain. The heavy chain linkers are known as L ₃ , which is located between the C-terminus of the V _H1 domain and the N-terminus of the V _H2 domain; and L ₄ , which is located between the C-terminus of the V _H2 domain and the N-terminus of the C _H1 domain.

The term "vector," as used herein, refers to any molecule (e.g., a nucleic acid, a plasmid, or a virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are sometimes used interchangeably herein, as plasmids are the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Although such progeny may not actually be identical to the parent cell, since certain modifications may occur in successive generations, either due to mutations or environmental influences, such cells are still included within the scope of the term "host cell" as used herein. A variety of host cell expression systems can be used to express the binding protein, including bacteria, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein, such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

The term "transformation" as used herein refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed to be genetically modified from its native state. After transformation, the transforming DNA can recombine with the cell's DNA by being physically integrated into the cell's chromosome, or it can be maintained transiently as an episomal element without being replicated, or it can be independently replicated as a plasmid. A cell is considered stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell; a cell is "transfected" when the exogenous DNA has been introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Using such techniques, one or more exogenous DNA molecules can be introduced into a suitable host cell.

The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide that can be isolated from a natural source and is present in an organism (including viruses) that has not been intentionally modified by humans is naturally occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by humans.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs, such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids, may also be suitable components for the polypeptide chain of a binding protein. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction, in accordance with standard usage and convention.

Naturally occurring residues can be divided into classes based on common side chain properties:
(1) Hydrophobicity: Met, Ala, Val, Leu, He, Phe, Trp, Tyr, Pro;
(2) Polar hydrophilicity: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;
(3) Aliphatic: Ala, Gly, He, Leu, Val, Pro;
(4) Aliphatic hydrophobicity: Ala, He, Leu, Val, Pro;
(5) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(6) Acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) Residues that affect chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and (10) aromatic hydrophobic: Phe, Trp, Tyr.

Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

One of skill in the art can determine suitable variants of the polypeptide chain of a binding protein using well-known techniques. For example, one may identify suitable sections of the polypeptide chain that can be altered without destroying activity by targeting regions that are not believed to be important for activity. Alternatively, one can identify residues and portions of molecules that are conserved among similar polypeptides. Additionally, even sections that may be important for biological activity or structure can be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure.

The term "patient" as used herein includes human and animal subjects.

The term "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with a disorder as well as those predisposed to having a disorder or in whom a disorder is to be prevented. In certain embodiments, the binding proteins can be used to treat humans with or susceptible to cancer or to bring cancer into remission in a human subject. The binding proteins can also be used to prevent cancer in human patients. In certain embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer, e.g., Her2+ breast cancer, germinal center B cell lymphoma, or B cell acute lymphoblastic leukemia, and in other embodiments, the binding proteins can be used to treat humans with or susceptible to an inflammatory disorder or to bring inflammatory disorders into remission in a human subject.

The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

The term "pharmacologically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

The terms "effective amount" and "therapeutically effective amount", when used in reference to a pharmaceutical composition comprising one or more binding proteins, refer to an amount or dosage sufficient to produce a desired therapeutic result. More particularly, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over some period of time. The effective amount may vary depending on the specific binding protein being used, as well as on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when a binding protein is administered in vivo, factors such as the age, weight, and health of the patient, as well as dose-response curves and toxicity data obtained in preclinical animal studies, are among the factors to be considered. The determination of an effective or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharma- ceutical acceptable carrier and a therapeutically effective amount of a binding protein.

Trispecific and/or trivalent binding proteins Certain aspects of the present disclosure relate to trispecific and/or trivalent binding proteins comprising four polypeptide chains forming three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein have dual variable domains with a crossover orientation and a second pair of polypeptides forming the binding protein have a single variable domain. Any CDR or variable domain of any of the antigen binding proteins described herein may have use in the trispecific binding proteins of the present disclosure.

In some embodiments, each of the three antigen-binding sites binds to a different target (e.g., a polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains that form the three antigen-binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is a second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_CL is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
_CH2 is the immunoglobulin _CH2 heavy chain constant domain;
_CH3 is the immunoglobulin _CH3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

It is contemplated that any of the antigen-binding sites described herein may have utility in the trispecific binding proteins of the present disclosure, which include, for example, four polypeptide chains having the structure described above. For example, in some embodiments, the trispecific binding proteins of the present disclosure include a _VH1 and _VL1 domain pair that forms a first antigen-binding site, a _VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and _VL3 domain pair that forms a third antigen-binding site. In some embodiments, the trispecific binding proteins of the present disclosure include a _VH1 and _VL1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a _VH2 and _VL2 domain pair that forms a second antigen-binding site that binds to a CD3 _polypeptide , and a _{VH3 and VL3 domain} pair that forms a third antigen-binding site. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V _H1 and V _L1 domain pair that forms a first antigen-binding site, a V H2 and V L2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a V _H3 and V _L3 domain pair that forms a third antigen-binding site that binds to a tumor targeting protein _. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V _H1 and V _L1 domain pair that forms a first antigen-binding site _that binds to a CD28 polypeptide, a V _H2 and V _L2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a V _H3 and V _L3 domain pair that forms a third antigen-binding site that binds to a tumor targeting protein. In some embodiments, a trispecific binding protein of the present disclosure comprises a V _H1 and V _L1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a V H2 and V L2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a V _H3 and V _L3 domain pair that forms a third antigen-binding site that binds to a CD38 polypeptide. In some embodiments, a trispecific binding protein of the present disclosure comprises a V _H1 and V _L1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a V _H2 and V _L2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a V _H3 and V _L3 domain pair that forms a third antigen-binding site that binds to _{a HER2} polypeptide.

In some embodiments, the binding proteins of the present disclosure bind to one or more tumor targeting proteins and one or more T cell targeting proteins. In some embodiments, the binding proteins can specifically bind to one tumor targeting protein and two different epitopes on a single T cell targeting protein. In some embodiments, the binding proteins can specifically bind to one tumor targeting protein and two different T cell targeting proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell targeting proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds to a tumor targeting protein. In some embodiments, the targeting protein is CD38 or HER2. Additional tumor targeting proteins are provided below. In some embodiments, the one or more T cell targeting proteins are one or more of CD3 and CD28. Exemplary, non-limiting polypeptides that may have use in any of the trispecific binding proteins described herein are provided in Table 1.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:156 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:156; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:157 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:157; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:158 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:158; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:159 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:159.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 160 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 161 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 162 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 163 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 163.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:164 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:164; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:165 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:165; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:166 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:166; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:167 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:167.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:168 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:168; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:169 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:169; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:170 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:170; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:171 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:171.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:172 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:172; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:173 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:173; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:174 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:174; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:175 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:175.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:176 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:176; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:177 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:177; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:178 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:178; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:179 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:179.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:181 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:181; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:182 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:182; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:183 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:183; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:184 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:184.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:185 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:185; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:186 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:186; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:187 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:187; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:188 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:188.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 100 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 101 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 102 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 103 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 103.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 104 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 105 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 106 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 107 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 107.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:112 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:112; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:113 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:113; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:114 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:114; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:115 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:115.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:116 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:116; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:117 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:117; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:118 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:118; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:119 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:119.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:120 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:120; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:121 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:121; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:122 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:122; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:123 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:123.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:124 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:124; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:125 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:125; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:126 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:126; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:127 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:127.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 128 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 129 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 130 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 131 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 131.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 132 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 133 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 134 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 135 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 135.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:136 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:136; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:137 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:137; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:138 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:138; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:139 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:139.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:140 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:140; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:141 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:141; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:142 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:142; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:143 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:143.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:144 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:144; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:145 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:145; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:146 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:146; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:147 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:147.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:148 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:148; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:149 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:149; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:150 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:150; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:151 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:151.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:152 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:152; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:153 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:153; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:154 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:154; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:155 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:155.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:286 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:287 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:288 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:289 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:289.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:290 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:291 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:292 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:293 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:293.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:294 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:295 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:296 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:297 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:297.

In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain comprising an amino acid sequence of SEQ ID NO:298 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprising an amino acid sequence of SEQ ID NO:299 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprising an amino acid sequence of SEQ ID NO:300 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO:301 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:301.

Anti-CD38 Binding Sites Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to a CD38 polypeptide. In some embodiments, the CD38 polypeptide is a human CD38 polypeptide, also known as ADPRC1. Human CD38 polypeptides are known in the art, including, but not limited to, the polypeptide represented by NCBI Accession No. NP_001766.2, or the polypeptide produced from NCBI Gene Identification No. 952. In some embodiments, the antigen-binding site binds to a human CD38 polypeptide, a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide, or a human CD38 polypeptide and a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD38 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.

In some embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding sites described below may be used in a monospecific antibody.

In other embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding sites described below may be used in any binding site of a trispecific binding protein comprising, for example, four polypeptides forming three antigen-binding sites as described above. In a particular embodiment, the binding protein comprising an antigen-binding site that binds to a CD38 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites as described above, with the pair of _VH3 and _VL3 domains forming a third antigen-binding site that binds to a CD38 polypeptide.

Various features of exemplary binding sites and binding proteins are described herein. For example, in some embodiments, an anti-CD38 binding site cross-reacts with human CD38 (e.g., human CD38 isoform A and/or isoform E polypeptides) and cynomolgus monkey CD38. In some embodiments, a binding protein comprising an anti-CD38 binding site induces apoptosis of CD38+ cells. In some embodiments, a binding protein comprising an anti-CD38 binding site recruits T cells to CD38+ cells and optionally activates the T cells (e.g., via TCR stimulation and/or costimulation).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO: 26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO: 27); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO: 28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42).

In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48).

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:79); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVLTQSPATHLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 79, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 79, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSETAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO:82). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 81, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 81, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 82.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 83, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 83, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 84.

In some embodiments, the binding site that binds to CD38 is
An antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87). and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO:88). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 87, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 87, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 88.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:89); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:90). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 89, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 89, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); and/or DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 85, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 85, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 86.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSA VYFCARYAYGYWGQGTTLTVSS (SEQ ID NO: 277); and/or DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO: 278). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 277, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 278. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 277, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 278. In some embodiments, the VH and/or VL domains are humanized.

In some embodiments, the binding site that binds to CD38 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEFYYSMDYWGQGTSVTVSS (SEQ ID NO: 279); and and/or DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK (SEQ ID NO: 280). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 279, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 280. In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 279, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 280. In some embodiments, the VH and/or VL domains are humanized.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds a CD38 polypeptide, an antigen-binding site that binds a CD28 polypeptide, and an antigen-binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein a polypeptide of Formula I and a polypeptide of Formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen-binding sites described above represent _VH3 and _VL3 , forming a third antigen-binding site that binds a CD38 polypeptide. In some embodiments, V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, V _H2 and V _L2 form a second antigen-binding site that binds to a CD3 polypeptide, and the VH and VL domains of any of the anti-CD38 antigen-binding sites described above and/or in Table 2 represent V _H3 and V _L3 and form a third antigen-binding site that binds to a CD38 polypeptide.

Exemplary anti-CD38 antigen binding site sequences are provided in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD38 antibody described in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises a VH domain sequence and/or a VL domain sequence of an anti-CD38 antibody described in Table 2.

Further provided herein are antibodies (e.g., monospecific antibodies) that contain any of the anti-CD38 CDRs and/or variable domains described above.

In some embodiments, the binding proteins of the present disclosure include an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen-binding site binds to an antigen are described herein and known in the art, including (but not limited to) ELISA, SPR, and flow cytometry assays.

Anti-HER2 Binding Sites Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds to a HER2 polypeptide. In some embodiments, the HER2 polypeptide is a human HER2 polypeptide, also known as NEU, NGL, ERBB2, TKR1, CD340, HER-2, MLN19, and HER-2/neu. Human HER2 polypeptides are known in the art and include, but are not limited to, the polypeptides represented by NCBI Accession Nos. XP_024306411.1, XP_024306410.1, XP_024306409.1, NP_001276867.1, NP_001276866.1, NP_001276865.1, NP_001005862.1, or NP_004439.2, or the polypeptides produced from NCBI Gene Identification No. 2064. In some embodiments, a binding protein comprising an antigen-binding site that binds to a HER2 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, the binding protein comprising an antigen-binding site that binds to a HER2 polypeptide is a trispecific binding protein comprising four polypeptides forming the three antigen-binding sites described above, and a _VH3 and _VL3 domain pair forming a third antigen-binding site that binds to a HER2 polypeptide.

In some embodiments, the binding site that binds to HER2 comprises a CDR-H1 sequence having the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence having the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence having the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or S an antibody heavy chain variable (VH) domain comprising a CDR-H3 sequence comprising the amino acid sequence of RWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds HER2 comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO:1) or GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), IYPTQGYT (SEQ ID NO:4), or IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), SRWGGEGFYAMDY (SEQ ID NO:7), or comprises an antibody heavy chain variable (VH) domain comprising a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGGFYAMDY (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO:10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12).

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGF NIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQ MNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAM DYWGQGTLVTVSS (SEQ ID NO:76); and/or an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of , or 100% identical to the amino acid sequence of VEIK (SEQ ID NO:77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:78.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 73, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 73, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 75, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 75, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:74), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 74, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 74, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 76, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 76, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the binding site that binds to HER2 is
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the binding site that binds HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the anti-HER2 antigen-binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of the anti-HER2 antibody trastuzumab, 30R/55Q/102E, 30R/56A/102S, 30R/55Q/102S, 30R/56A/102E, or 30Q. In some embodiments, the anti-HER2 antigen-binding site of the present disclosure comprises the VH domain sequence and/or the VL domain sequence of the anti-HER2 antibody trastuzumab, 30R/55Q/102E, 30R/56A/102S, 30R/55Q/102S, 30R/56A/102E, or 30Q.

Exemplary anti-HER2 antigen binding site sequences are provided in Table 3. In some embodiments, an anti-HER2 antigen binding site of the disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-HER2 antibody described in Table 3. In some embodiments, an anti-HER2 antigen binding site of the disclosure comprises a VH domain sequence and/or a VL domain sequence of an anti-HER2 antibody described in Table 3.

Other Anti-Tumor Target Binding Sites In some embodiments, the binding proteins of the disclosure comprise an antigen binding site that binds to a tumor targeting protein. In some embodiments, the tumor targeting protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor targeting protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, the tumor targeting protein of the disclosure includes A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CC1 (also known as CC2), CC3 (also known as CC4), CC5 (also known as CC5), CC6 (also known as CC7), CC8 (also known as CC9), CC10 (also known as CC11), CC12 (also known as CC13), CC14 (also known as CC15), CC15 (also known as CC16), CC16 (also known as CC17), CC18 (also known as CC19), CC20 (also known as CC21), CC22 (also known as CC23), CC24 (also known as CC25), CC25 (also known as CC26), CC26 (also known as CC27), CC28 (also known as CC29), CC30 (also known as CC31), CC32 (also known as CC33), CC34 (also known as CC35), CC36 (also known as CC37), CC38 (also known as CC39), CC40 (also known as CC41), CC42 (also known L5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2) , CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 ( CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, C SF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF F, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25) known as the receptor for IL33), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTP Dase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33 ), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

Anti-CD28 Binding Sites Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide, also known as Tp44. Human CD28 polypeptides are known in the art and include, but are not limited to, polypeptides represented by NCBI Accession Nos. XP_011510499.1, XP_011510497.1, XP_011510496.1, NP_001230007.1, NP_001230006.1, or NP_006130.1, or polypeptides produced from NCBI Gene Identification No. 940. In some embodiments, binding proteins comprising an antigen-binding site that binds to a CD28 polypeptide are monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the antigen-binding sites binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the antigen-binding sites binds to a CD3 polypeptide and one of the antigen-binding sites binds to a CD38 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a HER2 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor targeting protein.

In some embodiments, the binding site that binds to CD28 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54). In some embodiments, the binding site that binds to CD28 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO: 53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54).

In some embodiments, the binding site that binds to CD28 is
8. An antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO:91). and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92). In some embodiments, the binding site that binds CD28 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 91, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, the binding site that binds CD28 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 91, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 92.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds a tumor targeting protein (such as, but not limited to, CD38 or HER2), an antigen-binding site that binds a CD28 polypeptide, and an antigen-binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above represent _VH1 and _VL1 and form a first antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above and/or in Table 4 represent _VH1 and _VL1 , which form a first antigen binding site that binds to a CD28 polypeptide, _VH2 and _VL2 , which form a second antigen binding site that binds to a CD3 polypeptide, and _VH3 and _VL3 , which form a third antigen binding site that binds to a tumor target protein (such as, but not limited to, CD38 or HER2).

Exemplary anti-CD28 antigen binding site sequences are provided in Table 4. In some embodiments, an anti-CD28 antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD28 antibody described in Table 4. In some embodiments, an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and/or a VL domain sequence of an anti-CD28 antibody described in Table 4.

Anti-CD3 Binding Sites Certain aspects of the present disclosure relate to binding proteins that comprise an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, such as CD3-delta (also known as T3D, IMD19, and CD3-delta), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-gamma). Human CD3 polypeptides are known in the art and include, but are not limited to, the polypeptides represented by NCBI Accession Nos. XP_006510029.1 or NP_031674.1, or the polypeptides produced from NCBI Gene Identification Nos. 915, 916, or 917. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the antigen-binding sites binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the three antigen-binding sites binds to a CD3 polypeptide, and one of the antigen-binding sites binds to a CD38 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a HER2 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor targeting protein.

In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57 ₎ ; and/or QSLVHX ₁ NX ₂ X _{3 TY} , where X ₁ is E or Q, X ₂ is A or L, and X ₃ is Q, R, _or F. In some embodiments, the CDR-L1 sequence of the V L2 domain comprises an antibody light chain variable (VL) domain comprising a CDR- _L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62).

In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).

In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).

In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).

In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65).

In some embodiments, the binding site that binds to CD3 is
At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149 ... and/or an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98. In some embodiments, the binding site that binds to CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

In some embodiments, the binding site that binds to CD3 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the binding site that binds to CD3 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:96). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 96.

In some embodiments, the binding site that binds to CD3 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 97.

In some embodiments, the binding site that binds to CD3 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 98.

In some embodiments, the binding site that binds to CD3 is
an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302); and/or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95). In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site that binds CD3 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds a tumor targeting protein (such as, but not limited to, CD38 or HER2), an antigen-binding site that binds a CD28 polypeptide, and an antigen-binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD3 antigen-binding sites described above represent _VH2 and _VL2 and form a second antigen-binding site that binds to a CD3 polypeptide. In some embodiments, _VH1 and _VL1 form a first antigen-binding site that binds to a CD28 polypeptide, the VH and VL domains of any of the anti-CD3 antigen-binding sites described above and/or in Table 5 represent _VH2 and _VL2 and form a second antigen-binding site that binds to a CD3 polypeptide, and _VH3 and _VL3 form a third antigen-binding site that binds to a tumor target protein (such as, but not limited to, CD38 or HER2).

Exemplary anti-CD3 antigen binding site sequences are provided in Table 5. In some embodiments, an anti-CD3 antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD3 antibody described in Table 5. In some embodiments, an anti-CD3 antigen binding site of the present disclosure comprises a VH domain sequence and/or a VL domain sequence of an anti-CD3 antibody described in Table 5.

Linkers In some embodiments, the linkers _L1 , _L2 , _L3 , and _L4 range in length from zero amino acids (length=0) to about 100 amino acids, or are less than 100, 50, 40, 30, 20, or 15 amino acids, or less. The linkers may be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. The _L1 , _L2 , _L3 , and _L4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.

Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71), and DKTHT (SEQ ID NO: 66), as well as those disclosed in International Publications WO2017/074878 and WO2017/180913. The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable in binding proteins.

The identity and sequence of amino acid residues in the linker can be varied depending on the type of secondary structural element that needs to be achieved in the linker. For example, glycine, serine, and alanine are best for linkers with maximum flexibility. If a more rigid and longer linker is required, some combinations of glycine, proline, threonine, and serine are useful. Any amino acid residue can be considered as a linker in combination with other amino acid residues to build larger peptide linkers as needed depending on the desired properties.

In some embodiments, L ₁ is at least twice as long as L _3. In some embodiments, L ₂ is at least twice as long as L _4. In some embodiments, L ₁ is at least twice as long as L ₃ and L ₂ is at least twice as long as L _4. In some embodiments, L ₁ is 3-12 amino acid residues long, L ₂ is 3-14 amino acid residues long, L ₃ is 1-8 amino acid residues long, and L ₄ is 1-3 amino acid residues long. In some embodiments, L ₁ is 5-10 amino acid residues long, L ₂ is 5-8 amino acid residues long, L 3 is 1-5 amino acid residues long, and L ₄ is 1-2 amino acid residues long. _In some embodiments, L ₁ is 7 amino acid residues long, L ₂ is 5 amino acid residues long, L ₃ is 1 amino acid residue long, and L ₄ is 2 amino acid residues long.

In some embodiments, _L1 , _L2 , _L3 , and _L4 are each independently zero amino acid long or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71). In some embodiments, _L1 , _L2 , _L3 , and _L4 are each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71). In some embodiments, L ₁ comprises the sequence GQPKAAP (SEQ ID NO:67), L ₂ comprises the sequence TKGPS (SEQ ID NO:68), L ₃ comprises the sequence S, and L ₄ comprises the sequence RT.

In some embodiments, at least one of L ₁ , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO: 66).In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ comprise the sequence DKTHT (SEQ ID NO: 66).

Fc Regions and Constant Domains In some embodiments, the binding proteins of the disclosure comprise a second polypeptide chain further comprising an Fc region linked to the C _H1 , where the Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domains. In some embodiments, the binding proteins of the disclosure comprise a third polypeptide chain further comprising an Fc region linked to the C _H1 , where the Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the disclosure comprises a second polypeptide chain further comprising an Fc region linked to C _H1 , where the Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to C _H1 , where the Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domains.

In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region comprises an antibody hinge, C _H1 , C _H2 , C _H3 , and optionally a C _H4 domain. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the mutations described below. In some embodiments, the Fc region is the Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the heavy chain constant region is the constant region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the light chain constant region is the constant region of one of the light chain polypeptides of the binding proteins shown in Table 4 (eg, polypeptide 1 or 4).

In some embodiments, the binding proteins of the present disclosure include one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" may include a molecule or sequence that has been humanized from a non-human native Fc. Additionally, the native Fc includes regions that can be removed to provide structural features or biological activity that are not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences lacking one or more native Fc sites or residues or having one or more altered Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, a binding protein of the disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation in the second polypeptide chain and a "hole" mutation in the third polypeptide chain. In some embodiments, a binding protein of the disclosure comprises a "knob" mutation in the third polypeptide chain and a "hole" mutation in the second polypeptide chain. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to 407, and optionally 349, 366, and/or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y407V or Y407T and optionally Y349C, T366S, and/or L368A. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y349C, T366S, L368A, and Y407V.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being T366W or T366Y and optionally S354C; and the third polypeptide chain comprises The antibody further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 407 and optionally positions 349, 366, and/or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being Y407V or Y407T and optionally Y349C, T366S, and/or L368A.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 407 and optionally 349, 366, and/or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y407V or Y407T and optionally Y349. C, T366S, and/or L368A; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W or T366Y and optionally S354C.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to positions 366, 368, and/or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and/or Y407V.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366, 368, and/or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and/or Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being an Fc region of human IgG4 comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y4 07V, and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being an Fc region of human IgG4 comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L366S, Y366C, Y366D, Y366E, Y366F, Y366H, Y366I, Y366J, Y366N ... 68A, and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W.

In some embodiments, the binding proteins of the disclosure comprise one or more mutations that can reduce an effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to _CH1 , the first Fc region comprising an immunoglobulin hinge region and the _CH2 and _CH3 immunoglobulin heavy chain constant domains; the third polypeptide chain further comprises a second Fc region linked to _CH1 , the second Fc region comprising an immunoglobulin hinge region and the _CH2 and _CH3 immunoglobulin heavy chain constant domains; the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, respectively, the amino acid substitutions being L234A and L235A. In some embodiments, the Fc region of the second and third polypeptide chains is a human IgG1 Fc region, wherein the Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to _CH1 , the first Fc region comprising an immunoglobulin hinge region and the _CH2 and _CH3 immunoglobulin heavy chain constant domains; the third polypeptide chain further comprises a second Fc region linked to _CH1 , the second Fc region comprising an immunoglobulin hinge region and the _CH2 and _CH3 immunoglobulin heavy chain constant domains; the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, respectively, where the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc region of the second and third polypeptide chains is a human IgG1 Fc region, which comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1, respectively, according to the EU index, and the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc region of the second and third polypeptide chains is a human IgG4 Fc region, which comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, respectively, according to the EU index, and the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to _CH1 , the first Fc region comprising an immunoglobulin hinge region and _{a CH2} and _CH3 immunoglobulin heavy chain constant domain, and a third polypeptide chain further comprising a second Fc region linked to _CH1 , the second Fc region comprising an immunoglobulin hinge region and a _CH2 and _CH3 immunoglobulin heavy chain constant domain; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being an Fc region of human IgG4 comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y4 07V, and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K.

In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and/or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and/or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 228 and/or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 234 and/or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at positions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletions at 236; and/or R409K.

In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector functions of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).

In some embodiments, the Fc region is a human IgG1 Fc region that contains one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region that contains amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve stability, e.g., stability of the hinge region and/or dimer interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C _H1 , the first Fc region comprising an immunoglobulin hinge region and a C _H2 and C _H3 immunoglobulin heavy chain constant domain, and a third polypeptide chain further comprising a second Fc region linked to C _H1 , the second Fc region comprising an immunoglobulin hinge region and a C _H2 and C _H3 immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG4 Fc regions; and the first and second Fc regions comprise amino acid substitutions at positions corresponding to 228 and 409 of human IgG4 according to the EU index, respectively, the amino acid substitutions being S228P and R409K. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the Fc region is a human IgG1 Fc region that contains one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region that contains amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

Nucleic Acids Another aspect of the present disclosure pertains to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.

Another aspect of the disclosure relates to a kit of polynucleotides, wherein the polynucleotides encode one or more polypeptides of a binding protein, e.g., as described herein. In some embodiments, the kit of polynucleotides of the disclosure includes: (a) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 191; (d) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:210, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:211. (g) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:216; (h) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:220; (i) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:221, (j) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:228; (k) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:232; (l) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:228. (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244. (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, (r) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:260; (s) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:264; (t) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:266, (u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:272; or (v) a kit of polynucleotides comprising a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:276.

Other aspects of the present disclosure relate to vector systems comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, e.g., as shown in the polynucleotides of Table 6. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system includes a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system may be any vector described herein. In some embodiments, the one or more vectors are expression vectors. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g., one, two, three, or four expression vectors.

Standard recombinant DNA methodologies are used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Unless specific definitions are provided, the nomenclature utilized in the context of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical manufacturing, formulation, delivery, and treatment of patients.

In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the binding protein-encoding nucleic acid sequence. A promoter can refer to a nucleic acid control sequence that directs transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a binding protein coding sequence if the promoter affects the transcription or expression of the coding sequence. Examples of promoters include genomes of viruses (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)), heterologous eukaryotic promoters (e.g., actin promoter, immunoglobulin promoter, and heat shock promoter), CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK)-promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acid Res. 33:e43, 2005), lac system, trp system, tac system, trc system, phage lambda major operator and promoter regions, 3-phosphoglycerate kinase promoter, yeast acid phosphatase promoter, and yeast alpha mating factor promoter. The polynucleotide encoding the binding protein of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily recognized by one of skill in the art.

In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. An expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.), as well as enhancer sequences from eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

Host Cells Other aspects of the disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein. In some embodiments, the isolated host cell of the disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells can include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Exemplary mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HBsAg, HBV ... 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2/0 cells).

Other aspects of the present disclosure relate to methods of producing any of the binding proteins described herein. In some embodiments, the methods include a) culturing a host cell (e.g., any of the host cells described herein) containing an isolated nucleic acid, vector, and/or vector system (e.g., any of the isolated nucleic acid, vector, and/or vector system described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions for protein expression are well known to those of skill in the art. Methods of isolating proteins from cultured host cells are well known to those of skill in the art and include, for example, affinity chromatography (e.g., two-step affinity chromatography including Protein A affinity chromatography followed by size exclusion chromatography).

Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions can include a therapeutically effective amount of the binding protein, or binding protein-drug conjugate, in combination with a pharma- ceutical or physiologically acceptable formulation agent selected for suitability with the mode of administration.

Acceptable formulation materials are non-toxic to recipients at the dosages and concentrations used.

Pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, ammonium chloride, sodium lauryl sulfate ... Examples of suitable pharmaceutical agents include, but are not limited to, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, e.g., sodium or potassium chloride, or mannitol, sorbitol), delivery vehicles, diluents, excipients, and/or pharmaceutical adjuvants (e.g., REMINGTON'S PHARMACEUTICAL COMPOSITIONS, incorporated herein by reference for any purpose). PHARMACEUTICAL SCIENCES (18th Ed., edited by A.R. Gennaro, Mack Publishing Company 1990), and subsequent editions thereof).

Optimal pharmaceutical compositions can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection can be water, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the binding protein composition can be prepared for storage by mixing the selected composition having the desired purity with optional formulating agents in the form of a lyophilized cake or aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

The pharmaceutical compositions of the present disclosure can be selected for parenteral or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, e.g., orally. The preparation of such pharma- ceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable for the site of administration. For example, buffers are used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

When parenteral administration is contemplated, the therapeutic composition for use may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution containing the desired binding protein in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a properly preserved, sterile, isotonic solution. Yet another product may involve the formulation of the desired molecule with agents that provide controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, that can be delivered via depot injection. Hyaluronic acid may also be used, which may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, binding proteins administered in this manner may be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

Another pharmaceutical composition can involve an effective amount of the binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another suitable vehicle, the solution can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders, such as starch, gelatin, or acacia; or lubricants, such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions of the present disclosure will be apparent to those of skill in the art, including formulations with binding proteins in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyrate. Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

Pharmaceutical compositions used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be performed either prior to, or after, lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Additionally, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

The present disclosure also encompasses kits for producing single dose administration units. The kits can each contain both a first container having a dry protein and a second container having an aqueous formulation. Kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of the present disclosure.

The effective amount of a binding protein pharmaceutical composition used therapeutically will depend, for example, on the therapeutic context and purpose. Those skilled in the art will appreciate that appropriate dosage levels for treatment will therefore vary depending in part on the molecule being delivered, the indication for which the binding protein is being used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and general health) of the patient. Thus, clinicians can titrate dosages and modify routes of administration to obtain optimal therapeutic effects.

The frequency of dosing depends on the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may thus be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely made by, and is within the scope of tasks routinely performed by, those of skill in the art. The appropriate dosage may be ascertained through the use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is according to known methods, for example, orally; by injection, intravenous, intraperitoneal, intracerebral (intracerebral), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system; or by implantation device. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implantation device.

The compositions can also be administered locally via implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed release bolus, or continuous administration.

The pharmaceutical compositions can be used to prevent and/or treat HIV infection. The pharmaceutical compositions can be used as a stand-alone therapy or in combination with standard antiretroviral therapy.
The present disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in a biological sample. Such reagents may include detectable labels, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and/or host cells described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition alone or in combination with another composition effective for treating, preventing, and/or diagnosing a condition (e.g., HIV infection) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and/or treat a selected condition. Alternatively, or additionally, the article or kit may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Methods and Uses of Binding Proteins Viruses Certain aspects of the present disclosure relate to methods of expanding virus-specific memory T cells. In some embodiments, the method comprises contacting virus-specific memory T cells with a binding protein of the present disclosure, such as a trispecific binding protein comprising a first antigen-binding site that binds to a CD28 polypeptide, a second antigen-binding site that binds to a CD3 polypeptide, and a third antigen-binding site that binds to a CD38 polypeptide.

In some embodiments, the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo.

In some embodiments, contacting the virus-specific memory T cells with the binding protein causes activation and/or proliferation of the virus-specific memory T cells.

Other aspects of the disclosure relate to methods of expanding T cells. In some embodiments, the methods include contacting T cells with a binding protein of the disclosure, e.g., a trispecific binding protein that includes a first antigen-binding site that binds to a CD28 polypeptide, a second antigen-binding site that binds to a CD3 polypeptide, and a third antigen-binding site that binds to a CD38 polypeptide.

In some embodiments, the T cells are memory T cells or effector T cells.

In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

Other aspects of the present disclosure relate to methods of treating a chronic viral infection, e.g., in an individual in need thereof. In some embodiments, the method includes administering to an individual in need thereof an effective amount of a binding protein of the present disclosure, e.g., a trispecific binding protein comprising a first antigen-binding site that binds to a CD28 polypeptide, a second antigen-binding site that binds to a CD3 polypeptide, and a third antigen-binding site that binds to a CD38 polypeptide.

In some embodiments, the individual is a human.

In some embodiments, the binding protein is administered to the individual in a pharmaceutical formulation that includes the binding protein and a pharma- ceutically acceptable carrier.

In some embodiments, administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual.

In any of the above methods, the memory T cells can be CD8+ or CD4+ memory T cells. In any of the above methods, the memory T cells can be central memory T cells (T _CM ) or effector memory T cells (T _EM ).

Certain aspects of the present disclosure relate to methods of preventing and/or treating cancer in a patient, hi some embodiments, the methods comprise administering to the patient a therapeutically effective amount of a binding protein or pharmaceutical composition of the present disclosure.

In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof for the treatment or prevention of cancer. In some embodiments, the disclosure relates to a method of preventing and/or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to the patient a therapeutically effective amount of at least one of the binding proteins described herein, or pharmaceutical compositions associated therewith. In some embodiments, the disclosure relates to the use of at least one of the binding proteins described herein, or pharmaceutical compositions associated therewith, for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the disclosure relates to at least one of the binding proteins described herein, or pharmaceutical compositions associated therewith, for use in the manufacture of a medicament for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the patient is a human.

In some embodiments, the at least one binding protein is (or will be) administered in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art, e.g., chemotherapeutic agents or therapies). In some embodiments, the at least one binding protein is (or will be) administered prior to one or more anti-cancer therapies. In some embodiments, the at least one binding protein is (or will be) administered in parallel with one or more anti-cancer therapies. In some embodiments, the at least one binding protein is (or will be) administered after one or more anti-cancer therapies.

In some embodiments, the binding protein comprises one or two antigen-binding sites that bind to a T cell surface protein and another antigen-binding site that binds to the extracellular domain of a human HER2 polypeptide. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of a human HER2 polypeptide, an antigen-binding site that binds to a human CD28 polypeptide, and an antigen-binding site that binds to a human CD3 polypeptide.

In some embodiments, cancer cells from an individual express HER2. In some embodiments, a patient is selected for treatment based on the cells of the cancer expressing human HER2 polypeptide. Assays known in the art suitable for detecting HER2 expression by cancer cells include, but are not limited to, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) assays.

In some embodiments, the cancer (e.g., a HER2-positive cancer) is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

In some embodiments, the binding protein comprises one or two antigen-binding sites that bind to a T cell surface protein and another antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide, an antigen-binding site that binds to a human CD28 polypeptide, and an antigen-binding site that binds to a human CD3 polypeptide.

In some embodiments, cancer cells from an individual express CD38. In some embodiments, the cancer cells express human CD38 isoform A polypeptide on their cell surface. In some embodiments, the cancer cells express human CD38 isoform E polypeptide on their cell surface. In some embodiments, a patient is selected for treatment based on the cancer cells expressing human CD38 isoform E polypeptide on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.

In some embodiments, the cancer (e.g., a CD38-positive cancer) is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer, e.g., Her2+ breast cancer, prostate cancer, germinal center B-cell lymphoma, or B-cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

In certain embodiments, the cancer is multiple myeloma. Anti-CD38 antibodies, such as daratumumab, have been tested for the treatment of multiple myeloma. However, while multiple myeloma is considered treatable, relapse is inevitable in nearly all patients, leading to the development of treatment-refractory disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a previous multiple myeloma treatment. In some embodiments, the binding proteins of the present disclosure are administered to the patient as a first, second, or third line treatment for multiple myeloma. Without wishing to be bound by theory, it is believed that the anti-CD38 x anti-CD28 x anti-CD3 binding proteins of the present disclosure may be useful in the treatment of multiple myeloma, for example, by recruiting T cells to tumor cells via anti-CD38 (or anti-CD28/anti-CD38), activating engaged T cells via anti-CD3/anti-CD28, and/or killing tumor cells through a perforin/granzyme-based mechanism. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, J. R. et al. (2011) J. Immunol. 187:1243-1253.

Any of the binding proteins described herein may have use in the methods of the present disclosure.

In some embodiments of any of the methods of the present disclosure, the patient has been treated with daratumumab prior to administration of the binding protein. As described herein, the present disclosure provides anti-CD38 binding proteins and moieties that do not compete with daratumumab for binding to CD38. Without wishing to be bound by theory, this is believed to be advantageous since patients previously treated with daratumumab can be treated with the binding proteins of the present disclosure, for example, without a washout period prior to treatment.

The binding proteins can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantification of one or more target antigens. The binding proteins bind to one or more target antigens with an affinity appropriate for the assay method being used.

For diagnostic applications, in certain embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be any moiety that can generate, either directly or indirectly, a detectable signal. For example, the detectable moiety can be a radioisotope, such as ^3H , ^14C , ^32P , ^35S , ^125I , ^99Tc , ^111In , or ^67Ga ; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

The binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host can be assayed. The binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

For clinical or research applications, in certain embodiments, the binding protein can be conjugated to a cytotoxic agent. Various antibodies linked to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate agents to antibodies are known in the art; see, e.g., Parslow, A. C. et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

Binding Protein Therapeutic Compositions and Administration Thereof Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions can include a therapeutically effective amount of the binding protein, or binding protein-drug conjugate, in combination with a pharma- ceutical or physiologically acceptable formulating agent selected for suitability with the mode of administration. These pharmaceutical compositions can have use in any of the methods and uses described herein (e.g., ex vivo, in vitro, and/or in vivo).

Acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used.

Pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoate, or benzoyl chloride), and the like. and/or other suitable pharmaceutical agents, including, but not limited to, fragrance acids, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, or mannitol, sorbitol), delivery vehicles, diluents, excipients, and/or pharmaceutical adjuvants (e.g., see REMINGTON'S Pharmaceuticals, Inc., incorporated herein by reference for any purpose). PHARMACEUTICAL SCIENCES (18th Ed., edited by A.R. Gennaro, Mack Publishing Company 1990), and subsequent editions thereof).

Optimal pharmaceutical compositions can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection can be water, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the binding protein composition can be prepared for storage by mixing the selected composition having the desired purity with optional formulating agents in the form of a lyophilized cake or aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

The pharmaceutical compositions of the present disclosure can be selected for parenteral or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, e.g., orally. The preparation of such pharma- ceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable for the site of administration. For example, buffers are used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

When parenteral administration is contemplated, the therapeutic composition for use may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution containing the desired binding protein in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a properly preserved, sterile, isotonic solution. Yet another preparation may involve formulation of the desired molecule with agents that provide controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, that can be delivered via depot injection. Hyaluronic acid may also be used, which may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, binding proteins administered in this manner may be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

Another pharmaceutical composition can involve an effective amount of the binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another suitable vehicle, the solution can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders, such as starch, gelatin, or acacia; or lubricants, such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions of the present disclosure will be apparent to those of skill in the art, including formulations with binding proteins in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyrate. Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

Pharmaceutical compositions used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be performed either prior to, or after, lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Additionally, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

The present disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in a biological sample. Such reagents may include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and/or host cells described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition alone or in combination with another composition effective to treat, prevent, and/or diagnose a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and/or treat a selected condition. Alternatively, or additionally, the article or kit may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The present disclosure also encompasses kits for producing single dose administration units. The kits can each contain both a first container having a dry protein and a second container having an aqueous formulation. Kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of the present disclosure.

The effective amount of a binding protein pharmaceutical composition used therapeutically will depend, for example, on the therapeutic context and purpose. Those skilled in the art will appreciate that appropriate dosage levels for treatment will therefore vary depending in part on the molecule being delivered, the indication for which the binding protein is being used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and general health) of the patient. Thus, clinicians can titrate dosages and modify routes of administration to obtain optimal therapeutic effects.

The frequency of dosing depends on the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may thus be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely made by, and is within the scope of tasks routinely performed by, those of skill in the art. The appropriate dosage may be ascertained through the use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is according to known methods, for example, orally; by injection, intravenous, intraperitoneal, intracerebral (intracemdrally), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system; or by implantation device. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implantation device.

The compositions can also be administered locally via implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed release bolus, or continuous administration.

The following examples are illustrative of specific embodiments of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Development of trispecific HER2/CD28xCD3 antibodies and variant anti-CD3 binding sites Immuno-oncology is a promising emerging therapeutic approach to disease management in cancer. The immune system is the first line of defense against cancer development and progression. There is now growing evidence that T cells can control tumor growth and prolong the survival of cancer patients both in the early and late stages of the disease. However, tumor-specific T cells can be limited in various ways that prevent control of the disease.

To remove the restriction of T cells induced by uncontrolled tumors, novel antibodies were developed in a trispecific antibody format depicted in FIG. 1A that specifically activates T cells to engage cancer cells expressing HER2. These novel trispecific antibodies can bind three targets: HER2, CD3, and CD28. The anti-HER2 and anti-CD3 binding sites were further optimized for high affinity binding and reduction of possible manufacturing inconveniences.

HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, as well as in gastric, ovarian, lung and prostate cancer. Optimal activation of T cells requires two factors: (1) antigen recognition and (2) costimulation. Using the trispecific HER2/CD28×CD3 trispecific binding proteins described herein, signal 1 is provided by the agonistic anti-CD3 binding site and signal 2 is provided by the agonistic anti-CD28 binding site (see, e.g., FIG. 1D). The trispecific antibodies described in the Examples below are believed to recruit T cells to tumors via HER2 and activate the engaged T cells by binding to CD3 and CD28. The resulting activation induces the killing potential of immune cells against nearby tumor cells.

Materials and Methods Antibody Production and Characterization Trispecific antibody variants were produced by transient transfection of expression plasmids into Expi293 cells. Five days after transfection, supernatants from transfected cells were collected and quantified with Nano Drop and normalized by absorbance at 280 nm. Binding of the supernatants to the corresponding antigens was determined by ELISA, and the absorbance of the parental HER2WT triple Ab was set as 1.0. The fold change of other variants was calculated by dividing the corresponding absorbance by the absorbance of the parental Ab.

Trispecific antibody variants were purified using Protein A affinity purification followed by SEC purification. Binding of purified antibodies to the corresponding antigens was determined by ELISA. EC50 was determined based on binding curves generated by Graphpad Prism 7.

Results Trispecific Ab variants were produced with several mutations in the binding arms to reduce possible manufacturing inconveniences, such as deamidation sites. Binding ELISA assays were performed to evaluate the binding of the indicated trispecific antibodies to each of the three targets: HER2, CD3, and CD28. In FIG. 1B, HER2/CD3×CD28 trispecific antibodies with the indicated anti-HER2 or anti-CD3 variants were compared to the parent trispecific Ab. Introducing some sets of mutations (e.g., 32/33QQ and 33/35QQ) into the VL domain of the anti-CD3 binding site dramatically reduced binding to CD3, whereas the 32/35QQ mutation nearly retained wild-type binding. MS peptide analysis showed that the binding site with the DNAQ mutation in CDR-L1 (SEQ ID NO:63) was still subject to greater than 15% deamidation, whereas ENLQ (SEQ ID NO:281), ENLF (SEQ ID NO:282), and ENLR (SEQ ID NO:283) resulted in less than 5% deamidation. Importantly, these variants also retained binding to CD3.

In addition, Figure 1C provides binding curves for the indicated antibodies binding to human HER2, human CD28, and CD3. Table E provides EC50 values for selected trispecific antibody variants.

Without wishing to be limited by theory, it is believed that the HER2/CD3/CD28 trispecific antibody recruits T cells to cancer cells via the anti-HER2 and anti-CD3/CD28 arms, as depicted in FIG. 1D. Furthermore, the engaged T cells are believed to be activated by the anti-CD28/CD3 arms. Without wishing to be limited by theory, it is believed that the killing of cancer cells occurs via T cell-mediated mechanisms (e.g., perforin, granzymes). Without wishing to be bound by theory, it is contemplated that a similar mechanism may enable the killing of other types of tumors by replacing the antigen-binding site with one that recognizes other tumor target proteins.

Development of a Trispecific CD38/CD3xCD28 Antibody A trispecific CD38/CD3xCD28 antibody was developed and characterized for binding to CD38, CD3 and CD28 polypeptides.

Materials and Methods Generation of CD38/CD28xCD3 Trispecific Antibody A panel of anti-CD38, anti-CD3, and anti-CD28 antibodies, plus a human IgG4 Fc domain, was used to generate a CD38/CD28xCD3 trispecific antibody in the trispecific antibody format depicted in Figure 2A.

Trispecific binding proteins were produced by transient transfection of four expression plasmids into Expi293 cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 25% (w/w) of each plasmid was diluted in Opti-MEM, mixed with pre-diluted ExpiFectamine reagent for 20-30 min at room temperature (RT) and added to Expi293 cells (2.5× ¹⁰⁶ cells/ml). Transfection optimization was often used to determine the best ratio of plasmids to produce trispecific binding proteins with good yield and purity.

Four to five days after transfection, the supernatant from the transfected cells was collected and filtered through a 0.45 μm filter unit (Nalgene). The trispecific binding proteins in the supernatant were purified using a three-step procedure. First, Protein A affinity purification was used and the bound Abs were eluted using "IgG Elution Buffer" (Thermo Fisher Scientific). Second, the product was dialyzed overnight against PBS (pH 7.4) with two changes of PBS buffer. All precipitates were eliminated by filtration through a 0.45 μm filter unit (Nalgene) before the next step. Third, size exclusion chromatography (SEC) purification (Hiload 16/600 Superdex 200pg, or Hiload 26/600 Superdex 200pg, GE Healthcare) was used to remove aggregates and different species in the preparation. Fractions were analyzed by reducing and non-reducing SDS-PAGE to identify fractions containing monomeric trispecific binding protein, which were then combined. The purified antibodies can be aliquoted and stored long term at -80°C.

ELISA Binding Assay The binding affinity of CD38/CD28xCD3 T cell engagers to each target antigen was measured by ELISA. Briefly, each antigen was used to coat 96-well immunoplates (Thermo Fisher Scientific) overnight at 4°C using 200 ng/well of each antigen in PBS (pH 7.4). The coated plates were blocked with 5% skim milk + 2% BSA in PBS for 1 h at room temperature, followed by washing three times with PBS + 0.25% Tween 20 (Aqua Max 400, Molecular Devices). Serial dilutions of antibodies (trispecific and control Abs) were prepared and added (100 μl/well, in duplicate) onto the ELISA plate and incubated for 1 h at room temperature (RT), followed by washing five times with PBS + 0.25% Tween 20. After washing, HRP-conjugated secondary anti-human Fab (1:5000, Cat. No. 109-035-097, Jackson ImmunoResearch Inc) was added to each well and incubated for 30 min at RT. After washing five times with PBS + 0.25% Tween 20, 100 μl of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was terminated by adding 50 _μl of 1M _H2SO4 , and the OD450 was measured using a SpectraMax M5 (Molecular Devices) and analyzed using Softmax Pro 6.3 software (Molecular Devices). The final data was transferred and plotted in GraphPad Prism software (GraphPad Software, CA, USA). The EC50 was calculated using the same software.

Measurement of trispecific antibody binding using SPR Human CD38-His antigen was used for full kinetic analysis (Cambridge Biologics, Cambridge, MA). Kinetic characterization of purified antibodies was performed using SPR technology in a BIACORE 3000 (GE Healthcare). Capture assays using capture with human IgG1 specific antibodies and configuration of the investigated antibodies were used. For capture of protein constructs containing Fc, a human antibody capture kit (GE Healthcare) was used. For capture of antigens with His tags, an anti-His antibody capture kit (GE Healthcare) was used. Capture antibodies were immobilized via primary amine groups (11000 RU) to research grade CM5 chips (GE Life Sciences) using standard procedures. The analyzed antibodies were captured at a flow rate of 10 μL/min, with an adjusted RU value expected to typically result in a maximum analyte binding signal of 30 RU. Binding kinetics for trispecific antibodies were measured. Assay buffer HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) was used at a flow rate of 30 μl/min. The chip surface was regenerated with the regeneration solution of the respective capture kit. Kinetic parameters were analyzed and calculated with the BIAevaluation program package v4.1 using a 1:1 Langmuir binding model with a flow cell without captured antibody and mass transfer as a reference.

Daratumumab Competitive Binding Assay For the daratumumab competitive binding assay, daratumumab was amine coupled to the active surface of a CM5 chip. A reference surface was left blank and used to subtract any non-specific binding of the injected molecule. Recombinant CD38-His (Sino Biological, part #10818-H08H) was injected over the daratumumab surface followed by the injection of the test antibody. If the monospecific anti-CD38 antibody recognized an epitope on CD38 different from that of daratumumab, injection of the antibody resulted in an increase in the SPR signal. If the antibody recognized an overlapping epitope as daratumumab, injection of the antibody did not increase the SPR signal.

Results The binding affinity of selected CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains to human CD38 was determined by SPR. The association rate constants ( _KOn ), dissociation rate constants ( _KOff ), and _KD of the selected trispecific antibodies are provided in Table A. The selected trispecific antibodies showed varying degrees of affinity to the human CD38 antigen.

The binding affinities of selected CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains to human CD3, human CD28, human CD38 and cynomolgus CD38 were then determined by ELISA as described above. As shown in Figures 2B-2E, selected CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains showed varying affinities to human (Figure 2B) and cynomolgus CD38 (Figure 2C), but similar affinities to human CD3 (Figure 2D) and CD28 (Figure 2E). EC50 values were then calculated by GraphPad Prism 7.02 using a variable slope model with a four-parameter logistic curve. The EC50 values of selected trispecific antibodies against human CD3, human CD28, human CD38 and cynomolgus CD38 are provided in Table B. The control antibody was a human IgG4 isotype control.

An SPR competition assay was performed to determine whether the anti-CD38 antibodies hhy6284, hhy992, hhy5379, or hhy1195 (tested in a monospecific antibody format) competed with daratumumab for binding to CD38. After CD38 injection onto daratumumab (immobilized on an SPR sensor chip), the test antibody (or daratumumab) was injected onto the daratumumab/CD38 complex. As shown in Figure 3, injection of Hyb6264, hhy992, Hyb5379, and Hhy1195 increased the SPR signal, indicating that these antibodies recognized epitopes on CD38 that were distinct from the epitope recognized by daratumumab. As expected, injection of free daratumumab (competitive binding control) did not increase the SPR signal.

Table B2 summarizes the binding of anti-CD38 antibodies to human or cynomolgus CD38 polypeptides.

Anti-CD38 antibodies were also tested for competitive binding to daratumumab in an SPR assay. For the daratumumab competitive binding assay, daratumumab was amine coupled to the active surface of a CM5 chip. A reference surface was left blank and used to subtract out any non-specific binding of the injected molecule. Recombinant CD38-His (Sino Biological, part #10818-H08H) was injected over the daratumumab surface, followed by injection of the test antibody. If the antibody recognized an epitope on CD38 different from that of daratumumab, injection of the antibody resulted in an increase in the SPR signal. If the antibody recognized an overlapping epitope as daratumumab, injection of the antibody did not increase the SPR signal. The results of these assays showed that the antibodies tested, hhy992, hyb6284, hhy1195 and hhy1370, did not compete with daratumumab.

Trispecific CD38/CD3xCD28 antibodies promote lysis of human multiple myeloma and lymphoma tumor cells.
An in vitro cytolytic assay was used to determine whether the trispecific CD38/CD3xCD28 antibody had anti-tumor cell activity using human multiple myeloma and lymphoma cells.

Materials and Methods In vitro killing assay for tumor cells using human T cells. Target tumor cells were labeled with the membrane dye PKH-26 (Sigma) and co-cultured with human PBMCs or enriched CD8 T cells as effector cells at an E:T ratio of 10:1 (E:T=3:1 using enriched CD8 T cells) for 24 h in the presence of the indicated concentrations of trispecific or relevant control antibodies. Peripheral blood mononuclear cells were isolated from normal human donors by Ficoll separation and enriched for autologous CD8+ or pan T cells using a kit from Miltenyi Biotech (San Diego, CA). The extent of cytolysis in target cells was determined by staining with the LIVE/DEAD™ Fixable Violet Dead cell staining kit (Life Technologies) and measured by the number of dead cells in the labeled target cell population by running samples on an LSRFortessa instrument (BD Biosciences) and subsequently analyzed using Flowjo software (Treestar).

In vitro killing assay against tumor cells using human T cells in the presence of daratumumab. Daratumumab at 5 nM or isotype control antibody was pre-incubated with PKH-26 labeled target tumor cells ( ¹⁰⁵ cells/well) for 30 min, followed by addition of trispecific TCE at the indicated concentrations, and human PBMCs (E:T=10:1). After 24 h, the extent of cytolysis in target cells was determined by staining with LIVE/DEAD™ Fixable Violet Dead cell staining kit (Life Technologies) and measured by the number of dead cells in the labeled target cell population by sample running on an LSRFortessa instrument (BD Biosciences) followed by analysis using Flowjo software (Treestar).

Results The in vitro cell killing activity of the CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with alternative anti-CD38 binding domains was determined using the human multiple myeloma cell line NCI-H929, which expresses both CD38 and CD28. Assays were performed in the presence of 5 nM daratumumab or an isotype control antibody (present for the duration of the assay). As shown in Figures 4A-4B, all tested trispecific antibodies caused cell lysis in a concentration-dependent manner in the presence and absence of daratumumab. EC50 values were then calculated in the presence and absence of daratumumab (Table C). The cell killing activity of the trispecific antibody CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA with CD38VH1 or CD38hhy1370 anti-CD38 binding domains was reduced by daratumumab, whereas trispecific antibodies with CD38hyb5739, CD38hyb6284, or CD38hhy1195 anti-CD38 binding domains exhibited a 3-8 fold reduction in cell killing activity in the presence of daratumumab (Table C).

In addition, an in vitro cytolysis assay was used to measure the cell killing activity of selected CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains using the human lymphoma cell line OCI-LY19, which expresses CD38 but not CD28. The assay was performed in the presence of 5 nM daratumumab or isotype control antibody present during the assay period. As shown in Figures 5A-5B, all tested trispecific antibodies caused cell lysis in a concentration-dependent manner in the presence and absence of daratumumab. EC50 values were then calculated in the presence and absence of daratumumab (Table D). The cell killing activity of the CD38/CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with a CD38VH1 anti-CD38 binding domain was reduced approximately 24-fold by daratumumab, whereas trispecific antibodies with CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195, or CD38hhy1370 anti-CD38 binding domains also exhibited reduced cell killing activity in the presence of daratumumab (Table D).

CD38/CD28×CD3 trispecific antibodies promote CMV-specific immune responses As part of adaptive immunity, T cell immunity plays an important role in controlling viral infections and cancer, and in eliminating infected and malignant cells, which in some cases results in the clearance of viral infections or the cure of cancer. In chronic infectious diseases such as herpesvirus infections (HSV, CMV, EBV, etc.), HIV and HBV viruses establish their persistence in humans by various mechanisms, including immune suppression, T cell exhaustion, and establishment of latency. Nevertheless, viral infections generally induce viral antigen-specific immunity, including antigen-specific CD8 T cells that can easily recognize infected cells for control or killing via cytokine release or cytotoxic T cell (CTL)-mediated killing processes. Thus, activation and/or expansion of viral antigen-specific T cells in vivo and/or ex vivo provides a therapeutic strategy against chronic viral infections.

Anti-CD38/CD28xCD3 trispecific antibodies were developed and evaluated for their potential in activating T cells and promoting proliferation and/or expansion of antigen-specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen-specific CD8 T central memory and effector memory cells, in vitro. Specifically, in vitro expansion of CMV- and EBV-specific CD8 central memory and effector memory cells was demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibit novel properties by engaging CD3/CD28/CD38 and providing a signaling pathway to stimulate and expand T cells, which may provide an effective strategy to treat chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.

In this example, the ability of a CD38/CD28xCD3 trispecific antibody to promote activation and expansion of CMV-specific T cells was determined.

Materials and Methods In Vitro T Cell Proliferation Assay T cells were isolated from human PBMC donors by negative selection using a magnetic pan T cell isolation kit (Miltenyi Biotec GmbH, Germany). Antibodies were coated onto 96-well cell culture plates by preparing the antibodies in sterile PBS and dispensing 50 μL into each well (350 ng/well). The plates were then incubated at 37° C. for at least 2 hours and then washed with sterile PBS. Untouched T cells were added to the antibody-coated plates (5×10 ⁵ cells/mL) and incubated at 37° C. for several days. On day 4, the cells were passaged onto fresh antibody-coated plates with new cell culture medium. In certain experiments with 7-day incubation, only fresh medium was added without replacement to fresh antibody-coated plates. Cells were harvested at specific time points and cell numbers were calculated using CountBright™ counting beads.

In Vitro T Cell Proliferation Assays and T Cell Subset Determination Peripheral blood mononuclear cells were isolated from blood of healthy human donors collected by Research Blood Components, LLC (Boston, Mass.). PBMCs were added (350 ng/well) ( ^5x105 cells/mL) to antibody-coated plates as previously described above and incubated at 37°C for 3 and 7 days. Cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naive (CCR7+CD45RO-), Tcm (CCR7+CD45RO+), Tem (CCR7-CD45RO+), Treg (CD4+Foxp3+CD25hi). CMV pp65-specific and EBV BMLF-specific CD8+ T cells were detected using fluorescently conjugated pentamers restricted to the HLA/viral peptides (A ^* 02:01/NLVPMVATV, SEQ ID NO: 284), (A ^* 02:01/GLCTLVAML, SEQ ID NO: 285) of the PBMC donors, respectively (ProImmune, Oxford, UK). PBMCs from donors with known CMV or EBV infection were obtained from HemaCare (Van Nuys, CA). As negative controls, PBMCs from donors negative for restrictive HLA types were used. Staining was performed according to the manufacturer's protocol.

Quantification of CMV-specific T cells. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of human donors with known CMV infection as described above and added to plates containing the trispecific antibody or control antibody. Plates were incubated at 37° C. Cells were harvested at specific time points and analyzed by flow cytometry.

Results The CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with surrogate anti-CD38 binding domains ΔVH1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195, and CD38hhy1370 were tested using PBMCs isolated from CMV-infected human donor D (Figures 6A-6J) and CMV-infected human donor E (Figures 7A-7J) as described above. All tested CD38 trispecific Abs activated and promoted the proliferation of CMV-specific T cells, resulting in increases (cells/well) in CMV-specific CD8 + T cells in a dose-response manner over the 7-day experiment with different potencies and kinetics (CMV donor D, Figs. 6A-6B; CMV donor E, Figs. 7A-7B). In addition, all tested CD38 trispecific Abs promoted the expansion (cells/well) of CMV-specific central memory (T _cm ) (CMV donor D, Figs. 6C-6D; CMV donor E, Figs. 7C-7D) and effector memory (T _em ) CD8 + T cells (CMV donor D, Figs. 6E-6F; CMV donor E, Figs. 7E-7F), both of which were dramatically expanded over 7 days. Figures 6G-6J (CMV donor D) and Figures 7G-7J (CMV donor E) provide a time course showing the percentage of CMV-specific T _cm and T _em cells at days 0, 3, and 7 of the 7-day experiment described above.

Taken together, these data indicate that CD38/CD28×CD3 trispecific antibodies promote the activation and expansion of CMV-specific T cells, including CMV-specific CD8 + T cells, CMV-specific effector memory (T _<em> ) CD8 + T cells, and CMV-specific central memory (T _<em> ) CD8 + T cells.

CD38/CD28xCD3 Trispecific Antibody Promotes EBV-Specific Immune Responses Next, the ability of CD38/CD28xCD3 trispecific antibody to promote the activation and expansion of Epstein-Barr Virus (EBV)-specific T cells was determined.

Materials and Methods Quantification of EBV-specific T cells Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of human donors with known EBV infection as described above and added to plates containing the trispecific antibody or control antibody. Plates were incubated at 37° C. for up to 11 days. Cells were harvested at specific time points and analyzed by flow cytometry.

Results The CD38/CD28supxCD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with alternative anti-CD38 binding domains ΔVH1CD38 (control), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195, and CD38hhy1370 were also tested as described above using PBMCs isolated from EBV-infected donor C (Figures 8A-8J) and EBV-infected donor D (Figures 9A-12). All tested CD38 trispecific Abs activated T cells and promoted the proliferation of EBV-specific T cells, resulting in an increase (cells/well) in EBV-specific CD8 + T cells in a dose-response manner over the 7-day experiment with different potencies and kinetics (EBV donor C, Figs. 8A-8B; EBV donor D, Figs. 9A-9B). In addition, all tested CD38 trispecific Abs promoted the expansion (cells/well) of EBV-specific central memory (T _cm ) (EBV donor C, Figs. 8C-8D; EBV donor D, Figs. 9C-9D) and effector memory (T _em ) CD8 + T cells (EBV donor C, Figs. 8E-8F; EBV donor D, Figs. 9E-9F), both of which expanded dramatically over 7 days. Figures 8G-8J (EBV donor C) and Figures 9G-12 (EBV donor D) provide a time course showing the percentage of EBV-specific T _cm and T _em cells at days 0, 3, and 7 of the 7-day experiment described above.

Taken together, these data indicate that CD38/CD28×CD3 trispecific antibodies promote the activation and expansion of EBV-specific T cells, including EBV-specific CD8 + T cells, EBV-specific effector memory (T _<em> ) CD8 + T cells, and EBV-specific central memory (T _<em> ) CD8 + T cells.

Anti-tumor Effect of Her2/CD28xCD3 Trispecific Antibody in Tumor-Bearing Mice In this example, the Her2/CD28xCD3 trispecific antibody was tested for anti-tumor effect in a Nod scid gamma (NSG) mouse model bearing ZR-75-1 tumors engrafted with in vitro expanded T cells.

Materials and Methods NSG mice were divided into 5 groups of 10 mice each. On day 0, ZR-75-1 human breast cancer cells were implanted into the mammary fat pad of each mouse with 50% Matrigel at 5 million cells/mouse. On day 17/18, human CD3+ T cell expansion began. Mice were randomized on day 24 when tumors were approximately 150 ^mm3 . On day 25, all mice were inoculated with in vitro expanded human CD3+ T cells at 10 million cells/mouse in 300 μL (1 QW, 1 IP injection).

Beginning on day 25, one group of mice received a dose of vehicle alone (8% w/v sucrose, 0.05% w/v polysorbate 80, 10 mM histidine, pH 5.5), while the other four groups received the Her2/CD28xCD3 trispecific antibody, both at 10 mL/kg. Groups receiving the trispecific antibody were dosed at 100, 10, 1, or 0.1 μg/kg. Antibody or vehicle was administered intravenously in two doses (e.g., on days 25 and 32), 1 QW. Blood and tumor tissues were collected on days 38 or 39.

Results Her2/CD28xCD3 trispecific antibody (binding protein #2 from Table 1, corresponding to SEQ ID NOs: 104-107) was compared to vehicle control for its effect on human breast tumor growth in the NSG mouse model engrafted with in vitro expanded human T cells described above. Treatment with Her2/CD28xCD3 trispecific antibody at the highest dose (100ug/kg) resulted in the most significant inhibition of tumor growth and recovery, although the 10ug/kg dose also showed anti-tumor effects (Figures 13A and 13D). No significant weight loss was observed (Figure 13B). Individual tumor volumes over time from each trispecific antibody treatment group are provided in Figure 13C.

Next, the effect of trispecific antibody treatment on individual immune cell subsets was examined. Human CD45+, human CD8+, and human CD4+ cell populations were measured by flow cytometry, as well as mouse CD45+ cells (Figures 14A-14C). The highest dose of trispecific antibody (100ug/kg) resulted in depletion of human CD4+ cells, an effect that was dose-dependent (Figures 14B and 14C). Human CD8+ cell counts were not significantly affected by trispecific antibody treatment.

The effect of trispecific antibody treatment on tumor infiltrating lymphocytes (TILs) was also evaluated by immunohistochemical (IHC) staining for human CD45, CD4, and CD8. Using H&E staining, tumors from the low dose groups (1 ug/kg or 0.1 ug/kg trispecific antibody) were generally of comparable size to the vehicle control group. As shown in Figures 15A-15C, human TILs were increased in the groups receiving low doses of Her2/CD28xCD3 trispecific antibody, whereas human TILs were scattered in the high dose groups. IHC images were also quantitatively examined (Figures 16A-16C). These results showed a significant reduction in CD45+ and CD8+ cells in the higher trispecific antibody dose groups (100 ug/kg and 10 ug/kg).

Tumors from the high dose trispecific antibody treatment groups (100ug/kg or 10ug/kg) were characterized by scattered TILs when compared to vehicle controls. Moderate to numerous CD45+, CD4+, or CD8+ human TILs were observed in the 1ug/kg and 0.1ug/kg trispecific antibody treatment groups. These TILs were mostly present at the edge of the tumor, but sometimes extended deeper toward the tumor core.

In conclusion, these results demonstrate that treating NSG mice bearing ZR-75-1 breast tumors engrafted with in vitro activated T cells with two intravenous doses of T cell engaging trispecific antibody targeting HER2 at 100ug/kg or 10ug/kg resulted in a significant reduction in tumor volume, concomitantly with a significant reduction in TILs. The 1ug/kg trispecific antibody dose showed a non-significant but inconsistent trend for increased TILs compared to vehicle control.

Effect of anti-HER2 and anti-CD3 antigen-binding domain sequences in Her2/CD28xCD3 trispecific antibodies on cancer cell killing This example describes the effect of anti-Her2 and anti-CD3 variable domain sequences on target cell killing. In this example, a Her2/CD28xCD3 trispecific antibody having wild-type trastuzumab and anti-CD3 antigen-binding domains and no 32/35QQ mutation in the VL domain ("control") (see Example 1) was compared to Her2/CD28xCD3 trispecific antibodies #1-6 from Table 1, corresponding to SEQ ID NOs: 100-103, 104-107, 286-289, 290-293, 294-297, and 298-301, respectively.

Materials and Methods CD8+ T cells were isolated from human PBMCs from healthy donors using a magnetic bead isolation kit (Miltenyi Biotec). T cells were used as effector cells against breast cancer cell lines expressing various levels of HER2 at a ratio of 3:1 (effector:target). Cells were incubated with experimental or control trispecific antibodies for 2 days prior to acquisition by flow cytometry using a viability dye (Invitrogen) and PKH26 target cell stain (Sigma). The mean EC50 of target cell lysis was calculated for each trispecific Ab from 2-3 PBMC donors.

Results All trispecific antibodies were characterized for in vitro cytolysis of three HER2+ breast cancer target cell lines: HCC1954, BT20, and MDA-MB-231. HCC1954 breast cancer cells were found to express high levels of HER2 as assessed by flow cytometry (up to approx. 150,000 receptors/cell), IHC (3+), or HercepTest HER2 Expression Assay (3+) (FIG. 17A). BT20 breast cancer cells were found to express intermediate levels of HER2 as assessed by flow cytometry (approx. 60,000 receptors/cell), IHC (1+), or HercepTest HER2 Expression Assay (1+) (FIG. 17C). MDA-MD-231 breast cancer cells were found to express low levels of HER2 as assessed by flow cytometry (approximately 9,000 receptors/cell), IHC (0+), or HercepTest HER2 expression assay (0) (FIG. 17E). Figures 17B, 17D, and 17F, respectively, show the results of cell killing assays targeting HCC1954, BT20, or MDA-MB-231, comparing binding protein #2 to control or binding protein #1, and #5 to control. The results demonstrated that the Her2/CD28×CD3 trispecific antibody with 30R/55Q/102E mutations in the anti-HER2 arm and 32/35QQ mutations in the VL domain of the anti-CD3 arm showed improved target cell killing against all three cell lines, especially at lower antibody concentrations.

The average EC50 (pM) of in vitro cell killing of all trispecific antibodies targeting the three breast cancer cell lines mentioned above (HCC1954, BT20, and MDA-MB-231), as well as the gastric cancer cell lines OE19 (high HER2 expression) and GSU (moderate HER2 expression), was determined. Overall, the Her2/CD28×CD3 trispecific antibody with mutations in the VL domains of the anti-HER2 and anti-CD3 arms showed lower EC50 (and therefore superior cell killing) against all three breast cancer cell lines (FIG. 18A) and both gastric cancer cell lines (FIG. 18B). These results demonstrate that while all trispecific antibodies can induce cell killing of HER2+ cells, the mutated trispecific antibody consistently showed improved cell killing efficacy against multiple target cell types.

Claims (89)

A binding protein comprising four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L4-C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), the V _L2 domain comprising a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
_VH3 and _VL3 form a third antigen-binding site that binds to the tumor target protein.
The binding protein. The binding protein of claim 1, wherein the first antigen-binding site binds to a CD28 polypeptide. 3. The binding protein of claim 2, wherein the V _H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51); and the V _L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54). and/or the V _H1 Domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); The _L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92);
The binding protein of claim 3. The _VH2 domain is QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQL and/or the _VL2 Domain comprises the amino acid sequence of:
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPA SISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYL
SWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98),
The binding protein according to any one of claims 1 to 4. The binding protein according to any one of claims 1 to 5, wherein the tumor targeting protein is a human CD38 polypeptide. (a) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18);
(b) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24);
(c) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30);
(d) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36);
(e) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42); or 7. The binding protein of claim 6, wherein _{the H3} domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and the _VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). (a) the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); and/or The _L3 domain comprises the amino acid sequence of DIVLTQSPATHLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80);
(b) the V _H3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81), and/or the V _L3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82);
(c) the V _H3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83), and/or the V _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84);
(d) the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); and/or The _L3 domain comprises the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86);
(e) the _VH3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); and/or the _L3 domain comprises the amino acid sequence of: DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88); or (f) the _VH3 domain comprises the amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLY
and/or the _VL3 domain comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90);
The binding protein of claim 7. (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:159;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:167;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:171;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:175;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:179;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:184;
or (h) the binding protein of claim 6, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188. The binding protein according to any one of claims 1 to 5, wherein the tumor targeting protein is a human HER2 polypeptide. (a) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(b) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
(c) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12);
(d) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12);
(e) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12);
(f) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12); or (g) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO:1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), and the V The _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12);
The binding protein of claim 10. (a) the _VH3 domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVES GGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSC
AASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78);
(b) the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72); and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77);
(c) the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77);
(d) the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), and/or The _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77);
(e) the V _H3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), and/or the V _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP
Comprising the amino acid sequence of EDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77);
(f) the _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76); and/or the _L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77); or _{the H3} domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72); and/or the _VL3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78);
The binding protein of claim 11. (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:102; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:103;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:107;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:115;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:142; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:143;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:147;
(h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:155;
(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289;
(j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293;
(k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297;
or (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301. 13. The binding protein of any one of claims 1 to 8, 10 to 12, wherein at least one of L ₁ , L ₂ , L ₃ or L ₄ is independently 0 amino acids in length. 13. The binding protein of any one of claims 1-8, 10-12, wherein: (a) _L1 , _L2 , _L3 , and _L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71); or (b) _L1 , _L2 , _L3 , and _L4 are each independently GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71). The binding protein according to any one of claims 1 to 8 and 10 to 12, wherein _L1 comprises the sequence GQPKAAP (SEQ ID NO:67), _L2 comprises the sequence TKGPS (SEQ ID NO:68), _L3 comprises the sequence S, and _L4 comprises the sequence RT. 13. The binding protein of any one of claims 1 to 8, 10 to 12, wherein at least one of L ₁ , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO:66). 18. The binding protein of claim 17, wherein _L1 , _L2 , _L3 and _L4 each independently comprise the sequence DKTHT (SEQ ID NO:66). 19. The binding protein of any _{one of} claims 1 to 8, 10 to 12, 14 to 18, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG4 hinge- _{C H2} -C _H3 domains, each comprising amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. 19. The _binding protein of any _one of claims 1 to 8, 10 to 12, 14 to 18, wherein the hinge-C _H2 -C _H3 Domains of the second and third polypeptide chains are human IgG4 hinge- _{C H2} -C _H3 Domains, each of which comprises amino acid substitutions at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236. 21. The binding protein of any _{one of} claims 1 to 8, 10 to 12, 14 to 20, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG4 hinge- _{C H2} -C _H3 domains, each comprising amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. 19. The binding protein of any _{one of} claims 1 to 8, 10 to 12, 14 to 18, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG1 hinge-C _H2 -C _H3 domains, each comprising amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. 19. The binding protein of any _{one of} claims 1 to 8, 10 to 12, 14 to 18, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG1 hinge-C _H2 -C _H3 domains, each comprising amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S. 24. The binding protein of any one of claims 1 to 8, 10 to 12, 14 to 23, wherein the hinge-C _H2 -C _H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; and the hinge-C _H2 -C _H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. 24. The binding protein of any one of claims 1 to 8, 10 to 12, 14 to 23, wherein the hinge-C _H2 -C _H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; and the hinge-C _H2 -C _H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. V _H1 and V _L1 form a first antigen-binding site that binds to a CD28 polypeptide, the V _H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the V _L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54);
_VH2 and _VL2 form a second antigen-binding site that binds to the CD3 polypeptide, and _VH2
the V L2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), the V _L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65); and
2. The binding protein of claim 1, wherein V _H3 and V _L3 form a third antigen-binding site that binds to a HER2 polypeptide, and the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO:12). and/or the V _H1 Domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); the _L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92);
The _VH2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93); The _L2 domain comprises the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95);
The _VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and the _VL3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77).
27. The binding protein of claim 26. 28. The binding protein of claim 26 or 27, wherein _L1 , _L2 , _L3 and _L4 each independently comprise the sequence DKTHT (SEQ ID NO:66). The hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG4 hinge- _{C H2 -C H3} domains, each of which comprises amino acid substitutions at positions corresponding to positions 228, 234, 235 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A and _R235B .
The binding protein according to any one of claims 26 to 28, which has an affinity of 409K. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding protein according to any one of claims 1 to 29. An expression vector comprising the nucleic acid molecule of claim 30 . An isolated host cell comprising the nucleic acid molecule of claim 30 or the expression vector of claim 31. The isolated host cell of claim 32, which is a mammalian or insect cell. A pharmaceutical composition comprising the binding protein according to any one of claims 1 to 29 and a pharma- ceutical acceptable carrier. Use of at least one binding protein according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 34 in the manufacture of a medicament for treating cancer in a patient. 36. The use of claim 35, wherein at least one binding protein or composition is administered in combination with a chemotherapeutic agent. The use according to claim 35 or claim 36, wherein the patient is a human. The use according to any one of claims 35 to 37, wherein the tumor targeting protein is a human CD38 polypeptide and the cancer cells from the patient express CD38. The use according to claim 38, wherein the cancer is multiple myeloma. The use of claim 38, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. The use according to any one of claims 38 to 40, wherein prior to administration of at least one binding protein or composition, the patient has been treated with daratumumab without a washout period. The use according to any one of claims 35 to 37, wherein the tumor targeting protein is a human HER2 polypeptide and the cancer cells from the patient express HER2. The use of claim 42, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC). The pharmaceutical composition of claim 34 for use in treating cancer in a patient. The pharmaceutical composition for use according to claim 44, wherein the pharmaceutical composition is administered in combination with a chemotherapeutic agent. The pharmaceutical composition for use according to claim 44 or 45, wherein the patient is a human. The tumor targeting protein is human CD38 polypeptide, and the cancer cells from the patient are CD3
8. The pharmaceutical composition for use according to any one of claims 44 to 46, wherein the compound expresses The pharmaceutical composition for use according to claim 47, wherein the cancer is multiple myeloma. The pharmaceutical composition for use according to claim 47, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. The pharmaceutical composition for use according to any one of claims 47 to 49, wherein prior to administration of the pharmaceutical composition, the patient has been treated with daratumumab without a washout period. The pharmaceutical composition for use according to any one of claims 44 to 46, wherein the tumor targeting protein is a human HER2 polypeptide and the cancer cells from the patient express HER2. 52. The pharmaceutical composition for use according to claim 51, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC). 1. A method of expanding T cells in vitro or ex vivo, comprising contacting a T cell with a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain has the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to the CD28 polypeptide;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), the V _L2 domain comprising a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
The method, wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide. 54. The method of claim 53, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR. The method of claim 53 or 54, wherein the T cells are memory T cells or effector T cells. A method of expanding virus-specific memory T cells in vitro or ex vivo, comprising contacting the virus-specific memory T cells with a binding protein comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to the CD28 polypeptide;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the V _L2 domain comprises the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQ
a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHENLFTY (SEQ ID NO:60), QSLVHENLRTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising an amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising an amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
The method, wherein V _H3 and V _L3 form a third antigen-binding site that binds to the CD38 polypeptide. 57. The method of claim 56, wherein contacting the virus-specific memory T cells with the binding protein causes activation and/or proliferation of the virus-specific memory T cells. 1. Use of a binding protein in the manufacture of a medicament for treating a chronic viral infection in a patient in need thereof, the binding protein comprising four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site which binds a CD28 polypeptide; V _H2 and V _L2 form a second antigen-binding site which binds a CD3 polypeptide, the V _{H2 domain comprising a CDR-H1} sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); the _L2 domain comprises a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
_VH3 and _VL3 form a third antigen-binding site that binds to the CD38 polypeptide.
The above uses. The use according to claim 58, wherein the patient is a human. The use of claim 58 or claim 59, wherein the binding protein is administered to the patient in a pharmaceutical formulation comprising the binding protein and a pharma- ceutically acceptable carrier. The use according to any one of claims 58 to 60, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the patient. The method of any one of claims 55 to 57, or the use of claim 61, wherein the memory T cells are CD8+ or CD4+ memory T cells. The method according to any one of claims 55 to 57, 62, or the use according to claim 61 or 62, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM). The method according to any one of claims 56, 57, 62 and 63, or the use according to any one of claims 58 to 63, wherein the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2). The method according to any one of claims 53 to 57 and 62 to 64, or the use according to any one of claims 58 to 64, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide. 1. A binding protein for use in the expansion of T cells, said binding protein contacting a T cell, said binding protein comprising four polypeptide chains forming three antigen binding sites, a first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to the CD28 polypeptide;
_VH2 and _VL2 form a second antigen-binding site which binds to the CD3 polypeptide;
the V _H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); the V _L2 domain comprises a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
A binding protein for said use, wherein V _H3 and V _L3 form a third antigen-binding site which binds to a CD38 polypeptide. 67. The binding protein for use according to claim 66, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR. The binding protein for use according to claim 66 or claim 67, wherein the T cell is a memory T cell or an effector T cell. 1. A binding protein for use in the expansion of virus-specific memory T cells, the binding protein contacting a virus-specific memory T cell, the binding protein comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to the CD28 polypeptide;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), the V _L2 domain comprising a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
A binding protein for said use, wherein V _H3 and V _L3 form a third antigen-binding site which binds to a CD38 polypeptide. The binding protein for use according to claim 69, wherein the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo. The binding protein for use according to claim 69 or claim 70, wherein contacting virus-specific memory T cells with the binding protein causes activation and/or proliferation of the virus-specific memory T cells. 1. A binding protein for use in the treatment of a chronic viral infection, the binding protein being administered to a patient in need thereof, the binding protein comprising four polypeptide chains which form three antigen binding sites, the first polypeptide chain having the formula:
V _L2 -L ₁ -V _L1 -L ₂ -C _L [I]
The structure includes:
The second polypeptide chain has the formula:
V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge- _{C H2} -C _H3 [II]
The structure includes:
The third polypeptide chain has the formula:
V _H3 -C _H1 -hinge-C _H2 -C _H3 [III]
The structure includes:
The fourth polypeptide chain has the formula:
V _L3 -C _L [IV]
The structure includes:
Where:
_VL1 is the first immunoglobulin light chain variable domain;
_VL2 is the second immunoglobulin light chain variable domain;
_VL3 is the third immunoglobulin light chain variable domain;
_VH1 is the first immunoglobulin heavy chain variable domain;
_VH2 is the second immunoglobulin heavy chain variable domain;
_VH3 is the third immunoglobulin heavy chain variable domain;
_C is an immunoglobulin light chain constant domain;
C _H1 is the immunoglobulin C _H1 heavy chain constant domain;
C _H2 is the immunoglobulin C _H2 heavy chain constant domain;
C _H3 is the immunoglobulin C _H3 heavy chain constant domain;
Hinge is the immunoglobulin hinge region connecting the C _H1 and C _H2 domains;
L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;
a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair;
V _H1 and V _L1 form a first antigen-binding site that binds to the CD28 polypeptide;
V _H2 and V _L2 form a second antigen-binding site which binds to the CD3 polypeptide, the V _H2 domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), the V _L2 domain comprising a CDR-L1 sequence comprising an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65);
A binding protein for said use, wherein V _H3 and V _L3 form a third antigen-binding site which binds to a CD38 polypeptide. The binding protein for use according to claim 72, wherein the patient is a human. The binding protein for use according to claim 72 or claim 73, wherein the binding protein is administered to a patient in a pharmaceutical formulation comprising the binding protein and a pharma- ceutically acceptable carrier. The binding protein for use according to any one of claims 72 to 74, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in a patient. The binding protein for use according to any one of claims 68 to 71 and 75, wherein the memory T cells are CD8+ or CD4+ memory T cells. The binding protein for use according to any one of claims 68 to 71, 75 and 76, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM). The binding protein for use according to any one of claims 69 to 77, wherein the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2). The binding protein for use according to any one of claims 66 to 78, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide. The V _H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the V _L1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52).
80. The binding protein for use according to any one of claims 66 to 79, comprising a CDR-L1 sequence, a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54). and/or the V _H1 Domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); The _L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92);
81. A binding protein for use as described in claim 80. The _VH2 domain is QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQL and/or the _VL2 Domain comprises the amino acid sequence of:
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97); and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98),
82. A binding protein for use as described in claim 81. (a) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18);
(b) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and the V _L3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22).
-L1 sequence, a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24);
(c) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30);
(d) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36);
(e) the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and the V _L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42); or 83. The binding protein for use according to any one of claims 66 to 82, wherein _{the H3} domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45), and the _VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). (a) the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); and/or The _L3 domain comprises the amino acid sequence of DIVLTQSPATHLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80);
(b) the V _H3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81), and/or the V _L3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82);
(c) _VH3 domain is QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQR
PGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO:83), and/or the _VL3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84);
(d) the _VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); and/or The _L3 domain comprises the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86);
(e) the _VH3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); and/or the _L3 domain comprises the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88); or The _H3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89), and/or 84. A binding protein for use as described in claim 83, wherein _{the L3} domain comprises the amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90). (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:159;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:167;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:171;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:175;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:179;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:184;
or (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186; and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18
the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188;
A binding protein for use according to any one of claims 66 to 84. A vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of the binding protein according to any one of claims 1 to 29. 87. The vector system of claim 86, comprising a first vector encoding a first polypeptide chain of a binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein. A kit of polynucleotides comprising:
(a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:192;
(b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:196;
(c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204;
(d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220;
(e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228;
(f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232;
(g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236;
(h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244;
(i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248;
(j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252;
(k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256;
(l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260;
(m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264;
(n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268;
(o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; or (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276. The kit of claim 88, wherein the first, second, third, and fourth polynucleotides are present on one or more expression vectors.

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