AU2018366219B2

AU2018366219B2 - Using targeted radiotherapy (TRT) to drive anti-tumor immune response to immunotherapies

Info

Publication number: AU2018366219B2
Application number: AU2018366219A
Authority: AU
Inventors: Peter Carlson; Joseph Grudzinski; Reinier HERNANDEZ; Zachary MORRIS; Ravi PATEL; Paul Sondel; Jamey Weichert
Original assignee: Wisconsin Alumni Research Foundation
Current assignee: Wisconsin Alumni Research Foundation
Priority date: 2017-11-10
Filing date: 2018-11-09
Publication date: 2025-09-11
Anticipated expiration: 2038-11-09
Also published as: WO2019094657A1; IL274518B1; CN120550150A; AU2025271049A1; KR20200088374A; IL325659A; EP3706808A1; IL274518A; AU2018366219A1; JP7839135B2; JP2023179556A; JP2021502368A; KR102758660B1; CN111565762A; CA3082056A1

Description

WO 2019/094657 A1 Published: with with international international search search report report (Art. (Art. 21(3)) 21(3))

- before the expiration of the time limit for amending the

- claims and to be republished in the event of receipt of amendments (Rule 48.2(h))

USING TARGETED RADIOTHERAPY (TRT) TO DRIVE ANTI-TUMOR IMMUNE RESPONSE TO IMMUNOTHERAPIES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 15/809,427, filed

November 10, 2017, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under OD024576 and

CA197078 awarded by the National Institutes of Health. The government has certain

rights in the invention.

FIELD OF THE DISCLOSURE

[0003] This disclosure relates generally to methods of treating cancer. In particular,

the disclosure is directed to methods of treating a cancer comprising one or more

malignant solid tumors in a subject by (1) systemically administering to the subject an

immunomodulatory dose of a targeted radiotherapy (TRT) agent, such as a radioactive

metal chelate compound, a radiohalogenated compound, radiolabeled antibody, or a

radiosiotope that is differentially taken up by and retained within solid tumor tissue; and

(2) systemically administering to the subject one or more immunostimulatory agents,

such as one or more immune checkpoint inhibitors.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

BACKGROUND

[0004] Current cancer treatment typically involves systemic chemotherapy whereby

non-targeted small molecule or antibody directed cytotoxic agents preferentially enter, or

bind to (in the case of antibody directed agents) and kill cancer cells by a variety of

mechanisms. External beam radiation therapy (xRT), which is often combined with

chemotherapy, kills cancer cells by inducing nuclear DNA double strand breaks resulting

in cell-cycle death. Unlike systemic chemotherapy, xRT depends on the ability to

accurately determine the anatomic location of the tumor. Surgical resection of tumors

also depends on the ability to see the tumor and on complete removal, since residual

tumor cells will quickly reestablish the tumor following surgery. Surgery and xRT are

generally limited to the local treatment of malignant tumors and thus are limited in

treating disseminated or metastatic disease, which is why chemotherapy is often used in

conjunction with these treatment modalities. Although systemic chemotherapy is capable

of reaching many distant metastatic sites, with the possible exception of brain metastases,

for all too many patients, responses are typically short-lived (months to several years) and

ultimately result in tumor recurrence.

[0005] Because the body's natural immune system is also capable of destroying

cancer cells following their recognition, immunologic approaches are rapidly becoming

more prevalent in cancer treatment paradigms. However, some cancer cells, and to a

greater extent cancer stem cells, manage to initially avoid immune-surveillance and

actually acquire the ability to evolve and ultimately survive by remaining relatively

immune invisible [Gaipi et al, Immunotherapy 6:597-610, 2014].

[0006] One specific immunologic approach that is being increasingly investigated is

"in situ vaccination," a strategy that seeks to enhance tumor immunogenicity, generate

tumor infiltrating lymphocytes (TIL) and drive a systemic anti-tumor immune response

directed against "unvaccinated," disseminated tumors. In in situ vaccination, a malignant

solid tumor is injected with (or treated with) one or more agents that facilitate the release

of tumor antigens while simultaneously providing pro-inflammatory signals to reverse the

immune-tolerizing microenvironment of the tumor [Pierce et al, Human Vaccines &

Immunotherapoeutics 11(8):1901-1909, 11(8): 1901-1909,2015; 2015;Marabelle Marabelleet etal, al,Clin. Clin.Cancer CancerRes. Res.

20(7):1747-56, 2014; Morris et al, Cancer Res; 76(13); 3929-41, 2016].

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[0007] A second and quite different approach is systemically-administered

immunotherapy. In systemically-administered immunotherapy, an immunostimulatory

agent, such as an immune checkpoint inhibitor, is administered to circulate through the

entire body (e.g., intravenously), rather than being locally injected into the tumor. Such

agents can be used to treat tumors in which an anti-tumor immune response is present,

but has been "exhausted" or rendered ineffective. In the case of checkpoint inhibitors, the

tumor cells express "checkpoint ligands" or other checkpoint molecules that interact with

"checkpoint receptors" on the existing anti-tumor immune cells, triggering the

inactivation of these cells. By blocking this interaction, systemically-administered

checkpoint inhibitors turn on the exhausted, pre-existing immune response in cancer

patients, facilitating a more effective attack on the cancer cells by the patient's own

immune system.

[0008] Although recent data from clinical trials and pre-clinical models illustrate the

potential of these approaches, there is a great need in the art for systemically-

administered immunotherapy methods exhibiting improved systemic efficacy.

[0009] Radiation hormesis is a decades-old hypothesis that low doses of ionizing RT

can be beneficial by stimulating the activation of natural protective repair mechanisms

that are not activated in the absence of ionizing RT [Cameron and Moulder, Med. Phys.

25:1407, 1998]. The reserve repair mechanisms are hypothesized to be sufficiently

effective when stimulated as to not only cancel the detrimental effects of ionizing RT but

also inhibit disease not related to RT exposure. Perhaps related, the abscopal effect is a

phenomenon reported in the 1950's, whereby, xRT treatment of one tumor actually

causes shrinkage of another tumor outside the RT treatment area. Although rare, this

phenomenon is thought to be dependent on activation of the immune system. Together,

hormesis and the abscopal effect support the potential interaction and stimulation of the

immune system by low dosage (immune stimulatory but non-cytotoxic) RT, which may

then be combined with other immunologic approaches, such as systemically-administered

immunotherapy.

[0010] We have previously published that the combination of local xRT + in situ

vaccination and/or systemic checkpoint inhibitor immunotherapy are potently synergistic

in treating large established tumors in mice, when there is a single tumor present [Morris

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

et al, Cancer Res; 76(13); 3929-41, 2016]. However, we have surprisingly discovered

that the combination of in situ vaccination and xRT does not result in inhibited tumor

growth in the presence of a second, non-radiated tumor. Apparently, the non-radiated

tumor exhibits a dampening effect (which we have designated as "concomitant immune

tolerance") on the immunomodulatory effect of the xRT and in situ vaccine on the

radiated tumor.

[0011] This concomitant immune tolerance can be overcome, enabling efficacy of in

situ vaccination, when xRT is given to all areas of tumor. However, xRT cannot be

effectively used in combination with in situ vaccination methods in the presence of

multiple tumors, particularly if the tumors are not few in number, or if the location of one

or more of the tumors is not precisely known, or if it is not feasible to deliver xRT to all

sites of tumor. Furthermore, administering xRT to all tumor sites in patients with

metastatic disease would likely result in systemic immune suppression, defeating the

central purpose of systemically-administered immunotherapy.

[0012] Accordingly, in combination with systemically-administered immunotherapy,

there is a need for improved methods of delivering an immunomodulatory dose of RT to

all tumors within a subject, regardless of their number and anatomic location.

BRIEF SUMMARY

[0013] We have previously shown that certain alkylphosphocholine analogs are

preferentially taken up and retained by malignant solid tumor cells. In U.S. Patent

Publication No. 2014/0030187, which is incorporated by reference herein in its entirety,

Weichert et al. disclose using analogs of the base compound 18-(p-iodopheny1)octadecyl 18-(p-iodophenyl)octadecyl

phosphocholine (NM404; see Figure 1) for detecting and locating, as well as for treating,

a variety of malignant solid tumors. If the iodo moiety is an imaging-optimized

radionuclide, such as iodine-124 ([1241]-NM404), the analog ([¹²I]-NM404), the analog can can be be used used in in positron positron

emission tomography-computed tomography (PET/CT) or single-photon emission

computed tomography (SPECT) imaging of solid tumors. Alternatively, if the iodo

moiety is a radionuclide optimized for delivering therapeutic doses of RT to the solid

tumors cells in which the analog is taken up, such as iodine-125 or iodine-131 ([1251]- ([¹²I]-

[¹³¹I]-NM404), NM404 or [131 I]-NM404), the the analog analog can can be be used used to to treat treat the the solid solid tumors. tumors.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[0014] Such analogs not only target a wide variety of solid tumor types in vivo, but

also undergo prolonged selective retention in tumor cells, thus affording high potential as

RT agents. Moreover, tumor uptake is limited to malignant cancer and not premalignant

or benign lesions.

[0015] However, there are metal isotopes that have better properties for optimized

imaging and/or RT than the radioactive iodine isotopes used in the previously disclosed

alkylphosphocholine analogs. For example, as an imaging isotope, I-124 suffers from

poor positron output (only about 24% of the emissions are positrons), and it suffers

further from a confounding gamma emission (600 KeV), which actually interferes with

normal 511 KeV PET detection. Certain positron emitting metals have better imaging

characteristics. As another example, as an RT isotope, I-131 produces other non-

therapeutic emissions at other energies, which add undesired radiation dosimetry to

neighboring normal tissue, including bone marrow. The beta particle range of I-131 is

also quite long, which contributes to off target toxicity. Several metallic radiotherapy

isotopes offer a cleaner emission profile and shorter pathlength and thus less potential

toxicity.

[0016] We have developed improved alkylphosphocholine analogs that include a

chelated radioactive metal isotope instead of a radioactive iodine isotope (see, e.g., U.S.

Patent Publication No. 2018/0022768, which is incorporated by reference herein in its

entirety). The analogs include the same backbone as the previously disclosed

radioiodinated radioiodinated compounds, compounds, SO so they they are are still still selectively selectively taken taken up up and and retained retained in in tumor tumor

cells. However, the chelated radioactive metal isotope provides improved emissions for

imaging and/or radiotherapy applications. Such agents are well suited for delivering a

sub-cytotoxic but immunomodulatory dose of ionizing RT to all malignant tumors

present within a subject, regardless of whether their number and locations are known.

[0017] Accordingly, in a first aspect, the disclosure encompasses a method of treating

a cancer comprising one or more malignant solid tumors in a subject. The method

includes the steps of systemically administering to the subject (a) an immunomodulatory

dose of a targeted radiotherapy (TRT) agent that is differentially taken up by and retained

within the malignant solid tumor tissue; and (b) one or more immunostimulatory agents.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[0018] In some embodiments, the one or more immunostimulatory agents are

immune checkpoint inhibitors capable of targeting one or more checkpoint molecules.

[0019] Non-limiting examples of the one or more immune checkpoint inhibitors

include includeagents agentsthat areare that capable of targeting capable one or one of targeting more or of more the following checkpoint checkpoint of the following

molecules: A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator),

CTLA4 (cytotoxic T lymphocyte-associated protein 4), KIR (killer cell immunoglobulin-

like receptor), LAG3 (Lymphocyte Activation Gene 3), PD-1 (programmed death

receptor 1), PD-L1 (programmed death ligand 1), CD40 (cluster of differentiation 40),

CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD137 (cluster

of differentiation 137), OX40 (CD134; cluster of differentiation 134), OX40L (OX40

ligand; cluster of differentiation 252), GITR (glucocorticoid-induced tumor necrosis

factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor

receptor-related protein ligand), ICOS (inducible T-cell costimulatory), ICOSL (inducible

T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4

(VTCN1; V-set domain-containing T-cell activation inhibitor 1), IDO (Indoleamine 2,3-

dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), Gal-9

(galectin-9), or VISTA (V-domain Ig suppressor of T cell activation).

[0020] In some embodiments, the one or more immune checkpoint inhibitors include

one or more anti-immune checkpoint molecule antibodies. In some such embodiments,

the one or more anti-immune checkpoint molecule antibodies include at least one

monoclonal antibody.

[0021] In some embodiments, the one or more immune checkpoint inhibitors include

one or more small molecules capable of inhibiting or blocking one or more immune

checkpoint molecules. Non-limiting examples of such small molecule checkpoint

inhibitors include CA-170 and CA-327, which both target PD-L1.

[0022] In some embodiments, the one or more anti-immune checkpoint molecule

antibodies include an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1

antibody, an anti-LAG3 antibody, an anti-KIR antibody, an anti-A2AR antibody, and

anti-BTLA antibody, an anti-CD40 antibody, an anti-CD27 antibody, an anti-CD28

antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-OX40L antibody, an

anti-GITR antibody, an anti-GITRL antibody, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-IDO antibody, an anti-

TIM-3 antibody, an anti-Gal-9 antibody, or an anti-VISTA antibody.

[0023] In some embodiments, the TRT agent is metaiodobenzylguanidine (MIBG),

where the iodine atom in the MIBG is a radioactive iodine isotope.

[0024] In some embodiments, the TRT agent is a radiolabeled tumor-targeting

antibody.

[0025] In some embodiments, the TRT agent is radioactive isotope of radium, such as

Ra-223. Ra-223.

[0026] In some embodiments, the TRT agent is a radioactive phospholipid ether

metal chelate having the formula:

O R

or a salt thereof. R1 includesaachelating R includes chelatingagent agentthat thatis ischelated chelatedto toaametal metalatom, atom,wherein wherein

the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater

than 6 hours and less than 30 days; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1;

Y is -H, -OH, -COOH, -COOX, -OCOX, or -OX, -0X, wherein X is an alkyl or an aryl; R2 is R is

-N+H3,-NHZ, -NH, -N*H2Z, -N*HZ2, -NHZ, or -N*Z3, or -NZ, wherein wherein eacheach Z is Z is independently an independently an alkyl alkyl or or an an

aroalkyl; and b is 1 or 2. Non-limiting examples of metal isotopes that could be used

include Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223,

Ac-225, Pb-212, or Th-227.

[0027] In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-

1,4,7-triacetic 1,4,7-triacetic acid acid (DO3A) (DO3A) or or one one of of its its derivatives; derivatives; 1,4,7-triazacyclononane-1,4-diacetic 1,4,7-triazacyclononane-1,4-diacetic

acid acid (NODA) (NODA)oror oneone of of its its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic derivatives; acid 1,4,7-triazacyclononane-1,4,7-triaceticacid

(NOTA) (NOTA) or orone oneofof itsits derivatives; ; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic derivatives; ,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic

acid (DOTA) or one of its derivatives; 4,7-triazacyclononane,1-glutaric acid-4,7 1,4,7-triazacyclononane,1-glutaric 7- a acid-4,7-

diacetic acid (NODAGA) or one of its derivatives; ,4,7,10-tetraazacyclodecane, 1- 1,4,7,10-tetraazacyclodecane,1-

glutaric acid-4,7,10-triacetic acid (DOTAGA) or one of its derivatives; 1,4,8,11-

tetraazacyclotetradecane-1,4,8,11-tetraacetic acid tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) (TETA) or or one one of of its its derivatives; derivatives; wo 2019/094657 WO PCT/US2018/059927

1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) ,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) or or one one of of its its

derivatives; diethylene triamine pentaacetic acid (DTPA), its diester, or one of its

derivatives; 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A"-DTPA) or one

of its derivatives; deforoxamine (DFO) or one of its derivatives; 1,2-[[6-carboxypyridin-

2-y1]methylamino]ethane (H2dedpa) or one (Hdedpa) or one of of its its derivatives; derivatives; and and DADA DADA or or one one of of its its

derivatives, wherein DADA comprises the structure:

O NH NH NH HN NHHN HS SH SH

[0028] In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In other

embodiments, a is 0 (aliphatic alkyl chain).

[0029] In some embodiments, m is 1 (acylphospholipid series). In some such

embodiments, n is an integer between 12 and 20. In some embodiments, Y is -OCOX,

-COOX or -COOX or -OX. -OX

[0030] In In some some embodiments, embodiments,X is -CH2CH3 X is -CHCHoror -CH3. -CH.

[0031] In some embodiments, m is 0 (alkylphospholipid series).

[0032] In some embodiments, b is 1.

[0033] In some embodiments, n is 18.

[0034] In some embodiments, R2 is-NZ. R is -N+Z3. In In some some such such embodiments, embodiments, each each Z is Z is

independently independently-CH2CH3 -CHCH or or -CH3. -CH. In In some somesuch embodiments, such eacheach embodiments, Z is Z-CH3. is -CH.

PCT/US2018/059927

[0035] In some embodiments, the chelating agent chelated to the metal atom is:

OH IZ N H N N N N OH H HO HO IZ IN H I N N N N HO OH OH N N N N N HO O HO

2019/04457 OM PCT/US2018/059927 OM

HO O N OH N N O O HO O HO HO N N O NH N N O OH OH OH O HO

N à- NH

OH N NH N O o HO

10 or

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

O OH OH N O N N N O HO O HO HO OH N N N N HO O

N N N HO2C CO2H HOC COH HOC COHCO2H HO2C CO2HH H COH

N N N HO2C HOC CO2H COH CO2HCOH H COH CO2H HOC

O N N N HOC CO2H HO HO2C COH HOC

O HN

HO NH N 5 O HO-N HN 5 NH

HON HO O

12

NH HN N N N

OH HO , or O

O NH NH HN NHHN O O HS SH

WO wo 2019/094657 PCT/US2018/059927

[0036] In some embodiments, the radioactive phospholipid ether metal chelate is one

of the following compounds, wherein the selected compound is chelated to the metal

atom:

o O OH ZI N H N (CH2)18 + N DPOCH2CH2NMe3 (CH)OPOCHCHNMe N O N OH HO Ho O

o O OH N ZI H O II + N N -(CH2)18OPOCHCHNMe3 N N O O N OH HO O

HO ZI IN H O + N (CH2)18 DPOCH2CH2NMe3 N (CH)OPOCHCHNMe N N O O

HO HO O ZI N H N- II

+ N (CH2)18' POCH2CH2NMe3 (CH)OPOCHCHNMe N N O O o 1

HO Ho

PCT/US2018/059927

O OH OH

N O N O II + (CH2)18 POCH2CH2NMe3 N N (CH)OPOCHCHNMe O HO Ho O HO

O OH OH

N O N (CH2)18 + DPOCH2CH2NMe3 (CH)OPOCHCHNMe N N

O HO O HO ,

OH O N N O II + HO (CH2)18 DPOCH2CH2NMe3 N (CH)OPOCHCHNMe O O OH

OH O N HO N Il + N (CH2)18 DPOCH2CH2NMe3 (CH)OPOCHCHNMe O O OH wo 2019/094657 WO PCT/US2018/059927 PCT/US2018/059927

O OH OH O + O (CH2)18 DPOCH2CH2NMe3 N N HN (CH)OPOCHCHNMe N N

O Ho HO O HO

O OH OH II

O HN-(CH2)18 + N N DPOCH2CH2NMe3 HN (CH)OPOCHCHNMe

N N O O HO O HO

OH O N + HN (CH2) 18 DPOCH2CH2NMe3 HO N (CH)OPOCHCHNMe N O O O OH

OH O N O II + HN-(CH2)- 18 I OPOCH2CH2NMe3 Ho HO N HN-(CH)OPOCHCHNMe N O o OH

O OH OH

N O O= N + + (CH2)18 OPOCH2CH2NMe3 (CH)OPOCHCHNMe N N

O HO Ho O HO .

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

O OH OH

N N II + (CH2)18 POCH2CH2NMe3 (CH)OPOCHCHNMe N N e O HO Ho o HO Ho

OH

N O N II + (CH2)18OPOCHCH2NMe3 N (CH)OPOCHCHNMe N e HO Ho O

OH

N N N O (CH2)18 OPOCH2CH2NMe3 + (CH)OPOCHCHNMe N N

HO Ho O

II + (CH2)18OPOCHCH2NMe (CH)OPOCHCHNMe N N N e HO2C CO2H COH HOC HO2C HOC CO2H CO2H COH COH

(CH2)18 OPOCHCHNMe++ (CH) Il OPOCH2CH2NMe3

N N N e HO2C HOC CO2H COH HOC COH COH CO2H COH

O II + (CH2)18OPOCHCH2NMe3 (CH)OPOCHCHNMe N N N e HO2C > CO2H HOC COH HO2C HOC CO2H CO2H COH COH

(CH2)18 + OPOCH2CH2NMe3 (CH) OPOCHCHNMe N N N HO2C HOC CO2H COH HOC CO2H CO2H COH COH ,

O O + + -(CH2)18OPOCH2CH>NMeg MeNCHCHOPO(CH)8 N N N (CH)OPOCHCHNMe HO2C HOC CO2H COH HOC

O O o HN () N+1 N 5 HO O HO-N + + MeNCHCHO--O(CH) HN X NH5 O O o N / HO Ho O ,

O O HN (-)

HO N+ N 5 O HO-N HO-N + + MeNCHCHO-P-O(CH)8 HN X NH5

O N Ho HO O

(CH2)18OPOCH2CH2NMe3 + (CH)OPOCHCHNMe NH HN //

N N

OH Ho HO O

O II + (CH2)18 (CH) OPOCH2CH2NMe3 OPOCHCHNMe NH HN //

N N OH HO HO O ,

O II + NH (CH2)18OPOCHCHNMe3 (CH)OPOCHCHNMe e NH HN O O o

HS SH , or ,

O OII + NH-(CH2)18OPOCH2CH2NMe3 NH-(CH)OPOCHCHNMe O © NH HN NHHN O O

HS SH

[0037] In some embodiments, in the phospholipid ether metal chelate structure, a is 1,

b is 1, m is 0, n is 18, and R2 is -N(CH). R is -N*(CH3)3. In some In some suchsuch embodiments, embodiments, the the phospholipid phospholipid

ether ether metal metalchelate is is chelate NM600 chelated NM600 to thetometal chelated the atom, metalsuch as (but atom, such not as limited (but notto)limited 90 Y- to) Y-

NM600.

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[0038] In some embodiments, the TRT agent is a radiohalogenated phospholipid

ether having the formula:

R1 a (CH)(OCHCHYCH)OPOCHCH-R , , R ,

or a salt thereof. R1 comprises aa radioactive R comprises radioactive halogen halogen isotope; isotope; aa is is 00 or or 1; 1; nn is is an an integer integer

from 12 to 30; m is 0 or 1; Y is selected from the group consisting of -H, -OH, -COOH, -

COOX, -OX, -0X, and -OCOX, wherein X is an alkyl or an arylalkyl; and R2 isselected R is selectedfrom from

the the group groupconsisting consistingof of -N+H3, -NH,-N*H2Z, -NHZ, -N*HZ2, and -NZ, -NHZ, and -N*Z3,wherein wherein each each Z is is

independently an alkyl or an aryl.

[0039] In some In some embodiments, embodiments,thethe radioactive halogen radioactive isotopeisotope halogen is ¹²³I,is¹²I, ¹²I, ¹³¹I,

211 At, ² 76Br, or 77Br. ²¹¹At, Br, or Br.

[0040] In some embodiments, a is 1 and m is 0.

[0041] In some embodiments, n is 18.

[0042] In In some someembodiments, embodiments,R2 R isis -N+(CH3)3. -N(CH). In In some somesuch suchembodiments, a isa 1, embodiments, ism 1, m is 0, and n is 18. In some such embodiments, the radioactive halogen isotope is 1231, ¹²³I, 1241, ¹²I,

1251, or ¹³¹I ¹²I, or 1311 (the (the radiohalogenated radiohalogenated phospholipid phospholipid ether ether is is [¹²³I]-NM404,

[1231]-NM404, [¹²I]-NM404,

[1241-NM404,

[1251]-NM404, [1311]-NM404,

[¹²I]-NM404, [¹³¹I]-NM404, [211At]-NM404, [76Br]-NM404,

[²¹¹At]-NM404, or [7B]]-NM404).

[Br]-NM404, or [Br]-NM404).

[0043] In some embodiments, the TRT agent, the immunostimulatory agent, or both,

are administered intravenously.

[0044] In some embodiments, the subject is a human.

[0045] Non-limiting examples of the cancers presenting as malignant solid tumors

that can be treated using the method include melanoma, neuroblastoma, lung cancer,

adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver

cancer, subcutaneous cancer, squamous cell cancer of the skin or head or neck, intestinal

cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft

tissue sarcoma, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms' tumor, and

pediatric brain tumors.

[0046] In some embodiments, the cancer is treated without administering to the

subject an antibody to a tumor antigen that is not a checkpoint molecule.

[0047] In some embodiments, an anti-GD2 antibody is not adminstered to the subject.

[0048] In a second aspect, the disclosure encompasses the use of a TRT agent and

one or more immunostimulatory agents for treating a cancer comprising one or more

malignant solid tumors in a subject, as further described above.

[0049] In a third aspect, the disclosure encompasses the use of a TRT agent and/or

one or more immunostimulatory agents for the manufacture of a medicament treating a

cancer comprising one or more malignant solid tumors in a subject, as further described

above.

[0050] Other objects, features and advantages of the present invention will become

apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawing(s) will be

provided by the Office upon request and payment of the necessary fee.

[0052] Fig. 1 shows the chemical structure of the base compound 18- (p-iodophenyl)

octadecyl phosphocholine (NM404).

[0053] Fig. 2A is a graph showing that xRT + IT-IC elicits in situ tumor vaccination.

More specifically, Fig. 2A shows tumor growth curves that show synergy between xRT

and IT-hu14.18-IL2. 71% (22/31) of mice treated with xRT + IT-IC are rendered disease-

free.

[0054] Fig. 2B is another graph showing that xRT + IT-IC elicits in situ tumor

vaccination. More specifically, Fig. 2B shows Kaplan-Meier survival curves that show

synergy between xRT and IT-hu14,18-IL2. IT-hu14.18-IL2. 71% (22/31) of mice treated with xRT + IT-

IC are rendered disease-free.

[0055] Fig. 2C is another graph showing that xRT + IT-IC elicits in situ tumor

vaccination. More specifically, Fig. 2C shows that 90% of the treated mice reject

subsequent engraftment with B78 melanoma.

[0056] Fig. 3 is a graph demonstrating concomitant immune tolerance. Primary tumor

response is shown. A distant un-treated tumor suppresses response to xRT + IT-IC in a 2-

21

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tumor B78 melanoma model, and this suppression can be overcome be radiating the

second tumor.

[0057] Fig. 4 is a graph showing that concomitant immune tolerance is due to Tregs.

Primary tumor response is shown. A distant un-treated tumor suppresses response to xRT

+ IT-IC in a 2-tumor B78 melanoma model and this suppression can be overcome by

depleting Tregs (using transgenic DEREG mice that express diphtheria toxin receptors on

their Tregs, and thus depleting Tregs by administering diphtheria toxin).

[0058] Fig. 5 is an image showing selective uptake of 124I-NM404 byB78 ¹²I-NM404 by B78melanoma. melanoma.

¹²I-NM404 and had serial PET/CT A mouse bearing a ~200mm³ B78 tumor received IV 24I-NM404

scans done. This image at 71h shows selective uptake by the tumor with some residual

background uptake by the heart and liver.

[0059] Fig. 6 is a graph demonstrating that in situ vaccination can be elicited in the

presence of residual levels of molecular targeted radiation therapy (TRT). Treatment with

combined xRT + IT-IC is equally effective in the presence or absence of 3 uCi µCi 131L- ¹³¹I-

NM404. This approximates the residual activity of TRT that will be present when we

deliver xRT (d0) followed by IT-IC (d6-10), as described in Example 4.

[0060] Fig. 7 shows a time course MRI image of a tumor-bearing mouse following

injection of Gd-NM600 showing enhancement of the tumor (T) by 24 hours.

[0061] Fig. 8A is a graph showing tumor-specific inhibition of primary tumor

response to the combination of local RT+IT-IC by a distant untreated tumor in murine

melanoma and pancreatic tumor models. C57BL/6 mice bearing a syngeneic,

disialoganglioside-expressing (GD2+), primary flank tumor +/- a secondary tumor on the

contralateral flank were treated to the primary tumor only, as indicated, with xRT on day

"1" and intra-tumor (IT) injection of 50 mcg of the anti-GD2 immunocytokine (IC),

hul4.18-IL2 (a(a hu 14. 18-IL2 fusion ofof fusion anti-GD2 mAb anti-GD2 and mAb IL2), and onon IL2), day 6-10. day Mean 6-10. primary Mean tumor primary tumor

volumes are displayed in Fig. 8A. More specifically, Fig. 8A shows that in mice bearing

a primary B78 melanoma tumor, the presence of an untreated secondary B78 tumor

antagonized primary tumor response to RT+IT-IC. We describe this effect as

"concomitant immune tolerance" - an antagonistic effect of a non-treated distant tumor

on the local response of a treated tumor to xRT + IT-IC.

[0062] Fig. 8B is another graph showing tumor-specific inhibition of primary tumor

melanoma and pancreatic tumor models. C57BL/6 mice bearing a syngeneic,

hul4.18-IL2 18-IL2(a (afusion fusionof ofanti-GD2 anti-GD2mAb mAband andIL2), IL2),on onday day6-10. 6-10.More Morespecifically, specifically,Fig. Fig.

8B shows Kaplan-Meier survival curves for mice plus replicate experiments. Nearly all

mice were euthanized due to primary tumor progression.

[0063] Fig. 8C is another graph showing tumor-specific inhibition of primary tumor

melanoma and pancreatic tumor models. C57BL/6 mice bearing a syngeneic,

hul4.18-IL2 14.18-IL2 (a (a fusion fusion of of anti-GD2 anti-GD2 mAb mAb and and IL2), IL2), on on day day 6-10. 6-10. More More specifically, specifically, Fig. Fig.

8C shows that in mice bearing a primary Panc02-GD2+ pancreatic tumor, with or without

a secondary Panc02-GD2-tumor Panc02-GD2- tumoron onthe theopposite oppositeflank, flank,the thepresence presenceof ofan anuntreated untreated

Panc02 secondary tumor suppressed the response of a primary Panc02-GD2+ tumor to

RT+IT-IC.

[0064] Fig. 8D is another graph showing tumor-specific inhibition of primary tumor

melanoma and pancreatic tumor models. C57BL/6 mice bearing a syngeneic,

hul4.18-IL2 14.18-IL2(a (afusion fusionof ofanti-GD2 anti-GD2mAb mAband andIL2), IL2),on onday day6-10. 6-10.More Morespecifically, specifically,Fig. Fig.

8D shows that in mice bearing a primary B78 melanoma tumor, a secondary B78 tumor

suppressed primary tumor response to RT+IT-IC but a secondary Panc02-GD2+

pancreatic tumor did not exert this effect.

[0065] Fig. 8E is another graph showing tumor-specific inhibition of primary tumor

melanoma and pancreatic tumor models. C57BL/6 mice bearing a syngeneic,

hul4.18-IL2 hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on day 6-10. More specifically, Fig.

8E shows that in mice bearing a primary Panc02-GD2+ tumor a secondary Panc02-GD2-

tumor suppressed primary tumor response to combined xRT and IT-hu14.18-IL2, while a

B78 secondary tumor did not. n=number of mice per group. NS=non-significant,

***p<0.001.

[0066] Fig. 9A includes immunohistochemistry images (left and center) and graphs

(right) showing that concomitant immune tolerance is circumvented by specific depletion

of regulator T cells (Tregs). More specifically, Fig. 9A shows immunohistochemistry for

the Treg marker, FoxP3 (representative 400x images are shown) for tumors evaluated on

day 6 after xRT in mice with one (Fig. 9A, leftmost panels A1 and A2) or two (Fig. 9A,

center panels A3 and A4) tumors. Mice received no xRT, or xRT only to the primary

tumor. The primary tumor is shown in Fig. 9A, panels_Al-A3 panels_A1-A3 and the secondary is shown

in Fig. 9A, panel A4. Small arrows point out some of the FoxP3+ cells (brown nuclei =

FoxP3+, blue = hematoxylin counterstain). The graphs on the right display blinded

quantification of FoxP3+ cells per 200x field, corresponding to the conditions shown in

Fig. 9A, panels A1, A2, A3 and A4, respectively.

[0067] Fig. 9B is another graph showing that concomitant immune tolerance is

circumvented by specific depletion of regulator T cells (Tregs). More specifically, Fig.

9B shows that DEREG mice express diphtheria toxin receptor under control of the Treg-

specific FoxP3 promoter, enabling specific depletion of Tregs upon IP injection of

diphtheria toxin. DEREG mice bearing primary and secondary B78 melanoma tumors

were treated with xRT+IT-IC to the primary tumor and IP injection of either diphtheria

toxin or PBS (the first of replicate experiments are shown). Concomitant immune

tolerance is eliminated following depletion of Tregs in these mice, resulting in improved

(Fig. 9B) primary tumor response. n=number of mice per group. **p<0.01, ***p<0.001.

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[0068] Fig. 9C is another graph showing that concomitant immune tolerance is

9C shows that DEREG mice express diphtheria toxin receptor under control of the Treg-

diphtheria toxin. DEREG mice bearing primary and secondary B78 melanoma tumors

toxin or PBS (the first of replicate experiments are shown). Concomitant immune

(Fig. 9C) secondary tumor response. n=number of mice per group. **p<0.01,

***p<0.001. ***p<0.001.

[0069] Fig. 10A and is a graph showing that concomitant immune tolerance is

overcome by delivering xRT to both tumor sites. In mice bearing primary and secondary

B78 tumors, the secondary tumor suppresses primary tumor response to primary tumor

treatment with xRT + IT-IC. This is overcome by delivering 12 Gy xRT to both the

primary and secondary tumors and IT-IC to the primary tumor, resulting in improved

(Fig. 10A) primary tumor response (the first of replicate experiments is shown) from

replicate experiments. n=number of mice per group. **p<0.01, ***p<0.001.

[0070] Fig. 10B is another graph showing that concomitant immune tolerance is

(Fig. 10B) aggregate animal survival from replicate experiments. n=number of mice per

group. **p<0.01,***p<0.001. **p<0.01, ***p<0.001.

[0071] Fig. 11A Fig. 11Aisisa agraph showing graph thatthat showing low dose low xRT dosealone xRT does notdoes alone elicit notinelicit situ in situ

vaccination but does overcome concomitant immune tolerance when delivered to distant

tumor sites together with 12 Gy + IT-IC treatment of an in situ vaccine site. More

specifically, Fig. 11A shows that in mice bearing a primary B78 tumor only, 12 Gy + IT-

IC elicits in situ vaccination (as shown previously) and results in complete tumor

regression in most mice (4/6 in this experiment) and a memory immune response (Morris,

Cancer Res, 2016). On the other hand no animals exhibit complete tumor regression following either IT-IC alone or low dose (2 Gy) xRT + IT-IC (0/6 in both groups) p<0.05.

[0072] Fig. 11B is another graph showing that low dose xRT alone does not elicit in

situ vaccination but does overcome concomitant immune tolerance when delivered to

distant tumor sites together with 12 Gy + IT-IC treatment of an in situ vaccine site. More

specifically, Fig. 11B shows that in mice bearing a primary and secondary B78

melanoma tumor, low dose xRT (2 Gy or 5 Gy) delivered to the secondary tumor is

comparable to 12 Gy in its capacity to overcome concomitant immune tolerance at the

primary tumor.

[0073] Fig. 11C is another graph showing that low dose xRT alone does not elicit in

specifically, Fig. 11C shows that in these same animals, it is apparent that overcoming

concomitant immune tolerance by delivery of low dose xRT to the secondary tumor

rescues a systemic response to IT-IC immunotherapy. In this context, when xRT is

delivered to all tumor sites then IT-IC injection of the primary tumor triggers a systemic

anti-tumor effect that renders secondary tumor response to 2 Gy or 5 Gy greater than the

response to 12 Gy xRT in absence of primary tumor IT-IC injection.

[0074] ¹³¹I-NM404 Fig. 12A is a PET image showing that low dose TRT with 131I-NM404

effectively depletes tumor infiltrating FoxP3+ Tregs without systemic leukopenia or

depletion of tumor infiltrating CD8+ effector T cells. In most clinical scenarios, it is not

feasible to deliver external beam, even low dose, to all tumor sites without eliciting

marked bone marrow depletion and leukopenia that would result in immunosuppression.

Here we tested whether TRT could be administered systemically to specifically deplete

tumor infiltrating suppressive immune cells (Tregs), without triggering systemic immune

cell depletion and leukopenia. More specifically, Fig. 12A shows dosimetry studies in

¹²I-NM404 confirm this B78 melanoma tumor model using positron-emitting 124I-NM404 confirm tumor- tumor-

selective uptake of NM404. C57BL/6 mice bearing B78 tumors were treated with 60 uCi µCi

¹³¹I-NM404. This 131I-NM404. This activity activity approximates approximates the the amount amount of of 131-N-404 ¹³¹I-NM404necessary necessarytotodeliver deliver(

2 Gy TRT to a B78 tumor. Peripheral blood and tumor samples were collected in

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

untreated control mice (designated "C") and at 8 day intervals (T1 = d8, T2 = d16, T3 =

d24, T4 = d32) thereafter.

[0075] Fig. 12B is a bar graph showing that low dose TRT with I-NM404 ¹³¹I-NM404

cell depletion and leukopenia. More specifically, Fig. 12B shows that this dose of TRT

did not result in any significant systemic leukopenia.

[0076] ¹³¹I-NM404 Fig. 12C is another bar graph showing that low dose TRT with 31-NM404

cell depletion and leukopenia. More specifically, Fig. 12C shows that this dose of TRT

did not significantly affect the level of tumor infiltrating CD8+ effector T cells (ANOVA

p=0.25).

[0077] ¹³¹I-NM404 Fig. 12D is another bar graph showing that low dose TRT with 13-I-NM404

cell depletion and leukopenia. More specifically, Fig. 12D shows that tumor infiltrating

FoxP3+ Tregs were significantly depleted by this dose of TRT (ANOVA p=0.03; *

p<0.05).

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[0078] Fig. 13A is a graph showing that low dose TRT with 131-N-404 ¹³¹I-NM404effectively effectively

overcomes concomitant immune tolerance and rescues the systemic anti-tumor effect of

in situ vaccination. Given the capacity of low dose 131I-NM404 ¹³¹I-NM404 TRT to deplete tumor-

131I- infiltrating Tregs without rendering a mouse leukopenic, we tested whether low dose ¹³¹I-

NM404 might effectively overcome concomitant immune tolerance. C57BL/6 mice

bearing two B78 tumors were treated with 60-mcCi 131I-NM404 ¹³¹I-NM404 on day 1 (NM404), as

indicated. After one half-life (day 8), animals received 12 Gy xRT or no xRT to the

131 I-NM404 primary tumor (in situ vaccine site). Control mice receiving no 31I-NM404 were were treated treated

to the secondary tumor as indicated (0, 2, or 12 Gy). Mice received daily IT injections of

IC to the primary tumor (in situ vaccine site), as indicated, on days 13-17. More

specifically, Fig. 13A shows that primary tumor response is shown and demonstrates that

administration of low dose TRT effectively overcomes concomitant immune tolerance

and rescues the systemic anti-tumor effect of in situ vaccination.

[0079] Fig. 13B is another graph showing that low dose TRT with 131I-NM404 ¹³¹I-NM404

effectively overcomes concomitant immune tolerance and rescues the systemic anti-

tumor effect of in situ vaccination. Given the capacity of low dose 131 I-NM404 TRT ¹³¹I-NM404 TRT to to

deplete tumor-infiltrating Tregs without rendering a mouse leukopenic, we tested whether

low dose 131-NM404 ¹³¹I-NM404might mighteffectively effectivelyovercome overcomeconcomitant concomitantimmune immunetolerance. tolerance.

C57BL/6 mice bearing two B78 tumors were treated with 60-mcCi 131-N-404 ¹³¹I-NM404on onday day1 1

(NM404), as indicated. After one half-life (day 8), animals received 12 Gy xRT or no

xRT to the primary tumor (in situ vaccine site) site).Control Controlmice micereceiving receivingno no131-N-404 ¹³¹I-NM404

were treated to the secondary tumor as indicated (0, 2, or 12 Gy). Mice received daily IT

injections of IC to the primary tumor (in situ vaccine site), as indicated, on days 13-17.

More specifically, Fig. 13B shows that secondary tumor response is shown and

demonstrates that administration of low dose TRT effectively overcomes concomitant

immune tolerance and rescues the systemic anti-tumor effect of in situ vaccination.

[0080] Figure 14 shows the chemical structure of an exemplary alkylphosphocholine

metal chelate (64Cu-NM600). Other (Cu-NM600). Other metals metals may may bebe used used inin place place ofof Cu. Cu.

[0081] Figure 15 is a PET/CT image of two single tumor B78 mice from a scan taken

48 hours post-injection with 86Y-NM600. Y-NM600.

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[0082] Figure 16 is a PET/CT image of two two-tumor B78 mice from a scan taken

48 hours post-injection with 86Y-NM600. Y-NM600.

[0083] Figure 17 includes PET/CT images for a U87MG mouse from scans taken 3

hours (left panel), 24 hours (center panel) and 48 hours (right panel) post-injection with

Cu-NM600. 4Cu-NM600.The Theimages imagesshow showtissue tissueactivity activitycalculated calculatedasasa apercent percentofofinjected injecteddose/g dose/g

tissue (%ID/g, scale shown on far right).

[0084] Figure 18 includes PET/CT images for a 4T1 mouse from scans taken 3 hours

(left panel), 24 hours (center panel) and 48 hours (right panel) post-injection with Cu-

NM600. The images show tissue activity calculated as a percent of injected dose/g tissue

(%ID/g, scale shown on far right).

[0085] Figure 19 includes PET/CT images for an HCT-116 mouse from scans taken 3

Cu-NM600. TheThe 64Cu-NM600. images show images tissue show activity tissue calculated activity as as calculated a percent of of a percent injected dose/g injected dose/g

tissue (%ID/g, scale shown on far right).

[0086] Figure 20 includes PET/CT images for an A549 mouse from scans taken 3

Cu-NM600. The 64Cu-NM600. images The show images tissue show activity tissue calculated activity asas calculated a a percent ofof percent injected dose/g injected dose/g

tissue (%ID/g, scale shown on far right).

[0087] Figure 21 includes PET/CT images for a PC-3 mouse from scans taken 3

64Cu-NM600. The Cu-NM600. The images images show show tissue tissue activity activity calculated calculated asas a a percent percent ofof injected injected dose/g dose/g

tissue (%ID/g, scale shown on far right).

[0088] Figure 22 includes PET/CT images for an HT-29 mouse from scans taken 3

64Cu-NM600. Cu-NM600. The The images images show show tissue tissue activity activity calculated calculated asas a a percent percent ofof injected injected dose/g dose/g

tissue (%ID/g, scale shown on far right).

[0089] Figure 23 includes PET/CT images for a MiaPaca mouse from scans taken 3

Cu-NM600. The 4Cu-NM600. images The show images tissue show activity tissue calculated activity as as calculated a percent of of a percent injected dose/g injected dose/g

tissue (%ID/g, scale shown on far right).

[0090] Figure 24 includes PET/CT images for a 4T1 mouse from scans taken 3 hours

Y- (left panel), 24 hours (center panel) and 48 hours (right panel) post-injection with 6Y-

(%ID/g, scale shown on far right).

[0091] Figure 25 includes PET/CT images for a 4T1 mouse from scans taken 3 hours

(left panel), 24 hours (center panel) and 48 hours (right panel) post-injection with 89Zr- Zr-

(%ID/g, scale shown on far right).

[0092] Figure 26 includes PET/CT images for an HT-29 mouse from scans taken 4

hours (left panel) and 1 day (right panel) post-injection with 52Mn-NM600. Theimages ²Mn-NM600. The images

show tissue activity calculated as a percent of injected dose/g tissue (%ID/g, scale shown

on far right).

[0093] Figure 27 includes PET/CT images for a PC-3 mouse from scans taken 4

hours (left hours (leftpanel) panel)andand 1 day (right 1 day panel) (right post-injection panel) with 5²Mn-NM600. post-injection with The The images images

to the right of each image).

[0094] Figure 28 includes PET/CT images for an HT-29 mouse from scans taken 2

days (left panel), 3 days (second panel from the left), 5 days (second panel form the right)

and 7 days (right panel) post-injection with 52Mn-NM600. The images ²Mn-NM600. The images show show tissue tissue

activity calculated as a percent of injected dose/g tissue (%ID/g, scale shown to the right

of the images).

[0095] Figure 29 includes PET/CT images for a PC-3 mouse from scans taken 2 days

(left panel), 3 days (second panel from the left), 5 days (second panel form the right) and

7 days 7 days (right (rightpanel) post-injection panel) with with post-injection ²Mn-NM600. The images The images showshow tissue tissue activity activity

calculated as a percent of injected dose/g tissue (%ID/g, scale shown to the right of the

images).

[0096] Figure 30 is a graph showing PET quantitative region of interest data (chelate

uptake as a function of time) for 4T1 tumor tissue in 4T1 mice injected with 86 Y-NM600, Y-NM600,

Cu-NM600 4Cu-NM600 and and Zr-NM-600. Zr-NM-600.

[0097] Figure 31 is a graph showing PET quantitative region of interest data

(chelate uptake as a function of time) for heart tissue in 4T1 mice injected with 86Y- Y-

NM600, NM600, 6Cu-NM600 Cu-NM600 and and89Zr-NM-600. Zr-NM-600.

[0098] Figure 32 is a graph showing PET quantitative region of interest data (chelate

uptake as a function of time) for liver tissue in 4T1 mice injected with 86Y-NM600, 64Cu- Ý-NM600, Cu-

NM600 NM600 and and89Zr-NM-600. Zr-NM-600.

[0099] Figure 33 is a graph showing PET quantitative region of interest data (chelate

uptake as a function of time) for whole body in 4T1 mice injected with 86Y-NM600, Ý-NM600,

64Cu-NM600 Cu-NM600 and and 89Zr-NM-600. Zr-NM-600.

[00100] Figure 32 is a bar graph illustrating ex vivo chelate biodistribution in healthy

and tumor tissues in 4T1 mice 48 hours (86Y-NM600, 64Cu-NM600, (Y-NM600, Cu-NM600, 89Zr-NM-600 Zr-NM-600 and and

¹Lu-NM600) and 177Lu-NM600) 9696 and hours (¹Lu-NM600) hours post-injection (17 Lu-NM600) of the post-injection metal of the chelates. metal chelates.

[00101] Figure 35 shows the chemical structure of an exemplary alkylphosphocholine

(¹Lu-NM600). Other metal chelate (177Lu-NM600). metals Other may metals bebe may used inin used place ofof place ¹Lu. 177Lu.

[00102] Figure 36 is an audioradiographic image of three B78 mice taken 48 hours

after injection with 90Y-NM600. Xenografted Y-NM600. Xenografted B78 B78 tumors tumors are are seen seen asas large large dark dark spots spots atat

the lower right of each mouse image.

[00103] Figure 37 is an audioradiographic image of three B78 mice taken 96 hours

the lower right of each mouse image.

[00104] Figure 38 is an audioradiographic image of a B78 mouse taken on day 5 after

injection with 177Lu-NM600. Xenografted ¹Lu-NM600. Xenografted B78 B78 tumors tumors are are seen seen asas two two dark dark spots spots atat the the

bottom of the mouse image.

[00105] Figure 39 is an audioradiographic image of a B78 mouse taken on day 13 after

bottom of the mouse image.

[00106] Figure 39 is an audioradiographic image of a B78 mouse taken on day 13 after

¹Lu-NM600. Xenografted B78 tumors are seen as two dark spots at the injection with "Lu-NM600.

bottom of the mouse image.

31

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[00107] Figure 40 is an audioradiographic image of a MiaPaca mouse taken 10 days

after injection with 177Lu-NM600. The ¹Lu-NM600. The location location ofof the the xenografted xenografted MiaPaca MiaPaca tumor tumor isis

indicated by the arrow and dashed circle.

[00108] Figure 41 is an audioradiographic image of three 4T1 mice taken 48 hours

after injection with 177Lu-NM600. The ¹Lu-NM600. The locations locations ofof the the xenografted xenografted 4T1 4T1 tumors tumors are are

indicated by the arrows and dashed circles.

[00109] Figure 42 is an audioradiographic image of three 4T1 mice taken 96 hours

after injection with 177Lu-NM600 The locations ¹Lu-NM600. The locations of of the the xenografted xenografted 4T1 4T1 tumors tumors are are

indicated by the dashed circles.

[00110] Figure 43 is an audioradiographic image of three 4T1 mice taken 4 hours after

Y-NM600. injection with YON- The locations The locations of xenografted of the the xenografted 4T1 tumors 4T1 tumors are indicated are indicated by by

the arrows and dashed circles.

[00111] Figure 44 is an audioradiographic image of three 4T1 mice taken 48 hours

after injection with 90Y-NM600. The Y-NM600. The xenografted xenografted 4T1 4T1 tumors tumors are are seen seen asas large large dark dark

spots on the lower right of each mouse image.

[00112] Figure 45 is an audioradiographic image of three 4T1 mice taken 96 hours

spots on the lower right of each mouse image.

Figure

[00113] Figure 46 46 isisa agraph graph illustrating illustrating the theradiotherapeutic effect radiotherapeutic of 90Y-NM600 effect at of Y-NM600 at

two different doses (150 uCi µCi and 300 uCi) µCi) in a B78 xenograft mouse model, versus a

control (excipient only). Data is presented as measured tumor volume in mm³ as a

function of time in days after injection.

[00114] Figure 47 is a graph illustrating the radiotherapeutic effect of a single 500 uCi µCi

¹Lu-NM600 inin dose of 177Lu-NM600 a a B78 xenograft B78 mouse xenograft model, mouse versus model, a a versus control (excipient control only). (excipient only).

Data is presented as measured tumor volume in mm³ as a function of time in days after

injection.

Figure

[00115] Figure 48 48 isisa agraph graph illustrating illustrating the theradiotherapeutic effect radiotherapeutic of a single effect 400 uCi 400 µCi of a single

¹Lu-NM600 inin dose of 177Lu-NM600 a a MiaPaca xenograft MiaPaca mouse xenograft model, mouse versus model, a a versus control (excipient control (excipient

only). Data is presented as measured tumor volume in mm³ as a function of time in days

after injection.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[00116] Figure 49 is a graph illustrating the radiotherapeutic effect of a single 500 uCi µCi

dose of 177Lu-NM600 ¹Lu-NM600 inin a a 4T1 4T1 xenograft xenograft mouse mouse model, model, versus versus a a control control (excipient (excipient only). only).

injection. injection. * *P P< < 0.05;**P<0.01;***P<0.001. 0.05; ** P<0.01; *** P < 0.001.

[00117] Figure 50 is a graph illustrating the radiotherapeutic effect of two serial doses

of 177Lu-NM600 (500 ¹Lu-NM600 (500 uCi µCi and and 250 250 uCi) µCi) inin a a 4T1 4T1 xenograft xenograft mouse mouse model, model, versus versus a a

function of time in days after injection.

Figure

[00118] Figure 51 51 isisa agraph graph illustrating illustrating the theradiotherapeutic effect radiotherapeutic of 177Lu-NM600 effect at of ¹Lu-NM600 at

two different doses (500 uCi µCi and 250 uCi) µCi) in a 4T1 xenograft mouse model, versus a

function of time in days after injection.

[00119] Figure 52 is a graph illustrating the impact of tumor mass on the comparative

therapeutic efficacy of 90Y-NM600 and Y-NM600 and 131I-NM404 ¹³¹I-NM404 inin conventional conventional TRT. TRT.

Figure

[00120] Figure 53 aisbar 53 is a bar graph graph comparing comparing average average albumin albumin binding binding energies energies of three of three

different metal chelate analogs of NM404, along with an amine analog. For comparison,

the binding energy of I-NM404 is shown as a dotted line.

Figure

[00121] Figure 54 a 54 is isgraph a graph illustrating illustrating tumor tumor volume volume (mm³) (mm³) asfunction as a a function of time of time

(days) in B78 melanoma flank tumor mice treated with anti-CTLA4 immune checkpoint

inhibitor (CTLA4) and/or varying doses (25 uCi, µCi, 50 uCi, µCi, or 100 uCi) µCi) of the targeted

radiotherapy (TRT) agent Y90-NM600. Control mice were administered vehicle without

anti-CTLA4 or the TRT agent (PBS). After Day 18, combination treatment with 50 or

100 uCi µCi of Y90-NM600 with anti-CTLA4 had significantly (p < 0.05 <0.05by byANOVA) ANOVA)

reduced tumor growth compared to PBS, Y90-NM600 alone, or anti-CTLA4 alone. The

µCi Y90-NM600 combination treatment group with anti-CTLA-4 had an intermediate 25 uCi

growth delay response that showed a trend towards dose response.

Figure

[00122] Figure 55 a 55 is isgraph a graph showing showing aggregate aggregate animal animal survival survival for for mice mice administered administered

a combination of TRT (50 uCi µCi Y90-NM600) and checkpoint blockade (anti-CTLA4),

compared to mice administered TRT alone, checkpoint blockade alone (anti-CTLA4), or

PBS vehicle.

PCT/US2018/059927

Figure

[00123] Figure 56 a 56 is isgraph a graph showing showing aggregate aggregate animal animal survival survival for for mice mice administered administered

three different combinations of TRT (25 uCi, µCi, 50 uCi, µCi, and 100 uCi µCi Y90-NM600) with

checkpoint blockade (anti-CTLA4).

[00124] Figure 57 is a graph illustrating tumor volume (mm³) as a function of time

inhibitor (CTLA4) and/or varying doses (25 uCi, µCi, 50 uCi, µCi, or 100 uCi) µCi) of the molecularly

targeted targetedradiotherapy radiotherapy(MTRT) agent (MTRT) 90Y-NM600, agent Control Y-NM600. mice mice Control were administered were administered

vehicle without anti-CTLA4 or the MTRT agent (PBS). Combination treatment with 50

or or 100 100 uCi µCiofof90Y-NM600 Y-NM600 with withanti-CTLA4 anti-CTLA4hadhad significantly (p < 0.05 significantly (p < by ANOVA) 0.05 by ANOVA)

reduced reducedtumor tumorgrowth compared growth to PBS, compared 90Y-NM600 to PBS, alone, Y-NM600 or anti-CTLA4 alone, alone. alone. or anti-CTLA4

[00125] Figure 58 is a graph showing aggregate animal survival for B78 melanoma

flank tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and/or

varying doses (25 uCi, µCi, 50 uCi, µCi, or 100 uCi) µCi) of the molecularly targeted radiotherapy

(MTRT) (MTRT) agent agent90Y-NM600. Y-NM600. Mice Miceadministered a combination administered of MTRT a combination of (50 MTRTuCi 90 µCi (50 Y- Y-

NM600 NM600 or or100 100uCi 90Y-NM600) µCi Y-NM600)and andcheckpoint blockade checkpoint (anti-CTLA4) blockade exhibited (anti-CTLA4) exhibited

significantly increased survival as compared to other groups.

Figure

[00126] Figure 59 a 59 is isgraph a graph illustrating illustrating tumor tumor volume volume (mm³) (mm³) asfunction as a a function of time of time

(days) in NXS2 neuroblastoma tumor mice treated with anti-CTLA4 immune checkpoint

inhibitor (CTLA4) and/or 50 uCi µCi of the molecularly targeted radiotherapy (MTRT) agent

90Y-NM600. Control mice Y-NM600. Control mice were wereadministered administeredvehicle without vehicle anti-CTLA4 without or the or anti-CTLA4 TRTthe TRT

agent (PBS). Combination treatment of 90Y-NM600 MTRT Y-NM600 MTRT with with anti-CTLA4 anti-CTLA4 had had

Y-NM600 alone, significantly reduced tumor growth compared to PBS, 90Y-NM600 oror alone, anti-CTLA4 anti-CTLA4

alone.

Figure

[00127] Figure 60 a 60 is isgraph a graph illustrating illustrating tumor tumor volume volume (mm³) (mm³) asfunction as a a function of time of time

(days) in 4T1 breast cancer tumor mice treated with anti-CTLA4 immune checkpoint

inhibitor inhibitor (CTLA4) (CTLA4) and/or and/or 50 50 uCi µCi of of the the molecularly molecularly targeted targeted radiotherapy radiotherapy (MTRT) (MTRT) agent agent

alone.

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Figure

[00128] Figure 61 a 61 is isgraph a graph illustrating illustrating tumor tumor volume volume (mm³) (mm³) asfunction as a a function of time of time

(days) for the irradiated primary B78 tumor in B78 melanoma flank tumor mice having

both primary and secondary (distant) tumors. Mice were treated with various combinations

of EBRT of the primary tumor only (12 Gy, secondary tumor was shielded), anti-CTLA4

immune checkpoint inhibitor (CTLA4) and/or 50 uCi µCi of the molecularly targeted

radiotherapy (MTRT) agent 90Y-NM600. Combination Y-NM600. Combination treatment treatment ofof 1212 GyGy EBRT, EBRT, Y-90 Y.

NM600 MTRT, and anti-CTLA4 caused significantly reduced primary tumor growth

compared to other groups.

Figure

[00129] Figure 62 a 62 is isgraph a graph illustrating illustrating tumor tumor volume volume (mm³) (mm³) asfunction as a a function of time of time

(days) for the shielded secondary (distant) B78 tumor in B78 melanoma flank tumor mice

having both primary and secondary tumors. Mice were treated with various combinations

radiotherapy radiotherapy(MTRT) agent (MTRT) 90Y-NM600. agent Y-NM600.Combination treatment Combination of EBRT treatment of to the to EBRT primary the primary

tumor, 90Y-NM600 tumor, Y-NM600 MTRT, andanti-CTLA4 MTRT, and anti-CTLA4 caused caused significantly significantly reduced reduced secondary secondary tumor tumor

growth compared to other groups.

DETAILED DESCRIPTION I. I. IN GENERAL

[00130] It isItunderstood

[00130] is understood that that this this disclosure disclosure is limited is not not limited to particular to the the particular

methodology, protocols, materials, and reagents described, as these may vary. The

terminology used herein is for the purpose of describing particular embodiments only,

and is not intended to limit the scope of the present invention, which will be limited only

by any later-filed nonprovisional applications.

[00131] As used herein and in the appended claims, the singular forms "a", "an", and

"the" include plural reference unless the context clearly dictates otherwise. As well, the

terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

The terms "comprising" and variations thereof do not have a limiting meaning where

these terms appear in the description and claims. Accordingly, the terms "comprising",

"including", and "having" can be used interchangeably.

[00132] Unless defined otherwise, all technical and scientific terms used herein have

the same meanings as commonly understood by one of ordinary skill in the art to which

this invention belongs. Although any methods and materials similar or equivalent to

those described herein can be used in the practice or testing of the present invention, the

preferred methods and materials are now described. All publications and patents

specifically mentioned herein are incorporated by reference for all purposes including

describing and disclosing the chemicals, instruments, statistical analysis and

methodologies which are reported in the publications which might be used in connection

with the invention. All references cited in this specification are to be taken as indicative

of the level of skill in the art.

[00133] The The terminology terminology as set as set forth forth herein herein is for is for description description of the of the embodiments embodiments only only

and should not be construed as limiting of the invention as a whole. Unless otherwise

specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or

more than one.

[00134] TheThe disclosure disclosure is is inclusive inclusive of of thethe compounds compounds described described herein herein (including (including

intermediates) in any of their pharmaceutically acceptable forms, including isomers (e.g.,

diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, and the

like. In particular, if a compound is optically active, the invention specifically includes

each of the compound's enantiomers as well as racemic mixtures of the enantiomers. It

should be understood that the term "compound" includes any or all of such forms,

whether explicitly stated or not (although at times, "salts" are explicitly stated).

[00135] "Pharmaceutically acceptable" as used herein means that the compound or

composition or carrier is suitable for administration to a subject to achieve the treatments

described herein, without unduly deleterious side effects in light of the necessity of the

treatment.

[00136] The The term term "effective amount," "effective amount," as as used usedherein, refers herein, to the refers to amount of theof the the amount

compounds or dosages that will elicit the biological or medical response of a subject,

tissue or cell that is being sought by the researcher, veterinarian, medical doctor or other

clinician.

[00137] As used herein, "pharmaceutically-acceptable carrier" includes any and all dry

powder, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic

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agents, absorption delaying agents, and the like. Pharmaceutically-acceptable carriers are

materials, useful for the purpose of administering the compounds in the method of the

present invention, which are preferably non-toxic, and may be solid, liquid, or gaseous

materials, which are otherwise inert and pharmaceutically acceptable, and are compatible

with the compounds of the present invention. Examples of such carriers include, without

limitation, various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline,

polyethylene glycol, glycerin, polypropylene glycol, dimethylsulfoxide, an amide such as

dimethylacetamide, a protein such as albumin, and a detergent such as Tween 80, mono-

and oligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

[00138] The The term term "administering" or "administering" or "administration," "administration," as used herein, as used refersrefers herein, to to

providing the compound or pharmaceutical composition of the invention to a subject

suffering from or at risk of the diseases or conditions to be treated or prevented.

[00139] A route of administration in pharmacology is the path by which a drug is

taken into the body. Routes of administration may be generally classified by the location

at which the substance is applied. Common examples may include oral and intravenous

administration. Routes can also be classified based on where the target of action is.

Action may be topical (local), enteral (system-wide effect, but delivered through the

gastrointestinal tract), or parenteral (systemic action, but delivered by routes other than

the GI tract), via lung by inhalation.

[00140]

[00140]InInananenteral administration, enteral the desired administration, effect is the desired systemic effect (non-local), is systemic (non-local),

substance is given via the digestive tract. In a parenteral administration, the desired

effect is systemic, and substance is given by routes other than the digestive tract.

[00141] Enteral administration may be administration that involves any part of the

gastrointestinal tract and has systemic effects. The examples may include those by mouth

(orally), many drugs as tablets, capsules, or drops, those by gastric feeding tube, duodenal

feeding tube, or gastrostomy, many drugs and enteral nutrition, and those rectally, various

drugs in suppository.

Examples

[00142] Examples of parenteral of parenteral administrations administrations may may include include intravenous intravenous (into (into a vein), a vein),

e.g. many drugs, total parenteral nutrition intra-arterial (into an artery), e.g., vasodilator

drugs in the treatment of vasospasm and thrombolytic drugs for treatment of embolism,

intraosseous infusion (into the bone marrow), intra-muscular, intracerebral (into the

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brain parenchyma), intracerebroventricular (into cerebral ventricular system), intrathecal

(an injection into the spinal canal), and subcutaneous (under the skin). Among them,

intraosseous infusion is, in effect, an indirect intravenous access because the bone

marrow drains directly into the venous system. Intraosseous infusion may be

occasionally used for drugs and fluids in emergency medicine and pediatrics when

intravenous access is difficult.

[00143] The The following following abbreviations abbreviations are are used used in this in this disclosure: disclosure: ADCC, ADCC, Antibody Antibody

dependent cell-mediated cytotoxicity; anti-CTL4, an antibody that targets cytotoxic T

lymphocyte-associated antigen 4 (CTLA4), which is found on cytotoxic T lymphocytes

(CTLs); B16, A melanoma syngeneic to C57B1/6 mice; B78, A variant of B16 that

expresses GD2, due to transfection with GD2 synthase; D, day; Hu14.18-IL2, The

primary immunocytokine (reacts against GD2) used in the studies disclosed in the

examples; IC, Immunocytokine (a fusion protein of a tumor-reactive mAb linked to IL2);

ICI, ICI, immune immune checkpoint checkpoint inhibitor; inhibitor; IL2, IL2, Interleukin Interleukin 2; 2; IT, IT, Intratumoral; Intratumoral; IV, IV, Intravenous; Intravenous;

mAb, Monoclonal antibody; MAHA, Mouse anti-human antibody; NM404, used to

designate the phospholipid ether shown in Figure 1, which is selectively taken up by most

tumors and used for TRT in the studies disclosed in the examples; NM600, used to

designate the phospholipid ether shown in Figure 14, which can be chelated with any

metal, and which is also selectively taken up by most tumors and used for TRT in the

studies disclosed in the examples; NXS2, A neuroblastoma syngeneic to AJ mice;

Panc02-GD2, A pancreatic cancer syngeneic to C57B1/6 mice, expressing GD2, due to

transfection with GD2 synthase; PLE, Phospholipid ether; RT, Radiation therapy; TRT,

Targeted radiotherapy; W, week; 9464D-GD2, A neuroblastoma syngeneic to C57B1/6

mice, expressing GD2, due to transfection with GD2 synthase.

II. THE INVENTION

[00144] This disclosure is directed to methods of treating any cancer that presents as

one or more malignant solid tumors. The disclosed methods combine two treatment steps,

with an unexpected synergy resulting in a much improved effect against the malignant

solid tumors. Specifically, an immunomodulatory dose of a radioactive phospholipid

metal chelate compound or radiohalogenated phospholipid compound that is

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differentially taken up by and retained within malignant solid tumor tissue is

administered to the patient, and further immunomodulation is performed by systemically

administering (e.g., by IV injection) a composition that includes one or more agents

capable of stimulating specific immune cells, either with or without additional xRT to at

least one of the malignant solid tumors being treated with immune-stimulating agents.

[00145] The immunomodulatory dose of the radioactive phospholipid metal chelate or

radiohalogenated compound likely reduces Treg levels (and other immune-suppressive

elements) and prevents the immune system dampening (concomitant immune tolerance)

that occurs when xRT is used against a tumor and one or more additional tumors are not

radiated, although an understanding of the mechanism is not necessary to practice the

invention and the invention is not limited to any particular mechanism of action.

A. Systemically-administered immunotherapy: immune checkpoint

inhibitors as exemplary immunostimulatory agents.

In direct

[00146] In direct contrast contrast to methods to methods of immunostimulation of immunostimulation by administering by administering an an

immunomodulatory agent directly into a tumor (such as intratumoral immunization by in in

situ vaccination, as illustrated in some of the examples below), systemically-administered

immunotherapy is performed by administering an immunostimulatory agent systemically.

The immunostimulatory agent circulates through the whole body of the subject,

stimulating the body's natural immune response.

[00147] Immune checkpoint inhibitors are non-limiting examples of such

immunostimulatory agents. Activated T cells express multiple immune co-inhibitory

receptors, such as lymphocyte-activation gene 3 (LAG-3), programmed cell death protein

1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). These and other

immune checkpoint molecules have been shown to modulate T cell responses to tumor

antigens in the tumor microenvironment through unique and non-redundant pathways.

[00148] More specifically, cancer growth is partly mediated by immune suppression

induced by cancers. Tumors can activate suppressive immune checkpoint pathways in

order to diminish the general immune response to the tumor. Accordingly, blockade of

key key immune immune checkpoint checkpoint pathways pathways can can induce induce anti-tumor anti-tumor immunity, immunity, facilitated facilitated by by the the

patient's own immune system.

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[00149] CTLA4 was was CTLA4 the the first immune first checkpoint immune molecule checkpoint to be molecule to clinically targeted, be clinically by by targeted,

administering CTLA4-targeting (anti-CLA4) mAbs. To date, the most promising

immune checkpoint inhibitor strategies for the treatment of cancers involve administering

mAbs targeting CTLA-4 and/or PD-1/PD-L1. Other immune checkpoint inhibitor

strategies are currently in development, and the disclosed combination method is not

limited to targeting any specific immune checkpoint pathway.

[00150] A series of reviews covering checkpoint inhibitors and cancer immunotherapy

was recently published in volume 276 of Immunological Reviews. These reviews,

including the introductory overview, Sharpe, A.H., "Introduction to checkpoint inhibitors

and cancer immunotherapy," Immunol Rev. 276 (4 March 2017): 5-8, are incorporated by

reference herein in their entirety.

B. Immunomodulatory dose of a radioactive phospholipid metal chelate

compound

[00151] The The radioactive radioactive phospholipid phospholipid metal metal chelate chelate compound compound used used should should selectively selectively

target a wide range of solid tumor cell types, such that the RT emitted by the metal

isotope chelated to the metal chelate compound is directed to malignant solid tumor

tissue without substantially exposing other tissue types to the emitted RT. The radioactive

metal isotope included in the radioactive phospholipid metal chelate compound may be

any radioactive metal isotope known to emit ionizing RT in a form that would result in

immunostimulation of the cells that take up the compound. Non-limiting examples of

radioactive metal isotopes that could be used include Lu-177, Y-90, Ho-166, Re-186, Re-

188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, or Th-227.

[00152] The immunomodulatory RT dose (as opposed to injected dose) of the

radioactive phospholipid metal chelate compound is much less than the dose that would

be used for conventional RT against malignant solid tumors. Specifically, the dose

should be sufficient to stimulate a response in immune cells within the tumor

microenvironment (likely by reducing immune-suppressing Treg levels and other

immunosuppressive cells or molecules), while not ablating the desired immune cells that

are responsible for the immunostimulatory effect.

[00153] The The proper proper immunomodulatory immunomodulatory dose dose can can be calculated be calculated from from imaging imaging data data

obtained after administering a "detection-facilitating" dose of a radioactive metal chelate

compound. The detection-facilitating dose may be quite different than the

immunomodulatory dose, and the radioactive metal isotope that is chelated into the

radioactive metal chelate compound may be different (although the rest of the compound

structure should be the same). The radioactive metal isotope used in the detection step

and dosimetry calculations may be any radioactive metal isotope known to emit RT in a

form that is readily detectable by conventional imaging means. Non-limiting examples

of "conventional imaging means" include gamma ray detection, PET scanning, and

SPECT SPECT scanning. scanning. Non-limiting Non-limiting examples examples of of radioactive radioactive metal metal isotopes isotopes that that could could be be used used

include Ga-66, Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44, Ga-67, In-111,

or Tc-99m.

C. Metal chelates of PLE analogs

[00154] The The disclosed disclosed structures structures utilize utilize an alkylphosphocholine an alkylphosphocholine (APC) (APC) carrier carrier

backbone. Once synthesized, the agents should harbor formulation properties that render

them suitable for injection while retaining tumor selectivity as was demonstrated

previously with the related radiohalogenated compounds. The disclosed structures

include a chelating moiety to which the radioactive metal isotope will chelate to produce

the final imaging or therapeutic agent.

D. Methods of Synthesizing Exemplary M-PLE Analogs

Proposed

[00155] Proposed synthesis synthesis of compound of compound 1 shown 1 is is shown below. below. TheThe first first step step of the of the

synthesis is similar to described in Org Synth, 2008, 85, 10-14. The synthesis is started

from cyclen which is converted into DO3A tris-Bn ester. This intermediate is then

conjugated with NM404 in the presence of the base and Pd catalyst. Finally, benzyl

protecting groups are removed by the catalytic hydrogenation.

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O OBn O N BnO N H BrCH2CO2Bn BrCHCOBn + H HN + (CH2)18OPOCH2CH2NMe3 HN N (CH) OPOCHCHNMe NH AcONa, DMAc ZI H N e N N cyclen BnO O O OBn H2, Pd/C N O + (CH2)18 + Pd catalyst N DPOCH2CH2NMe3 (CH) 18OPOCHCHNMe N base O N e OBn BnO O O OH N II + (CH2)18OPOCH2CH2NMe3 N N (CH)OPOCHCHNMe O N e OH 1 HO O

Synthesis

[00156] Synthesis of compound of compound 2 is2 shown is shown below. below. It begins It begins withwith DO3ADO3A tris-Bn tris-Bn ester ester

which is alkylated with 3-(bromo-prop-1-ynyl)-trimethylsilane. After alkylation, 3-(bromo-prop-1-ynyl)-trimethylsilane After alkylation, the the

trimethylsilyl group is removed and the intermediate acetylene is coupled with NM404

by the Sonogashira reaction. The benzyl groups are removed and the triple bond is

hydrogenated hydrogenated simultaneously simultaneously in in the the last last step step of of the the synthesis. synthesis.

PCT/US2018/059927

O O OBn OBn O O N K2CO3 BnO N BrCH2-CEC-SiMe3 BnO KCO BrCH-CEC-SiMe N N MeOH N HN i-Pr2NEt i-PrNEt N N SiMe3 SiMe BnO BnO O O DO3A tris-Bn ester

O OBn O N O II PdCl2(PPh3)2 PdCl(PPh) BnO + + (CH2)18 DPOCH2CH2NMe3 N N (CH)OPOCHCHNMe Et3N, MeOH EtN, MeOH

N H BnO O O OBn N O + II H2, Pd/C H, Pd/C (CH2)18 OPOCH2CH2NMe3 N N (CH)OPOCHCHNMe. O N e OBn BnO O O OH N O II + (CH2)18OPOCHCH2NMe3 N N (CH)OPOCHCHNMe O N OH 2 HO O

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[00157] Compounds 55 and

[00157] Compounds and 6 6 can can be be synthesized synthesizedfrom same from precursors, same DTPA DTPA precursors,

dianhydride and 18-p-(3-hydroxyethyl-pheny1)-octadecyl 18-p-(3-hydroxyethyl-phenyl)-octadecyl.phosphocholine phosphocholineas asshown shownin inthe the

schemes below. schemes below.

CO2H COH O HO O + (1 (CH)OPOCHCHNMe II (CH2)18OPOCH2CH2NMe3 (1 eq) eq) N N + + N / O O DTPA dianhydride

O HOC N N N O + CO2H (CH2)18OPOCH2CH2NMe) HOC HOC HO2C COH (CH)OPOCHCHNMe HOC 5

CO2H COH O HO O + (2(2 eq) (CH2)18OPOCH2CH2NMe3 eq) N N o N N + (CH)OPOCHCHNMe O O e DTPA dianhydride

O o O

+ + o II MegNCH2CH2OPO(CH2)18 O N N N O o II

+ HOC HOC CO2H COH (CH)OPOCHCHNMe HO2O Oe HOC o o 6 1

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[00158] NOTA-NM404 conjugates can be synthesized in an analogous manner. One

example of NOTA-NM404 conjugate 7:

CO2H COH HO2C HOC N N N (CH2)+8OPOCH2CH2NMe3 (CH)OPOCHCHNMe CO2H COH 7

E. Dosage Forms and Administration Methods

[00159] For the synergistic targeted RT, any route of administration may be suitable.

In one embodiment, the disclosed alkylphosphocholine analogs may be administered to

the subject via intravenous injection. In another embodiment, the disclosed

alkylphosphocholine analogs may be administered to the subject via any other suitable

systemic deliveries, such as parenteral, intranasal, sublingual, rectal, or transdermal

administrations.

[00160] In another embodiment, the disclosed alkylphosphocholine analogs may be

administered to the subject via nasal systems or mouth through, e.g., inhalation.

In another

[00161] In another embodiment, embodiment, the the disclosed disclosed alkylphosphocholine alkylphosphocholine analogs analogs may may be be

administered to the subject via intraperitoneal injection or IP injection.

[00162] In certain embodiments, the disclosed alkylphosphocholine analogs may be

provided as pharmaceutically acceptable salts. Other salts may, however, be useful in the

preparation of the alkylphosphocholine analogs or of their pharmaceutically acceptable

salts. Suitable pharmaceutically acceptable salts include, without limitation, acid

addition salts which may, for example, be formed by mixing a solution of the

alkylphosphocholine analog with a solution of a pharmaceutically acceptable acid such as

hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic

acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or

phosphoric acid.

[00163] Where the disclosed alkylphosphocholine analogs have at least one

asymmetric center, they may accordingly exist as enantiomers. Where the disclosed

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alkylphosphocholine analogs possess two or more asymmetric centers, they may

additionally exist as diastereoisomers. It is to be understood that all such isomers and

mixtures thereof in any proportion are encompassed within the scope of the present

disclosure.

[00164] The disclosure also includes methods of using pharmaceutical compositions

comprising one or more of the disclosed alkylphosphocholine analogs in association with

a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage

forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or

suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or

suppositories; for parenteral, intranasal, sublingual or rectal administration, or for

administration by inhalation or insufflation.

[00165] For For preparingsolid preparing solid compositions compositions such suchasas tablets, the the tablets, principal activeactive principal

ingredient is mixed with a pharmaceutically acceptable carrier, e.g. conventional

tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid,

magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents,

e.g. water, to form a solid preformulation composition containing a homogeneous

mixture for a compound of the present invention, or a pharmaceutically acceptable salt

thereof. When referring to these preformulation compositions as homogeneous, it is

meant that the active ingredient is dispersed evenly throughout the composition SO so that

the composition may be easily subdivided into equally effective unit dosage forms such

as tablets, pills and capsules. This solid pre-formulation composition is then subdivided

into unit dosage forms of the type described above containing from 0.1 to about 500 mg

of the active ingredient of the present invention. Typical unit dosage forms contain from

1 to 100 mg, for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The

tablets or pills of the novel composition can be coated or otherwise compounded to

provide a dosage affording the advantage of prolonged action. For example, the tablet or

pill can comprise an inner dosage and an outer dosage component, the latter being in the

form of an envelope over the former. The two components can be separated by an enteric

layer which, serves to resist disintegration in the stomach and permits the inner

component to pass intact into the duodenum or to be delayed in release. A variety of

materials can be used for such enteric layers or coatings, such materials including a

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number of polymeric acids and mixtures of polymeric acids with such materials as

shellac, cetyl alcohol and cellulose acetate.

[00166] The liquid forms in which the alkylphosphocholine analogs may be

incorporated for administration orally or by injection include aqueous solutions, suitably

flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such

as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar

pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous

suspensions include synthetic and natural gums such as tragacanth, acacia, alginate,

dextran, sodium caboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or

gelatin.

[00167] The The disclosed disclosed alkylphosphocholine alkylphosphocholine analogs analogs are are particularly particularly useful useful when when

formulated in the form of a pharmaceutical injectable dosage, including in combination

with an injectable carrier system. As used herein, injectable and infusion dosage forms

(i.e., parenteral dosage forms) include, but are not limited to, liposomal injectables or a

lipid bilayer vesicle having phospholipids that encapsulate an active drug substance.

Injection includes a sterile preparation intended for parenteral use.

[00168] Five distinct classes of injections exist as defined by the USP: emulsions,

lipids, powders, solutions and suspensions. Emulsion injection includes an emulsion

comprising a sterile, pyrogen-free preparation intended to be administered parenterally.

Lipid complex and powder for solution injection are sterile preparations intended for

reconstitution to form a solution for parenteral use. Powder for suspension injection is a

sterile preparation intended for reconstitution to form a suspension for parenteral use.

Powder lyophilized for liposomal suspension injection is a sterile freeze dried preparation

intended for reconstitution for parenteral use that is formulated in a manner allowing

incorporation of liposomes, such as a lipid bilayer vesicle having phospholipids used to

encapsulate an active drug substance within a lipid bilayer or in an aqueous space,

whereby the formulation may be formed upon reconstitution. Powder lyophilized for

solution injection is a dosage form intended for the solution prepared by lyophilization

("freeze drying"), whereby the process involves removing water from products in a

frozen state at extremely low pressures, and whereby subsequent addition of liquid

creates a solution that conforms in all respects to the requirements for injections. Powder

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lyophilized for suspension injection is a liquid preparation intended for parenteral use that

contains solids suspended in a suitable fluid medium, and it conforms in all respects to

the requirements for Sterile Suspensions, whereby the medicinal agents intended for the

suspension are prepared by lyophilization. Solution injection involves a liquid

preparation containing one or more drug substances dissolved in a suitable solvent or

mixture of mutually miscible solvents that is suitable for injection.

[00169] Solution concentrate injection involves a sterile preparation for parenteral use

that, upon addition of suitable solvents, yields a solution conforming in all respects to the

requirements for injections. Suspension injection involves a liquid preparation (suitable

for injection) containing solid particles dispersed throughout a liquid phase, whereby the

particles are insoluble, and whereby an oil phase is dispersed throughout an aqueous

phase or vice-versa. Suspension liposomal injection is a liquid preparation (suitable for

injection) having an oil phase dispersed throughout an aqueous phase in such a manner

that liposomes (a lipid bilayer vesicle usually containing phospholipids used to

encapsulate an active drug substance either within a lipid bilayer or in an aqueous space)

are formed. Suspension sonicated injection is a liquid preparation (suitable for injection)

containing solid particles dispersed throughout a liquid phase, whereby the particles are

insoluble. In addition, the product may be sonicated as a gas is bubbled through the

suspension resulting in the formation of microspheres by the solid particles.

[00170] The The parenteral parenteral carrier carrier system system includes includes one one or more or more pharmaceutically pharmaceutically suitable suitable

excipients, such as solvents and co-solvents, solubilizing agents, wetting agents,

suspending agents, thickening agents, emulsifying agents, chelating agents, buffers, pH

adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents,

protectants, tonicity adjusters, and special additives.

[00171] The following examples are offered for illustrative purposes only, and are not

intended to limit the scope of the present invention in any way. Indeed, various

modifications of the invention in addition to those shown and described herein will

become apparent to those skilled in the art from the foregoing description and the

following examples and fall within the scope of the appended claims.

PCT/US2018/059927

III. III. EXAMPLES Introduction to the Examples

These

[00172] These examples examples demonstrate demonstrate the the potential potential of bringing of bringing together together two two very very

distinct cutting-edge disciplines in cancer treatment research, capitalizing on an

unexpected and very potent synergy. These disciplines are: 1) systemically administered

TRT; and 2) locally directed, antibody-mediated cancer immunotherapy or systemically

administered cancer immunotherapy. The data presented herein suggest that powerful

synergy results from combining these approaches. Together, these two strategies can be

used to destroy visible macroscopic tumor in a way that enables the destroyed cancer

cells to function as a potent immunostimulator that creates tumor-specific T cell

immunity able to eradicate persistent residual metastatic disease, for any type of solid

tumor in any location.

[00173] Our Our ongoing ongoing preclinical preclinical work work has has shown shown that that combination combination of tumor-specific of tumor-specific

mAb with IL2 (to activate innate immune cells) results in augmented antibody-dependent

cell-mediated cytotoxicity (ADCC) [1,2]; a process that has already been translated into

clinical benefit for children with neuroblastoma [3]. Recent preclinical data demonstrate

more potent antitumor efficacy when the mAb-IL2 fusion protein is injected

intratumorally (IT) [4,5]. Remarkably, large tumors that do not respond to these mAb/IL2

injections and continue growing if treated only with local xRT, can be completely

eradicated if the xRT is combined with the mAb/IL2 treatment. Most mice are cured and

develop T cell memory that rejects re-challenge with similar tumor cells [6];

demonstrating that the combined xRT + mAb/IL2 is acting as a potent "in situ" anti-

cancer vaccine.

[00174] A key limitation is that if there is another macroscopic tumor present in these

animals when they receive xRT+ mAb/IL2 treatment to the primary (first) tumor, the

second tumor will continue to grow and, to our surprise, suppress the immune response,

preventing any shrinkage of the 1st treated tumor. This "concomitant immune tolerance"

results, in part, from suppressive regulatory T cells (Tregs) in the 2nd tumor. 2 tumor. Delivering Delivering

RT alone to both tumors has minimal anti-tumor effect, but does deplete these Tregs.

Thus, when first tumors are treated with xRT + mAb/IL2, the addition of RT to the

second tumor circumvents this immune tolerance, enabling eradication of both tumors

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[7]. These observations indicate a limitation of in situ tumor vaccination in the metastatic

setting, but also suggest a robust capacity of RT to overcome this limitation.

[00175] xRT cannot typically be delivered to all metastatic sites without prohibitive

normal tissue toxicity and immune suppression. Yet not delivering xRT to all sites of

macroscopic disease may leave inhibitory immune lineages intact, which are capable of

suppressing the immunologic response to our local xRT + mAb/IL2 immunotherapy.

What is needed, therefore, is a means to deliver RT to all tumor sites in a cancer patient

in a targeted manner.

[00176] We have developed TRT vehicles capable of targeting systemically

administered RT to both primary and metastatic cancers. One such TRT agent, ¹³¹I-

NM404, an intravenously (IV) administered phospholipid ether (PLE) analog, has shown

nearly universal tumor targeting properties in over 60 in vivo cancer and cancer stem cell

models. This agent is currently being evaluated clinically in multiple imaging and therapy

trials [8,9]. A systemic injection of 131I-NM404 ¹³¹I-NM404 localizes in all tumors regardless of

anatomic location and internally provide sufficient RT to ablate intratumoral

immunosuppressive pathways that can prevent development of an effective, tumor-

eradicating, immune response. The unique attributes of this approach are the near

universal tumor targeting capability of NM404, as well as the ability to deliver

immunomodulatory sub-lethal doses of RT to all tumor sites, something that is not

typically feasible with xRT. What is new about this is that our TRT Agents may immuno-

modulate all tumors regardless of anatomic location, overcoming concomitant tolerance,

which will result in a long-term in situ tumor vaccination effect following local xRT

followed by injection of a tumor specific mAb + IL2. As an increasing number of tumor

specific mAbs are becoming approved for clinical use, this combination strategy provides

an expanded approach for any tumor type that can be targeted by a tumor-reactive mAb.

Furthermore, the approach can be readily generalized to all in situ tumor vaccination

strategies.

[00177] Recently, we have discovered that the iodine in 131 I-NM404 can ¹³¹I-NM404 can be be substituted substituted

with chelators capable of carrying a wide variety of metallic imaging (MRI and PET) and

TRT radiotherapy moieties. In these examples, we describe how to assess the ability of

131-N-404 ¹³¹I-NM404(and (andthus, thus,the therelated relatedmetal metalchelate chelateanalogs) analogs)to toinitiate initiatethe thesystemic systemic

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

immunomodulatory response necessary to enable combined xRT + immunotherapy

treatment to induce a potent radioimmune-facilitated response against cancerous solid

timuors. A similar approach can be used for combined PLE analog-delivered TRT with

other immunotherapy methods used against cancerous solid tumors. For example, we

have illustrated below that the combination method can use an immunodulation step that

is quite different from local in situ tumor vaccination: the systemic administration of an

immunostumulatory agent such as an immune checkpoint inhibitor.

[00178] In sum, we disclose In sum, herein we disclose therapeutic herein and and therapeutic research processes research thatthat processes combine combine

two different methods from seemingly disconnected cancer therapy disciplines into a

single unified treatment. The data presented in these examples indicate that the two

methods can be synergistically combined to effectively eliminate malignant solid tumors

and to prevent tumor recurrence.

[00179]

[00179] InIn Example 1,1, Example wewe present background present data background from data our from B78 our GD2+ B78 model GD2+ inin model

support of the method.

[00180] In Example 2, we provide guidance for determining the dose of xRT needed

for optimal in situ vaccine effect to a primary tumor, and the lowest dose of xRT to a

distant tumor needed to prevent concomitant immune tolerance.

¹³¹I-NM404dosing

[00181] In Example 3, we provide guidance for determining the 31I-NM404 dosing

that approximates the required dosing of xRT to metastases, as determined in Example 2,

and subsequently evaluating the effects of that 131-N-404 ¹³¹I-NM404dose doseon onin invivo vivoimmune immune

function. Such guidance can be similarly applied when using the disclosed radioactive

phospholipid metal chelate compounds as the TRT agent.

[00182] In Example 4, we provide guidance for using data from Examples 2 and 3 to

design/test/develop a regimen of ¹³¹I-NM404 + local 1-N-404 + local xRTxRT + IT-mAb/IL2 + IT-mAb/IL2 in in mice mice bearing bearing

two or more tumors in order to destroy the locally treated tumors and induce T-cell

mediated eradication of all distant tumors. Critical issues of TRT and xRT dose and time

are optimized for antitumor efficacy. Again, such guidance can be similarly applied when

using the disclosed radioactive phospholipid metal chelate compounds as the TRT agent.

[00183] In Example 5, we provide an exemplary synthesis that also finds use to the

synthesis of analogous compounds chelating radioactive metal isotopes.

[00184] In Example 6, we demonstrate that an analog having a chelating agent and

chelated metal substituted for the iodine moiety of NM404 (Gd-NM600) is taken up by

(and can be imaged in) solid tumor tissue, thus providing proof of concept for using the

disclosed metal chelates as a TRT agent.

In Examples

[00185] In Examples 7, 9 7, 8, 8,and 9 and 10, 10, we provide we provide information information and and specific specific data data from from

experimental studies performed in accordance with the guidance of Examples 1-4.

In Examples

[00186] In Examples 11 and 11 and 12, 12, we demonstrate we demonstrate that that additional additional analogs analogs having having a a

chelating agents and chelated metals substituted for the iodine moiety of NM404 are

taken up by, and can be imaged in, and can be used therapeutically for TRT in a range of

solid tumor in vivo models, thus providing additional proof of concept for using the

disclosed metal chelates as TRT agents in the disclosed methods.

[00187] In Example 13, we discuss how dosimetry in combination with known

radiosensitivities can be used by the skilled artisan to optimize treatment dosages for any

solid tumors.

[00188] In Example 14, we discuss differences and advantages in using

alkylphosphocholine metal chelates in the disclosed methods, rather than the iodinated

compounds exemplified in Examples 1-4 and 7-10.

[00189] In Examples 15 and 16, we demonstrate that TRT in combination with

systemically-administered immunotherapy, rather than in situ vaccination, is also

effective is treating solid tumors. The immunostimulatory agent that is systemically

administered may be an immune checkpoint blocker or inhibitor (in this case, anti-

CTLA4).

Example 1: Background Supporting Data

[00190] The The Sondel lab lab Sondel has has shown that shown tumor-specific that mAb mAb tumor-specific + IL2 activates + IL2 innate activates innate

immune cells to mediate ADCC in mice [2], with clinical benefit for children with

neuroblastoma [3]. In mice, IV administration of the u14.18-IL2 is is hu 14.18-IL2 more potent more than potent IV IV than

administration of anti-GD2 mAb + IL2 [2, 10]. This can provide dramatic antitumor

effects against very small recently established GD2+ tumors or very small microscopic

metastases, potentially accounting for the clinical use of this approach in patients in

remission but at great risk for relapse [3]. More potent antitumor efficacy against

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measurable, macroscopic tumors [i.e. ~ 50 mm³ GD2+ tumors] can be achieved when the

IC is injected intratumorally (IT-IC) rather than IV [4,5].

[00191] We are now focusing on ways to provide benefit in the setting of much larger,

macroscopic tumors. Mice bearing a moderately large (200 mm³) B78 melanoma tumor,

established five weeks earlier, show no response to IV-IC, and are slowed in their growth

by IT-IC, but the tumors continue to grow. These same 200 mm³ tumors also grow after

12 Gy of xRT. In contrast, when the IT-IC and xRT are combined, 73% of the animals

become tumor-free and appear cured of their disease (Figs. 2A and 2B). These mice then

show T-cell mediated rejection of rechallenge with the same tumor (Fig. 2C). Thus IT-IC

+ xRT synergize, inducing the tumor to become an "in situ tumor vaccine" [6].

[00192] In order to simulate clinical metastases, we inoculate mice with B78 in one

flank on d-1, and the other flank at week 2. At week 5, the first tumor is 200 mm³, and

the second is 50 mm³. We anticipated that xRT + IT-IC would destroy the first tumor and

that the resultant T cell response would then destroy the second. However, adding IT-IC

to the xRT had virtually no effect on either the 50 mm³ tumor or the 200 mm³ tumor (Fig.

3). This demonstrated a key limitation to the therapy we delivered; namely, if there is

another tumor present when these mice receive xRT + IT-IC to the first tumor, the second

tumor will cause a systemic tumor-specific concomitant immune tolerance effect,

preventing any shrinkage of either tumor. Importantly, we have found that local xRT (12

Gy) to the first and second tumor simultaneously, abrogates this tolerance effect,

allowing IT-IC to the first tumor to induce an immune response that eradicates both

tumors in most mice (Fig. 4) [7]. Recent data, using a Treg depleting mAb (not shown) or

transgenic mice that allow selective Treg depletion (Fig. 4) [7], demonstrate that this

immune tolerance is mediated, in part, by regulatory T cells (Tregs); RT to the first and

second tumors partially deplete these Tregs, potentially explaining how irradiating both

tumors circumvents the tolerance effect [7].

[00193] While local xRT to both the first and second tumors circumvents tolerance,

clinical metastatic disease is often in several locations. All macroscopic metastatic

disease must receive RT to block immune tolerance and enable xRT + IT-IC to

effectively eradicate all tumor sites. However, delivery of 12 Gy xRT to all sites of disease may be akin to "total body RT" with major dose-dependent (potentially lethal) toxicity and profound systemic immune suppression.

[00194] Previously, the Weichert lab has pioneered the development of TRT, in order

to deliver RT to all systemic tumor sites, while minimizing "off-target" RT to normal

tissue (especially marrow and immune tissue).

[00195] Based on the finding that tumor cells contain an overabundance of

phospholipid ethers (PLE) [11], we synthesized over thirty radioiodinated PLE analogs in

hopes of identifying analogs that would selectively target tumors [12]. One of these,

NM404, not only displayed near universal tumor uptake in all but three of over 70 in vivo

models examined regardless of anatomic location, including brain metastases and cancer

stem cells, but also underwent prolonged selective retention once it entered tumor cells

[8]. These diapeutic PLE analogs are unique in that they avoid premalignant and

inflammatory lesions. Surface membrane lipid rafts, which are overexpressed on cancer

cells relative to normal cells, serve as portals of entry for PLE's, including NM404, into

cancer and cancer stem cells [8]. Radioiodinated NM404 (I-124 and I-131), which has

now been evaluated in five phase 1 and 2 PET imaging trials and three phase 1 TRT

radiotherapy trials, respectively, affords similar tumor uptake and retention properties in

over a dozen human cancer types [8]. Excellent tumor uptake in the cancer models

relevant to these examples (the B78 GD2+ murine melanoma) have been confirmed with

124 I-NM404 ¹²I-NM404 PET PET imaging imaging (Fig. (Fig. 5). 5).

Example 2: Determining Dosages of xRT

[00196] Our Our data data suggest suggest these these four four hypotheses: hypotheses: (1) (1) the the dose dose of xRT of xRT we have we have used used to to

treat a single tumor causes modest direct in vivo tumor death and increases susceptibility

to immune mediated death (via both ADCC and T cells); (2) the strong T-cell response

provided by the addition of IT-IC, but not IT mAb, suggests that mAb binding to radiated

tumor cells, in the presence of IL2, facilitates antigen presentation and augmented

induction of adaptive immunity; (3) the presence of a second tumor prevents the xRT +

IT-IC to the first tumor from causing virtually any anti-tumor effect, due to tolerance

caused largely by the systemic actions of immunosuppressive cells present in the second

tumor [such as Tregs and possibly myeloid derived suppressor cells (MDSC)]; this

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tolerance can be circumvented by depletion of Tregs (Fig. 4) or irradiating the second

tumor (Fig. 3); (4) the dose of RT needed at the second tumor to circumvent tolerance

might be much lower than the xRT dose needed for the first tumor to become an "in situ

vaccine" [14].

Optimizing

[00197] Optimizing xRT xRT dose dose for for the the primary primary ("in ("in situ situ vaccine") vaccine") tumor tumor site. site.

[00198] Our Our in vivo in vivo studies studies of xRT of xRT + IT-IC + IT-IC have have focused focused on one on one dose dose of Gy of 12 12 to Gy the to the

first tumor. This is based on our data showing that in vitro RT induces a dose-dependent

functional upregulation of Fas on B78 tumor cells (nearing peak at >12 Gy), coupled to

our in vivo data demonstrating our in situ vaccine effect of xRT + IT-IC requires mice

with functional Fas-L (6). We conducted in vivo pilot studies prior to selecting the 12 Gy

dose, which showed higher dose (16 Gy) or increased fractionation flank RT had toxicity

(dermatitis, ulceration, and late limb edema) and no improvement in tumor response.

While we chose a 12 Gy single fraction of xRT for our in vivo studies, as we move

towards clinical translation, it will be beneficial to better understand the mechanism of

the local xRT effect and its dose requirements, in order to safely and effectively induce

the in situ vaccine effect.

[00199] Our mouse data (Figs. 2A, 2B and 2C) show that we can induce a potent

vaccine effect with 12 Gy xRT + IT-IC, even though 12 Gy of xRT alone causes no

shrinkage of the tumor; it merely slows the progressive growth. It is contemplated that we

might see just as potent an in situ vaccine effect using lower doses of RT. To test this, we

will evaluate a range of xRT doses (from 4 - 16 Gy) as a single fraction in mice bearing a

~ 200 mm³ B78 tumor, followed by our standard IT-IC regimen (50 mcg/d on days 6-10).

We will determine which xRT doses give optimal tumor eradication and T-cell memory,

when combined with IT-IC. If doses lower than 12 Gy are less toxic and show

comparable efficacy, such lower doses would be better targets for our xRT dose to the "in

situ vaccine" site in Examples 3 and 4. Similar approaches may be used to optimize

dosing for particular targets or subjects.

[00200] Optimizing xRT dose at a distant tumor to prevent tolerance from

blocking "in situ vaccination."

Treating

[00201] Treating both both the the first first and and second second tumors tumors with with 12 (Fig. 12 Gy Gy (Fig. 3) enables 3) enables IT-IC IT-IC to to

the first tumor to induce a potent response that eradicates both tumors. Our goal is to be able to accomplish this same in situ vaccine effect by providing xRT + IT-IC to a single tumor while using the minimal RT dose necessary at metastatic sites to circumvent tolerance. We recognize that xRT itself, especially if widespread, can be myelo/immunosuppressive. myelo/immunosuppressive. This This is is why why we we are are pursuing pursuing TRT TRT in in Examples Examples 33 and and 4. 4. Even Even though it is targeted, TRT does have some systemic delivery of RT. In order to minimize the systemic immune suppression from TRT, we wish to give as low of a dose of TRT as is needed to effectively inhibit the tumor-induced immune tolerance, while not causing systemic RT-induced global immune suppression. Therefore, it is best to select the lowest dose of xRT needed to be delivered to the distant tumor in order to enable a higher xRT dose to the first tumor to function as an in situ vaccine when combined with IT-IC to the first tumor.

[00202] As an exemplary optimization experiment, mice bearing a 200 mm³ first B78

tumor and a ~50 mm³ second B78 tumor will receive 12 Gy of xRT to the first tumor on

day-0 (~5 weeks after implantation of the first B78 tumor). This will be followed by our

standard regimen of IT-IC on days 6-10. Separate groups of mice will receive varying

doses of xRT to the second tumor. Based on data from the lab of B. Johnson

demonstrating that a total body xRT of 3 Gy can prevent an immunosuppressive effect in

a myeloma model (15), we will evaluate doses of 0, 1, 5 and 8 Gy (in addition to the 12

Gy dose we know is effective). We will see if doses substantially less than 12 Gy to the

second tumor can be as effective as the full 12 Gy dose at eliminating the immune

tolerance.

Once

[00203] Once we have we have selected selected the the critical critical dose dose of xRT of xRT where where we lose we lose the the beneficial beneficial

effect, we will perform subsequent analyses to better optimize the critical dose. For

example, if 5 Gy is as effective as 12 Gy, but 1 Gy is not much better than 0 Gy, we

would thencompare would then compare 2, 2, 3, and 3, and 4 Gy 4toGyidentify to identify the critical the critical lowest effective lowest effective RT dose RT dose

needed to eliminate tolerance and obtain efficacy in this two tumor model, receiving 12

Gy + IT-IC to the first tumor.

Repeat

[00204] Repeat studiesare studies are then then be be done done to toconfirm confirmif if this lowest this effective lowest dose to effective the to the dose

second tumor still enables an effective in situ vaccine when the dose to the first is the

lowest effective dose in the 1-tumor model (tested in Example 2, above) rather than the

12 Gy dose. In summary, the studies of Example 2 optimize what the lowest xRT doses are for the first and second tumors, without losing the efficacy we have demonstrated with 12 Gy to both.

[00205] Initiating studies of required xRT dose to first and second tumors in mice

bearing tumors other than B78.

[00206] To allow our mouse studies to suggest more clinical generalizability, we will

initiate analyses of RT + IT-IC in additional models of GD2+ tumors. We have published

on IT-IC with hu 18-IL2 IC in AJ mice bearing the GD2+ NXS2 neuroblastoma [5]. 14.18-IL2

We are also evaluating IT-IC with this same IC in C57BL/6 mice bearing the GD2+

9464D-GD2 neuroblastoma, and the Panc02-GD2 pancreatic cancer that express GD2 via

our insertion of the gene for GD2 synthase. As for Example 2, for each model we will

determine the lowest effective xRT dose needed to the primary and the secondary tumors

to retain the in situ vaccine effect.

Example 3:

Determining Dosage of 131I-NM404 ¹³¹I-NM404 and Evaluating Effects on Immune Function

Dosimetry with TRT and immunesuppression from TRT in C57BL/6 mice.

[00207] ¹³¹I-NM404

[00207] 131-N-404 has shown has shown selective selective uptake uptake in vitro in vitro in >95% in >95% of tumor of tumor lineslines

(human and mouse), with poor uptake by non-malignant cells, and with similar tumor

specificity seen in vivo. This includes selective uptake in vivo with the B78 tumor (Fig.

¹²I-NM404 to 5). In our preliminary dosimetry study, we gave 124I-NM404 to C57BL/6 C57BL/6 mice mice and and

characterized the time course of TRT exposure by serial PET/CT imaging (as in Fig. 5).

Monte Carlo dosimetry calculations [16-18] based on this study indicated that ~ 60 uCi µCi

¹³¹I-NM404 would be needed to deliver ~ 3 Gy to an established B78 tumor over a of 131I-NM404

four week period of decay. After those four weeks, the remaining TRT dose to the B78

tumor would be less than 0.25 Gy. We will replicate the data we obtained in our 2-tumor

model using xRT (Fig. 3), but use the lowest possible dose of targeted 131-N-404 ¹³¹I-NM404TRT TRT

to enable effective elimination of tumor-induced tolerance at all sites of distant disease.

However, unlike xRT, which delivers all dose in minutes and is then done, TRT deposits

dose over time, depending upon both the biological and physical half-life of the targeted

isotope (8 day t1/2 for t½ for 131I). ¹³¹D. We We want want an an initial initial TRTTRT effect effect at at thethe distant distant tumor tumor sites sites to to

eradicate immune tolerance; however we want the immunosuppressive TRT effect to

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then be minimal when we give the IT-IC to induce ADCC and the in situ vaccine anti-

tumor effects. This is essential to allow full tumor destruction at all sites.

[00208] Using the dosimetry calculations from our preliminary data, we estimated that

a dose dose of of3 3µCi of of uCi ¹³¹I-NM404, should 131-NM404, deliver should an equivalent deliver of ~0.2 of an equivalent Gy ~0.2 to theGytumor site, to the tumor site,

a dose that we hypothesized should not be immunosuppressive and should not prevent

lymphocyte-mediated tumor destruction. As noted above, this is the dose we estimated

¹³¹I-NM404dose would remain yet to be delivered 28 days after an initial 31I-NM404 doseof of60 60uCi. µCi.We We

thus evaluated groups of mice bearing a single 200 mm³ B78 tumor. On day 0, all mice

got 12 Gy xRT to their tumor, and on days 6-10, all got 50 mcg/d of IT-IC. One group

also got 3 uCi µCi of 131I-NM404 ¹³¹I-NM404 on d-0 (~0.2 Gy) Gy).Fig. Fig.6 6shows showsthat thatthe thegroup groupreceiving receivingthe the

¹³¹I-NM404had 131-NM404 hadthe thesame samedegree degreeof oftumor tumoreradication eradicationas asthe thegroup groupwithout withoutI-NM404, ¹³¹I-NM404,

demonstrating that this low dose of "residual" TRT in the tumor does not block immune

mediated destruction by the RT + IT-IC in situ vaccine. We thus hypothesize that if we

use an initial dose of 60 uCi µCi of 131I-NM404 ¹³¹I-NM404 TRT on day-22, it would effectively block

the tolerogenic effect of distant tumors, yet enable xRT on day 0 and IT-IC on days 6-10

(28d after the TRT) to the first tumor to function as an in situ vaccine, inducing an

adaptive response that then eradicates all tumors.

[00209] The experiments outlined in this example optimize the dose relationships

¹³¹I-NM404 tested in Fig. 6. In our 1-tumor B78 model, we will test a range of doses of 1-NM404

TRT to select the best TRT dose that results in enough unwanted systemic immune

suppression to interfere with the desired in situ vaccine effect (and thereby slow or

prevent eradication of the first tumor). This is important to Example 4, as it allows us to

make sure the residual radioactivity of the TRT has decayed to less than this value at the

time we initiate IT-IC to the first tumor in mice with distant disease. We will also

evaluate the kinetics of the TRT response after varying TRT doses to select an optimal

time period for how long we should wait after the "tolerance-preventing TRT dose" is

given to animals with multiple tumors to allow the RT + IT-IC treatment of the first

tumor to still induce the in situ vaccine effect and eradicate the primary as well as distant

tumors.

[00210] Related studies will also look at what dose of TRT, given as single agent

treatment, are most beneficial to cause slowing, versus shrinkage, versus eradication of a

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single B78 tumor. The dose of TRT that is most beneficial to eliminate the tumor-induced

immune tolerance will be substantially less than the TRT dose needed to actually induce

complete tumor destruction (from the TRT alone).

[00211] Finally, once the effects of various optimized doses of TRT are determined in

the 1-tumor model, we will evaluate the subtle immune-suppressive effects of TRT, by

evaluating sera from these subject for their immune response to the human IgG

component of the IC. We have shown that immunocompetent mice generate a readily

quantified level of Mouse Anti-Human Antibody (MAHA) following treatment with

these humanized ICs (19). We will use this as a means of determining at what dose we

are seeing the TRT cause a detectible dose-dependent decrease in the strength of the

murine immune response, to gauge the overall immunosuppressive effects from the

systemic doses of RT these mice will receive from this TRT. The low TRT dose that we

will need to block the tumor-induced immune tolerance will cause minimal systemic

immune suppression.

Example 4: Developing an optimal Regimen of 131-N-404 ¹³¹I-NM404++local localxRT xRT++IT- IT-

mAb/IL2 in Mice Bearing Two or More Tumors

[00212] Testingthe

[00212] Testing the efficacy efficacy of of TRT TRT+ +RTRT+ IT-IC in the + IT-IC 2-tumor in the B78 model. 2-tumor B78 model.

[00213] The The dose dose and and timing timing information information learned learned from from the the studies studies outlined outlined in in

Examples 2 and 3 will provide the information we need to optimize TRT dosing and

timing required for efficacy in our 2-tumor model. C57BL/6 mice will be inoculated with

B78 in the left (L) and right (R) flanks simultaneously. Each tumor should be - ~ 50 mm³

after two weeks and ~ 200 mm³ after five weeks. If we assume that our dosimetry

calculations in Example 3 suggest that we need to deliver 60 uCi µCi of TRT to approximate

3 Gy RT to the second tumor (to block the immune tolerance), our external beam xRT

studies predict that this dose should have minimal slowing effect on tumor growth. We

would plan to treat different groups of mice with 30, 60 or 90 uCi µCi at the 2 W time point

(when the tumors are ~ 50 mm³. mm³).Three Threeweeks weekslater laterthe thetumors tumorsshould shouldbe be- ~200 200mm³; mm³;at at

that time we will give xRT (dose determined as outlined in Example 2) followed six days

later later (~ (~ 28 28 dd after after the the TRT) TRT) by by five five daily daily injections injections of of IT-IC IT-IC to to the the tumor tumor in in the the LL flank, flank,

to induce the in situ vaccine effect. Control mice would get no TRT, and only the xRT

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and IT-IC to the L flank, anticipating no in situ vaccine due to tolerance from the distant

tumor. A separate group would get local xRT to both tumors and IT-IC to the L flank,

anticipating eradication of both tumors via the in situ vaccine effect. Another group get

TRT + IT-IC, but without local xRT, anticipating an incomplete vaccine effect.

Follow-up

[00214] Follow-up experiments experiments further further evaluate evaluate varying varying doses doses of TRT of TRT and and variations variations

in the timing between the TRT and the local xRT + IT-IC to the primary tumor (L flank).

The readouts will be: (A) eradication of the primary tumor; (B) eradication of the

secondary tumor; and (C) systemic immune suppression, via ELISA analyses of the

MAHA response. Our goal is to identify optimal TRT dose and timing with a particular

subject and disease model, to add to the local xRT + IT-IC regimen that can eradicate

both tumors in most subject, while minimizing systemic immunosuppression (as

measured by MAHA response).

[00215] Optimizing

[00215] Optimizing TRT TRT + + xRT xRT + + IT-IC IT-IC inin mice mice bearing bearing more more than than two two B78 B78

tumors. This

[00216] This sectionofofExample section Example 4 is is most mostanalogous analogousto to the the relevant clinical relevant setting; clinical setting;

namely, patients with an injectable tumor that could be used as an in situ vaccine site, but

with multiple distant metastases that could each be causing tumor-induced immune

tolerance. These studies will replicate the conditions found to be most effective in the

first part of Example 4 (above). The important difference is that these subject will each

have four separate tumors, in L and R flanks, and L and R para-scapular regions. The

TRT is given at the dose and timing found most effective in the studies outlined in the

first section of Example 4, with xRT + IT-IC subsequently given only to the L-flank

lesion. The goal here is to select TRT dose and timing issues to enable most effective in

situ vaccine, because the TRT would effectively eliminate the tumor-induced immune

tolerance caused by the three sites not getting xRT. The measure of efficacy is

elimination of all four tumors in most subjects. Modifications in TRT dose and timing are

tested in order to generate an optimized regimen that is most effective. Such a regimen

finds use in the clinic for patients with multiple distant metastases, that could not all be

irradiated via external beam, but could be irradiated via TRT, when combined with local

xRT + IT-IC to the "in situ vaccine" site.

Example 5: Synthesis of metal chelated NM600

[00217] In this Example, we show the synthetic scheme used to synthesize one

exemplary phospholipid chelate, Gd-NM600. Analogs incorporating various radioactive

isotopes could be synthesized in a similar manner, where the radioactive isotope in

questions is substituted for Gd.

2019/04457 OM PCT/US2018/059927

Gd): for substituted be could isotopes metal radioactive disclosed (the Gd-NM600 synthesizing for Scheme

[00218] Gd): for substituted be could isotopes metal radioactive disclosed (the Gd-NM600 synthesizing for Scheme

[00218] (CH)OPOCHCHNMe

(CH2)18OPOCHCH2NMe3 + O'Bu OBn CHCI, 80% CHCl3, 80% COMU, Et3N COMU, Et3N

O o O O O" O N N

N N OPOCH2CH2NMe3 18 (CH2) (CH)OPOCHCHNMe

BnO BnO BnO

Bn

+

OPOCH2CH2NMe3 (CH2)18 (CH)OPOCHCHNMc

NH II O 3152.303.P180116WO01 3152.303.P180116WO01 OH KCO, MeCN K2CO3, MeCN ++

}62 / {01273789.DOCX 62 {01273789.DOCX/ e N OO OtBu O'Bu

O O O 93% N N OF O Ho HO e O N o o Br OH O O o H2, Pd/C H, Pd/C

OBn OBn EtOH EtOH 80%

HBr H2N O HN

HN (CH2)18OPOCHCHNMe3 (CH)OPOCHCHNMe

N HN ZI + N N OBn + OH N N- Gd Gd BnO BnO

O O BnO BnO O o O N N N1 e O N N N O o NaOAc, DMAC NaOAc, DMAC O OBn OBn N N 75% 75% BnO

O BnO BnC O Br Py-HO Py-H2O GdCl3 GdCl 82%

IZ N H OBn OII dioxane dioxane

95% HCI NH HN NH NH HN HN

cyclen

N O O N N BnO BnO

N OBn

O o

Example 6: In vivo Imaging Proof of Concept

[00219] In this example, we demonstrate the successful in vivo MRI imaging of a

tumor, using Gd-NM600 as the MRI contrast agent. The data demonstrates that the

backbone phospholipid and chelating agent are taken up and retained by solid tumors,

demonstrating that such chelates incorporating various radioactive metals, as

disclosed herein, would exhibit similar properties

[00220] For proof-of-concept in vivo imaging of tumor uptake of the Gd-NM404

agent, nude athymic mouse with a flank A549 tumor (non small cell lung cancer)

xenograft was scanned. The Gd-NM600 agent (2.7 mg) was delivered via tail vein

injection. Mice were anesthetized and scanning performed prior to contrast

administration and at 1, 4, 24, 48, and 72 hours following contrast delivery. Imaging

was performed on a 4.7T Varian preclinical MRI scanner with a volume quadrature

coil. Tl-weighted T1-weighted images were acquired at all imaging time points using a fast spin

echo scan with the following pulse sequence parameters: repetition time (TR) = 206

ms, echo spacing = 9 ms, echo train length = 2, effective echo time (TE) = 9 ms, 10

averages, with a 40x40 mm² field of view, 192x192 matrix, 10 slices of thickness 1mm

each.

[00221] As As seenininFigure seen Figure 7, 7, MRI MRI imaging imagingofof thethe tumor was was tumor significantly enhanced significantly enhanced

by 24 hours post-injection.

[00222] These results demonstrate that the differential uptake and retention of

alkylphosphocholine analogs is maintained for the metal chelated analogs disclosed

herein. Thus, the disclosed metal chelates can readily be applied to clinical

therapeutic and imaging applications.

Example 7: Experiments determining the dose of xRT needed for optimal in situ

vaccine effect to a primary tumor, and the lowest dose of xRT to a distant tumor

needed to prevent concomitant immune tolerance

[00223] As aAs

[00223] a follow-up follow-up to Examples to Examples 1-4,1-4, dosedose titration titration experiments, experiments, evaluating evaluating a a

variety of xRT doses, to mice with 1 or 2 tumors have been performed. The first goal

has been to test the dose of xRT needed in mice with one tumor to facilitate synergy

and an "in situ vaccine" with IT-IC, tumor-reactive mAb linked to IL2. Initial

experiments have confirmed our prior observation that 12 Gy RT alone does not

eradicate or even regress the growth of established B78 melanoma tumors (0%

complete regression), whereas 12 Gy + IT-IC results in complete regression of most

PCT/US2018/059927

B78 tumors (66%) in mice bearing a single tumor. On the other hand, 2 Gy + IT-IC

slows tumor progression compared to IT-IC alone (mean tumor size day 32 = 472

mm³ VS 1214 mm³, respectively) but did not render any mice disease free (0%

complete regression).

[00224] In our "2-tumor model", we have previously shown that treatment of one

"primary" tumor with xRT + IT-IC is not effective in treating either the treated

primary tumor or the untreated "secondary" tumor. In fact, in this 2-tumor model we

have observed that the presence of the second tumor eliminates the efficacy of IT-IC

injection following xRT. We have designated this phenomenon as "concomitant

immune tolerance" (CIT), and demonstrated that this results, at least in part, from T

regulatory cells (Tregs) in the distant (non-irradiated) secondary tumor, which

circulate systemically and repopulate the xRT-treated/IT-IC xRT-treated /IT-ICinjected injectedprimary primarytumor. tumor.

These Tregs that return to the primary tumor appear to interfere with the desired "in

situ vaccine" effect.

[00225] We We have have nownow confirmed confirmed ourour prior prior observation observation that that CITCIT cancan be be overcome overcome

by delivering 12 Gy xRT to both the primary and the secondary tumor. Importantly,

given that Tregs are quite sensitive to RT, we hypothesized that a lower dose of RT

could be delivered to the secondary tumor in order to overcome CIT and rescue

response to in situ vaccination at the primary tumor (primary tumor treated with 12

Gy + IT-IC). We have now tested this and observed that xRT doses of 2 Gy or 5 Gy

to the secondary tumor are comparable to 12 Gy in their capacity to blunt CIT and

rescue response to primary tumor treatment with 12 Gy + IT-IC. These important

experiments have been repeated in duplicate, and suggest (as hypothesized) that the

dose of xRT that must be given to distant tumors to prevent CIT is much less than the

dose needed at the IT-IC injected primary tumor site for the purpose of generating an

in situ vaccine effect.

[00226] ThisThis

[00226] supports supports our our overarching overarching hypothesis hypothesis in this in this disclosure, disclosure, and and suggests suggests

that in animals bearing multiple tumors we will be able to deliver a relatively low

dose of RT to all sites of disease using the targeted radiotherapeutic (TRT) NM600,

and thereby overcome CIT when this is combined with local xRT and IT-IC injection

of a single tumor site (the in situ vaccine site).

wo 2019/094657 WO PCT/US2018/059927 PCT/US2018/059927

Example 8: Experiments determining the 131-N-404 ¹³¹I-NM404dosing dosingthat thatapproximates approximates

the required dosing of xRT to metastases, as determined above, and then

evaluating the effects of that 131I-NM404 ¹³¹I-NM404 dose on in vivo immune function

Based

[00227] Based ononthe thepreliminary preliminary data datadescribed describedabove in Examples above 1-4, studies in Examples 1-4, studies

have been done to move these concepts into in vivo testing using TRT. Dosimetry

studies have been performed on mice bearing 1 or 2 B78 tumors (the tumor model

that we have used to demonstrate best our in situ vaccine approach and the hurdle of

¹³¹I-NM404that CIT). This was done in order to estimate the amount of 131-NM404 thatwould wouldbe be

needed to approximate ~ 2 Gy of xRT.

[00228] In order

[00228] to then In order determine to then if aif determine ~2a Gy ~2 equivalent dosedose Gy equivalent of ¹³¹I-NM404 of 131-N-404

would have the desired effects against intratumor lymphoid cells (particularly Tregs),

2 separate approaches have been pursued. First, we administered this dose of 131 L ¹³¹I-

NM404 to mice bearing a radiosensitive lymphoma tumor, which exhibits comparable

NM404 uptake to B78 tumors. Following this we have documented potent lymphoid

tumor shrinkage/dose-dependent inhibition under conditions that did not cause either

substantial shrinkage/slowing of the B78 tumor or any evident depletion of circulating

lymphoid cells (as gauged by peripheral complete blood counts). These data are

consistent with the fact that lymphoid cells are much more sensitive to low-dose RT

than are typical solid tumor cells, and suggest that selective uptake of TRT in tumor

may enable intratumor lymphoid cell depletion without systemic lymphopenia. These

studies also suggest that such a lymphoid tumor could serve as an in vivo biological

"dosimeter" for identifying and monitoring the effect of TRT on intratumor lymphoid

cells.

A secondapproach

[00229] A second approach involved involved treating treatingmice with mice B78 B78 with tumors with these tumors with these

same doses of 1-NM404. ¹³¹I-NM404. These These animals animals were were then then sacrificed sacrificed at half-life at half-life (8d) (8d)

intervals, and after sufficient delay for radioactive decay, the tumors were stained for

the presence of effector T cells and Tregs by immunohistochemistry Intriguingly, the

¹³¹I-NM404in animals receiving 131-NM404 inthis thisinitial initialexperiment experimentshowed showedno nosystemic systemic

lymphopenia at any time point (by peripheral complete blood count) but did show a

decrease in intratumor FoxP3+ Tregs at 2 half-lifes following TRT administration. At

this 2-half-life time point, we also observed a decrease in intratumor effector CD8+ T

cells. Importantly, however at subsequent 3 and 4 half-life time points we observed an

increase in intratumor CD8+ effector T cells but a further decline in the levels of

and2 half-life intratumor Tregs, both compared to untreated baseline and2nd levels. half-life This levels. This

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

observation again supports our hypothesis that it will be feasible to use TRT to

overcome Treg-mediated CIT in order to rescue an in situ vaccine effect in animals

bearing multiple tumors.

Finally,

[00230] Finally,

[00230] to characterizing to characterizing the the immunological immunological effects effects of TRT of TRT on the on the

immune cells immune cellswithin tumors, within we have tumors, treated we have B78 bearing treated mice with B78 bearing 131 with mice I-NM404 and 131-N-404 and

collected tumor tissue at pretreatment and at half-life (8d) intervals thereafter. These

tissues were then analyzed by RT-PCR for gene expression of a panel of immune

signatures. The results indicate that TRT treatment alone causes striking changes in

expression of tumor cell markers of immunsusceptibility and in genes normally

expressed only by immune cells, with the latter showing a clear time course of

decreased expression followed by rebound over-expression.

Example 9: Experiments using data from Examples 5 and 6 to develop a regimen

of 131 I-NM404++local ¹³¹I-NM404 localxRT xRT++IT-mAb/IL2 IT-mAb/IL2in inmice micebearing bearingtwo twoor ormore moretumors tumors

and induce T-cell mediated eradication of all distant tumors

[00231] ThisThis

[00231] Example Example illustrates illustrates treating treating animals animals bearing bearing tumors tumors in least in at at least 2 2

locations. Our strategy involves using xRT and local IT-IC at the in situ vaccine site,

in combination with TRT systemically to inhibit CIT, in order to obtain enhanced

anti-tumor immune activity at all tumor sites. Critical issues of TRT and xRT dose

and timing will be optimized for antitumor efficacy.

Using

[00232] Using thedata the data summarized summarized in inExamples Examples7 and 8, a8,study 7 and was done a study was in micein mice done

bearing 2 separate B78 tumors. Mice received the estimated required systemic ¹³¹I-

NM404 dose followed by xRT and local immunotherapy to the in situ vaccine site.

With appropriate controls, this dose of ¹³¹I-NM404 didappear 31I-NM404 did appearto toattenuate attenuateCIT, CIT,as as

desired in mice with 2 tumors. In addition, in mice with one tumor, this TRT dose did

not appear to interfere with the local in situ vaccine effect (as hypothesized and

desired). Further testing, and modification of some of the experimental variables, is

underway in order to try to maximize the desired effect of blocking CIT without

suppressing the in situ vaccine effect. More details regarding these experiments are

disclosed ininExample disclosed 10 10 Example below. below.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

Example 10: Data from mice bearing two or more tumors

[00233] Tumor-specific inhibition of primary tumor response to the

combination of local xRT+IT-IC by a distant untreated tumor in murine

melanoma and pancreatic tumor models.

[00234] C57BL/6 mice bearing a syngeneic, GD2+ primary flank tumor +/- a secondary tumor on the contralateral flank were treated to the primary tumor only, as

indicated, with xRT on day "1" and IT injection of 50 mcg of the anti-GD2IC, anti-GD2 IC,hul4.18- hul 4.18-

IL2 on day 6-10.

[00235] In mice bearing a primary B78 melanoma tumor, the presence of an

untreated secondary B78 tumor antagonized primary tumor response to xRTTIT-IC xRT+IT-IC

(Figure 8A). We describe this effect as "concomitant immune tolerance" - an

antagonistic effect of a non-treated distant tumor on the local response of a treated

tumor to xRT + IT-IC. Kaplan-Meier survival curves were obtained for these mice plus

replicate experiments (Figure 8B). Nearly all mice were euthanized due to primary

tumor progression.

[00236] In In micebearing mice bearing aa primary primary Panc02-GD2+ Panc02-GD2+pancreatic tumor, pancreatic with or tumor, without with or without

a secondary Panc02-GD2- tumor on the opposite flank, the presence of an untreated

xRT+IT-IC (Figure 8C). In mice bearing a primary B78 melanoma tumor, a secondary

B78 tumor suppressed primary tumor response to xRT+IT-IC but a secondary Panc02-

GD2+ GD2+ pancreatic pancreatic tumor tumor did did not not exert exert this this effect effect (Figure (Figure 8D). 8D). In In mice mice bearing bearing aa primary primary

Panc02-GD2+ tumor a secondary Panc02-GD2- tumor suppressed primary tumor

response to combined xRT and IT-hu14.18-IL2, while a B78 secondary tumor did not

(Figure 8E).

Concomitant

[00237] Concomitant immune immune tolerance tolerance is circumvented is circumvented by specific by specific depletion depletion

of regulator T cells (Tregs).

[00238] Immunohistochemistry images were obtained for the Treg marker, FoxP3

for tumors evaluated on day 6 after xRT in mice with one or two tumors (Figure 9A).

Mice received no xRT, or xRT only to the primary tumor. DEREG mice express

diphtheria toxin receptor under control of the Treg-specific FoxP3 promoter, enabling

specific depletion of Tregs upon IP injection of diphtheria toxin (Figures 9B and 9C).

DEREG mice bearing primary and secondary B78 melanoma tumors were treated with

xRT+IT-IC to the primary tumor and IP injection of either diphtheria toxin or PBS.

Concomitant immune tolerance is eliminated following depletion of Tregs in these

WO wo 2019/094657 PCT/US2018/059927

mice, resulting in improved primary (Figure 9B) and secondary (Figure 9C) tumor

response.

[00239] Concomitant immune Concomitant tolerance immune is is tolerance overcome by by overcome delivering xRTxRT delivering to to both both

tumor sites.

[00240] In In micebearing mice bearing primary primary and andsecondary secondaryB78B78 tumors, the secondary tumors, tumor tumor the secondary

suppresses primary tumor response to primary tumor treatment with xRT + IT-IC.

This is overcome by delivering 12 Gy xRT to both the primary and secondary tumors

and IT-IC to the primary tumor, resulting in improved primary tumor response

(Figure 10A) and aggregate animal survival (Figure 10B) from replicate experiments.

[00241] LowLow dosexRT dose xRT alone alone does does not notelicit elicitin in situ vaccination situ but does vaccination but does

overcome concomitant immune tolerance when delivered to distant tumor sites

together with 12 Gy + IT-IC treatment of an in situ vaccine site.

[00242] In mice bearing a primary B78 tumor only, 12 Gy + IT-IC elicits in situ

vaccination (as shown previously) and results in complete tumor regression in most

mice (Figure 11A) and a memory immune response (Morris, Cancer Res, 2016). On

the other hand no animals exhibit complete tumor regression following either IT-IC

alone or low dose (2 Gy) xRT + IT-IC (0/6 in both groups) p<0.05.

[00243] In mice bearing a primary and secondary B78 melanoma tumor, low dose

xRT (2 Gy or 5 Gy) delivered to the secondary tumor is comparable to 12 Gy in its

capacity to overcome concomitant immune tolerance at the primary tumor (Figure

11B). In these same animals, it is apparent that overcoming concomitant immune

tolerance by delivery of low dose xRT to the secondary tumor rescues a systemic

response to IT-IC immunotherapy (Figure 11C). In this context, when RT is delivered

to all tumor sites then IT-IC injection of the primary tumor triggers a systemic anti-

tumor effect that renders secondary tumor response to 2 Gy or 5 Gy greater than the

response to 12 Gy RT in absence of primary tumor IT-IC injection.

[00244] Low dose TRT with 131-N-404 ¹³¹I-NM404effectively effectivelydepletes depletestumor tumorinfiltrating infiltrating

FoxP3+ Tregs without systemic leukopenia or depletion of tumor infiltrating

CD8+ effector T cells.

In most

[00245] In most

[00245] clinical clinical scenarios, scenarios, it not it is is not feasible feasible to deliver to deliver external external beam, beam, eveneven

low dose, to all tumor sites without eliciting marked bone marrow depletion and

leukopenia that would result in immunosuppression. Here we tested whether TRT

could be administered systemically to specifically deplete tumor infiltrating

suppressive immune cells (Tregs), without triggering systemic immune cell depletion and leukopenia. Dosimetry studies in this B78 melanoma tumor model were performed performedusing positron-emitting using 124I-NM404 positron-emitting confirm ¹²I-NM404 tumor-selective confirm uptake ofuptake of tumor-selective

NM404 (Figure 12A). C57BL/6 mice bearing B78 tumors were treated with 60 uCi µCi

¹³¹I-NM404. This 131-NM404. Thisactivity activityapproximates the amount approximates of ¹³¹I-NM404 the amount necessary of 131-N-404 to necessary to

deliver ~ 2 Gy TRT to a B78 tumor. Peripheral blood and tumor samples were

collected in untreated control mice (C) and at 8 day intervals (T1 = d8, T2 = d16, T3

= d24, T4 = d32) thereafter. This dose of TRT did not result in any significant

systemic leukopenia (Figure 12B) and did not significantly affect the level of tumor

infiltrating CD8+ effector T cells (Figure 12C). However, tumor infiltrating FoxP3+

Tregs were significantly depleted by this dose of TRT (Figure 12D).

[00246] Low dose TRT with 131-N-404 ¹³¹I-NM404effectively effectivelyovercomes overcomesconcomitant concomitant

immune immune tolerance toleranceandand rescues the systemic rescues anti-tumor the systemic effect of anti-tumor in situ effect of in situ

vaccination.

Given

[00247] Given

[00247] thecapacity the capacity of low low dose dose131 S-I-NM404 TRT ¹³¹I-NM404 TRT to todeplete depletetumor- tumor-

infiltrating Tregs without rendering a mouse leukopenic, we tested whether low dose

131-NM404 ¹³¹I-NM404might mighteffectively effectivelyovercome overcomeconcomitant concomitantimmune immunetolerance. tolerance.C57BL/6 C57BL/6

mice bearing mice bearingtwo B78B78 two tumors werewere tumors treated with 60-µCi treated 131 I-NM404 with 60-uCi on day on 31I-NM404 1 day 1

xRT to the primary tumor (in situ vaccine site). Control mice receiving no 131- ¹³¹I-

NM404 were treated to the secondary tumor as indicated (0, 2, or 12 Gy). Mice

received daily IT injections of IC to the primary tumor (in situ vaccine site), as

indicated, on days 13-17. Primary tumor (Figure 13A) and secondary tumor (Figure

13B) response demonstrates that administration of low dose TRT effectively

overcomes concomitant immune tolerance and rescues the systemic anti-tumor effect

of in situ vaccination.

References Cited in the Examples 1-4 and 7-10:

[1] Hank JA, Robinson RR, Surfus J, Mueller BM, Reisfeld RA, Cheung N--K and

Sondel PM. Augmentation of antibody dependent cell mediated cytotoxicity

following in vivo therapy with recombinant Interleukin-2. Cancer Res. 50:5234-9.

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[2] Neal ZC, Yang JC, Rakhmilevich AL, Buhtoiarov I, Lum HE, Imboden M, Hank

JA, Lode HN, Reisfeld RA, Gillies SD, Sondel PM. Enhanced activity of hul4.18-IL2 hu14.18-IL2

IC against the murine NXS2 neuroblastoma when combined with IL2 therapy. Clin

Cancer Res. 2004 Jul 15;10(14):4839-47.

[3] Yu AL, Gilman AL, Ozkaynak MF, London WB, Kreissman S, Chen H, Smith M,

Anderson B, Villablanca J, Matthay KK, Shimada H, Grupp SA, Seeger R, Reynolds

CP, Buxton A, Reisfeld RA, Gillies SD, Cohn SL, Maris JM, Sondel PM. Anti-GD2

antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J.

Med. 2010 Sep 30;363(14):1324-34. 30;363(14): 1324-34.

[4] Johnson EE, Yamane BH, Lum HD, Buhtoiarov IN, Rakhmilevich AL, Mahvi

DM, Gillies SD, Sondel, PM. Radiofrequency Ablation Combined with KS-IL2 IC

(EMD 273066) Results in an Enhanced Anti-tumor Effect Against Murine Colon

Adenocarcinoma. Clin Cancer Res. 2009 Aug 1;15(15):4875-84.

[5] Yang RK, Kalogriopoulos NA, Rakhmilevich AL, Ranheim EA, Seo S, Kim KM,

Alderson KL, Gan J, Reisfeld RA, Gillies SD, Hank JA, Sondel PM. Intratumoral

hul4.18-IL2 (IC) Induces Local and Systemic Antitumor Effects that Involve Both hu14.18-IL2

Activated T- and NK cells as well as Enhanced IC Retention. J Immunol. 2012 Sep

1;189(5):2656-64. 1;189(5):2656-64.

[6] Morris ZS, Emily I. Guy EI, Francis DM, Gressett MM, Carmichael LL, Yang

RK, Armstrong EA, Huang S, Navid F, Gillies SD, Korman A, Hank JA,

Rakhmilevich AL, Harari PM, Sondel PM. Combining Local Radiation and tumor-

specific antibody or IC to elicit in situ tumor vaccination. Cancer Research, e-pub

ahead of print, 2016.

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[7] Morris ZS, G.E., Francis DM, Gressett MM, Armstrong EA, Huan S, Gillies SD,

Korman AJ, Hank JA, Rakhmilevich AL, Harari PM, and Sondel PM., IC augments

local and abscopal response to radiation and CTLA-4 checkpoint inhibition in a

murine melanoma model. Am. Soc. Therapeutic Radiation Oncology. Abstract

accepted Oct. 2015 (and selected as the meeting's winning abstract in the basic---

translational science category).

[8] Weichert JP, Clark PA, Kandela IK, Vaccaro AM, Clarke W, Longino MA, Pinchuk

AN, Farhoud M, Swanson KI, Floberg JM, Grudzinski J, Titz B, Traynor AM, Chen

HE, Hall LT, Pazoles CJ, Pickhardt PJ, Kuo JS. Alkylphosphocholine Analogs for

Broad Spectrum Cancer Imaging and Therapy. Science Translational Medicine 6,

240ra75, 1-10. 2014.

[9] Morris ZS, JP Weichert, J Sakera, EA Armstrong, A Besemer, B Bednarz, R

Kimple, PM Harari. Therapeutic combination of radiolabeled NM404 with external

beam radiation in head and neck cancer model systems. Radiotherapy and Oncology. J.

Radiation Oncology, DOI: 10.1016. 2015.

[10] Lode HN, Xiang R, Dreier T, Varki NM, Gillies SD, Reisfeld RA. Natural killer

cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted

interleukin-2 therapy. Blood 91(5), 1706-1715. 1998.

[11] Snyder F, Wood R. Alkyl and alk-1-enyl ethers of glycerol in lipids from normal

and neoplastic human tissues. Cancer Res 29, 251-257. 1969.

[12] Pinchuk AN, Rampy MA, Longino MA, Skinner RW, Gross MD, Weichert JP,

Counsell RE, Synthesis and structure-activity relationship effects on the tumor avidity

of radioiodinated phospholipid ether analogues. J Med Chem 49, 2155- 2165. 2006. 2155-2165. 2006.

[13] Swanson KI, Clark PA, Pinchuk AN, Longino MA, Farhoud M, Weichert JP, Kuo

JS. Initial Studies on Novel Cancer-Selective Alkylphosphocholine Analogs CLR1501

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and CLR1502 for Fluorescence-guided Neurosurgery. Neurosurgery. 76(2): 115-123.

2015.

[14] Filatenkov A, Baker J, Mueller AM, Kenkel J, Ahn GO, Dutt S, Zhang N, Kohrt

H, Jensen K, Dejbakhsh-Jones S, Shizuru JA, Negrin RN, Engleman EG, Strober S.

Ablative Tumor Radiation Can Change the Tumor Immune Cell Microenvironment to

Induce Durable Complete Remissions. Clin Cancer Res. 21:3727-39. 2015.

[15] Jing W, Gershan JA, Weber J, Tlomak D, McOlash L, Sabatos-Peyton C, Johnson

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WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

Example 11:

In vivo uptake of multiple NM600 metal chelates in mice xenografted with eight

different solid tumor types, demonstrated by PET imaging

[00248] In In thisexample, this example, we we demonstrate demonstratethe differential the uptake differential of NM600 uptake chelated of NM600 chelated

with four different metals in a range of solid tumors in vivo, as demonstrated by

PET/CT imaging of such tumors. These data provide additional support for the use of

metal chelated alkylphosphocholine analogs as TRT agents for eliminating tumor-

induced immune tolerance, as disclosed herein. The structure of NM600 is shown in

Figure Figure 14, 14,asasanan example species example chelated species with 64 chelated Cu (64CU-NM600); with however, Cu (Cu-NM600); any any however,

metal can be readily chelated to NM600.

Specifically,

[00249] Specifically,

[00249] micemice werewere eacheach xenografted xenografted withwith one one of eight of eight different different solid solid

tumor cell lines (B78 (melanoma), U87MG (glioblastoma), 4T1 (breast carcinoma),

HCT-116 (colorectal carcinoma), A549 (lung carcinoma), PC-3 (prostate carcinoma),

HT-29 (colorectal adenocarcinoma), or MiaPaca (pancreatic carcinoma). For each of

the xenografted mice, cell suspension containing tumor cells was inoculated into

subcutaneous tissue of one or both flanks of the mouse. Once xenograft tumors

reached a minimum size, each mouse was injected with between 150-300 uCi µCi of

64 Zr, 89 64 NM600 radiolabeled with Cu, 89 or 52 Mn52via lateral tail vein injection. After 86 Y, 86 NM600 radiolabeled with Cu, Zr, Y, or Mn via lateral tail vein injection. After

an uptake period, PET imaging was performed in an Inveon micro PET/CT. Right

before each scan, mice were anesthetized with isoflurane (2%) and placed in a prone

position in the scanner. Longitudinal 40-80 million coincidence event static PET

scans were acquired at 3, 12, 24, and 48 hours post-injection of the radiotracer and the

images were reconstructed using an OSEM3D/MAP reconstruction algorithm.

[00250] Figure 15 shows the resulting images 48 hours post-injection-for single-

Y-NM600; Figure tumor B78 mice injected with 86Y-NM600; 1616 Figure shows the shows resulting the images resulting 4848 images

hours hours post-injection-for post-injection-fortwo-tumor B78 mice two-tumor B78 injected with 36Y-NM600; mice injected Figure 17 with Ý-NM600; Figure 17

shows the resulting images 3, 24 and 48 hours post-injection for a U87MG mouse

injected injectedwith with64Cu-NM600; Cu-NM600;Figure 18 18 Figure shows the the shows resulting imagesimages resulting 3, 24 and 3, 48 24 hours and 48 hours

post-injection post-injection forfor a 4T1 mouse a 4T1 injected mouse with 64 injected 4Cu-NM600; with Figure Cu-NM600; 19 shows Figure the 19 shows the

resulting images 3, 24 and 48 hours post-injection for an HCT-116 mouse injected

with 64Cu-NM600; with Cu-NM600; Figure Figure2020shows shows thethe resulting resulting images images 3, 24 3, and24 48 and 48post- hours hours post-

injection injectionfor foran an A549 mouse A549 injected mouse with 64 injected Cu-NM600; with Figure Cu-NM600; 21 shows Figure the 21 shows the

resulting images 3, 24 and 48 hours post-injection for a PC-3 mouse injected with wo 2019/094657 WO PCT/US2018/059927

64 Cu-NM600;Figure Cu-NM600; Figure 22 22 shows shows the theresulting resultingimages 3, 24 images 3,and 24 48 hours and post-injection 48 hours post-injection

for for aa HT-29 HT-29mouse injected mouse withwith injected 64 4Cu-NM600; Cu-NM600;Figure 23 shows Figure the resulting 23 shows images images the resulting

3, 3, 24 24 and and4848hours post-injection hours for afor post-injection MiaPaca mouse injected a MiaPaca with 64 Cu-NM600; mouse injected with Cu-NM600;

Figure 24 shows the resulting images 3, 24 and 48 hours post-injection for a 4T1

mouse mouse injected injectedwith 86Y-NM600; with Y-NM600;Figure 25 shows Figure the resulting 25 shows images images the resulting 3, 24 and 3, 48 24 and 48

hours hours post-injection post-injectionforfor a 4T1 mousemouse a 4T1 injected with 89Zr-NM600. injected with Zr-NM600.

²Mn-NM600, PET

[00251] For HT-29 and PC3 mice injected with 52Mn-NM600, PETimages imageswere were

obtained at 4 hours, and one day post-injection (Figure 26 for HT-29; Figure 27 for

PC3), as well as on days 2, 3, 5 and 7 post-injection (Figure 28 for HT-29; Figure 29

for PC-3).

[00252] As As seenininFigures seen Figures 15-29, 15-29, the thescanned scannedmice produced mice PET/CT produced three-three- PET/CT

dimensional volume renderings showing cumulative absorbed dose distribution

concentrated in the xenografted tumor. The results confirm the differential uptake of

metal chelated NM600 into the xenografted solid tumor tissue, and demonstrate the

potential use of NM600 analogs incorporating radioactive metal isotopes in the

disclosed treatment methods.

Quantitative

[00253] Quantitative

[00253] region-of-interest region-of-interest analysis analysis of images of the the images was performed was performed by by

manually contouring the tumor and other organs of interest. Quantitative data was

expressed as percent injected doe per gram of tissue (%ID/g). Exemplary data show

that 4T1 tumor tissue increased its uptake over time and effectively retained all three

tested tested NM600 NM600chelates (86Y-NM600, chelates (Y-NM600,64Cu-NM600 Cu-NM600and and89Zr-NM600, Zr-NM600, see seeFigure Figure30), 30),

while healthy heart (Figure 31), liver (Figure 32) and whole body tissue (Figure 33)

all exhibited significantly decreased uptake/retention over time.

Ex vivo

[00254] Ex vivo

[00254] biodistribution biodistribution analysis analysis was was performed performed after after the the lastlast longitudinal longitudinal

PET scan. Mice were euthanized and tissues harvested, wet-weighed, and counted in

an automatic gamma counter (Wizard 2480, Perkin Elmer). Exemplary

biodistribution data show significant uptake and retention in tumor tissue (4T1) for

different differentNM-600 NM-600chelates (86Y-NM600, chelates (Y-NM600,64Cu-NM600, Cu-NM600,89Zr-NM600 Zr-NM600 and and177Lu- ¹Lu-

NM600, see Figure 34),

Together,

[00255] Together,

[00255] these these results results demonstrate demonstrate thatthat the the disclosed disclosed metal metal chelates chelates can can

readily be used for the TRT step of the disclosed treatment methods.

WO wo 2019/094657 PCT/US2018/059927

Example 12:

Demonstrating anti-tumor activity and tumor autoradiography with two

different NM600 metal chelates against multiple solid tumor types in xenografted

mice In this

[00256] In this

[00256] example, example, using using three three different different solid solid tumor tumor models, models, we show we show thatthat

alkylphosphocholine metal chelate analogs can be effectively used to facilitate

conventional TRT. These results further demonstrate the potential for using the metal

chelates in the TRT step of the presently disclosed treatment methods.

[00257] B78, MiaPaca and 4T1 subcutaneous flank xenografts were induced in mice,

as described previously. Subsequently, the mice were administered therapeutic doses

(250-500 µCi) uCi) of Y-NM600, 177Lu-NM600, 90Y-NM600, ¹Lu-NM600, or ora acontrol controlsolution solutionvia vialateral lateraltail tailvein vein

injection.

[00258] Planar 2D phosphor images of the biodistribution of the agent were taken

using a Cyclone Phosphorimager (Perkin Elmer). Mice were anesthetized and place in

direct contact with the phosphor plate in a supine position, where they remained for a

period of 15-30 min; plates were then read in the phosphorimager. Various images were

recorded between 4 and 96 h post-injection of the radioactive dose. The resulting

autoradiography images demonstrate rapid and selective uptake and long term retention

of the chelates in all of the solid tumor tissues types tested (see Figures 40, 41, 42, 43,

44 and 45).

[00259] Tumor response was assessed by comparing tumor growth of the treated VS.

control mice. Tumor volume was determined by measuring tumor's length and width

with calipers and calculating the volume using the formula for the volume of the

ellipsoid. Mice weight was also recorded. Humane endpoints were defined as: tumor

volume >2500 m³ or significant weight lost below 13 g.

[00260] As seen in Figures 46, 47, 48, 49, 50 and 51, the results demonstrate that

the two tested NM600 chelates had a statistically significant in vivo therapeutic effect

when compared with the control, resulting in decreased mean tumor volumes for

double double doses dosesofof 177Lu-NM600 ¹Lu-NM600inin 4T14T1 xenografts (see (see xenografts Fig. 50), Fig. and reducing 50), growth togrowth to and reducing

near zero or slowing the growth rate of MiaPaca, 4T1 or B78 xenografts given a

single single dose doseofof 177Lu-NM600 ¹Lu-NM600(see Figures (see 47, 47, Figures 48, and 48, 49) andor49) B78or or B78 4T1 or xenografts 4T1 xenografts

given given aasingle singledose of of dose 90Y-NM600 Y-NM600(see Figures (see 46 and Figures 46 51). and 51).

WO wo 2019/094657 PCT/US2018/059927

[00261] These results further demonstrate the efficacy of using the disclosed

alkylphosphocholine metal chelates to deliver TRT to effectively treat solid tumors of

various types.

Example 13:

Coupling radiation dosimetry and radiosensitivity index to predict TRT response

in a wide range of solid tumor types

In this

[00262] In this

[00262] example, example, we discuss we discuss factors factors for for determining determining chelate chelate dosages dosages

appropriate for the TRT step of the disclosed methods in a range of solid tumor types.

[00263] Estimation of tumor absorbed doses

Whether

[00264] Whether

[00264] thethe amount amount of of 177Lu/90Y-NM600 ¹Lu/Y-NM600 that isthat is administered administered is immuno- is immuno-

stimulatory or cytotoxic depends on the tumor absorbed dose. The diapeutic property

of of NM600, NM600,that that64CU/66Y-NM600 can be Cu/Y-NM600 can be used usedasasanan imaging surrogate imaging for therapeutic surrogate for therapeutic

metals metals177Lu 90Y-NM600,respectively, ¹Lu/Y-NM600, respectively, was wasleveraged to to leveraged estimate tumortumor estimate dosimetry. dosimetry.

Ultimately, 64CU86Y-NM600 PET/CT Cu/Y-NM600 PET/CT waswas used used to to quantitatively quantitatively measure measure in in vivo vivo

biodistribution and estimate radiation dosimetry which can help identify dose limiting

organs and potential tumor efficacy of ¹Lu/Y-NM600 TRT. organs and potential tumor efficacy of TRT.

[00265]

[00265]The Thegeneral concept general is as concept isfollows: (1) the(1) as follows: concentration of 64Cu/86Y. the concentration of Cu/Y-

NM600 within the tumor is quantified over time using longitudinal PET/CT imaging,

(2) (2) the theconcentration concentrationof 64CU/66Y-NM600 of Cu/Y-NM600isisdecay corrected decay to account corrected for the to account for the

difference in in difference decay ratesrates decay betweenbetween the Cu/Y-NM600 and ¹Lu/Y-NM600, (3) the 64CU/66Y-NM600 and the (3) the concentration ofof concentration 177LU/90Y-NM600 ¹Lu/Y-NM600 within withinthe thetumor is is tumor time-integrated to compute time-integrated the to compute the

cumulative activity, or total number of decays, (4) the deposition of the radionuclide

decays is modeled within the tumor and quantified.

[00266] Steps (1) through (3) can be performed with any medical image processing

software package whereas step (4) requires sophisticated radiation dosimetry

software. OLINDA/EXM (Stabin, Sparks and Crowe 2005) is a dosimetry estimation

software with 510(k) approval that uses the formalism developed by the Medical

Internal Radiation Dose (MIRD) committee of the Society for Nuclear Medicine

(Bolch et al., 2009). The MIRD approach estimates the mean absorbed dose received

by a tissue or organ due to the radiation emitted from within the organ itself or from

another source organ. The simplest form of the MIRD equation,

D(t - s) = A,S(t - s), =

WO wo 2019/094657 PCT/US2018/059927

gives the absorbed dose, D [mGy], to a target region t from the radionuclide activity

within a source region S. The radionuclide activity of S is expressed as a cumulated

activity Às which is Å which is the the total total number number of of radionuclide radionuclide decays decays given given in in units units of of MBq-s. MBq-s.

The S-factor, S(t - s) s) [mGy/MBq-s],

[mGy/MBq-s],is is the fraction of the energy released by one

radionuclide decay within the source region S which is deposited within the target

region t normalized by the mass of the target region t, Mt. The S-factor m. The S-factor is is aa tabulated tabulated

value calculated using Monte Carlo in a set of standard phantoms and organs.

Typically, we are concerned with the dose per unit of injected activity, D

[mGy/MBq]. The

[mGy/MBq]. Theequation is written equation in terms is written of the of in terms residence time, 'n,[MBq- the residence time, [MBq-

s/MBqinj],

Th = Ainj'

which is the ratio of the cumulative activity and the injection activity, Ainj [MBq], A [MBq], as as

=

[00267] In In thethe case case of of calculating calculating tumor tumor dosimetry, dosimetry, OLINDA/EXM OLINDA/EXM models models thethe

tumor as an isolated unit density sphere whose volume was estimated from the tumor

region of interest (ROI) created as part of step (1). The concentration of NM600

(%ID/g) within the tumor was determined at each time point and decay corrected.

Cumulative activity was then calculated by integrating the concentrations over all

time using trapezoidal piecewise integration.

[00268] Radiation dosimetry results for many cell lines are shown in Table 1. This

information can be used to estimate the absorbed dose for radiotherapy studies aimed

to either eradicate tumors or stimulate the immune system.

PCT/US2018/059927

Table Table 1: 1:Dosimetry Dosimetryestimates for for estimates both both 177Lu-NM600 and 90Y-NM600 ¹Lu-NM600 (Gy/MBqinj) and Y-NM600 (Gy/MBqinj)

Y-NM600 PET using either Cu-NM600 or 86Y-NM600 imaging PET asas imaging a a surrogate surrogate

PC3 A549 HT-29 MiaPaca U87MG 4T1 B78 Lu-177 0.39 0.30 0.30 0.49 0.49 0.24 0.24 0.58 1.50 0.92 0.92

Y-90 Y-90 0.69 0.69 0.53 0.53 0.84 0.84 0.45 0.45 1.01 4.68 2.86 2.86

[00269] Radiosensitivity Index to Predict Dose-response

Intrinsic

[00270] Intrinsic

[00270] radiosensitivity radiosensitivity is ais a crucial crucial factor factor underlying underlying radiotherapy radiotherapy

response; and, knowing it a priori for a cancer type could help predict how it may

respond to radiation from TRT. However, since there is no method for its routine

assessment in tumors, radiosensitivity is measured as the surviving fraction (between

0 and 1) following irradiation with 2 Gy (SF2) by clonogenic assay. The relative

radiosensitivity of cancer cell phenotypes ranges from those that have very low

radiosensitivities (pancreas, colorectal, glioma and breast) to those with high

radiosensitivities (lymphomas). Cancers can be categorized or ranked by their

radiosensitivity indices (Table 2).

[00271] If If we we can candemonstrate demonstrategoodgood tumor uptake tumor and growth uptake inhibition and growth with APC with APC inhibition

metal chelates in a highly radiosensitive tumor like lymphoma and in a highly

radiation resistant tumor like glioma, breast, pancreatic or colorectal, then it can be

implied that these agents would be effective against any tumor with an SF2 value SF value

between that of lymphoma and glioma (0.3-0.82) if they are able to target the tumor in

vivo. It would also be expected then that the radiation dose needed to eradicate glioma

tumor cells would be higher than that needed to treat the more radiosensitive

lymphoma cells.

[00272] We We currently have currently have in in vivo vivo imaging imagingto to confirm tumor confirm selectivity tumor and selectivity and

therapy response (tumor growth inhibition) data in all the tumor cell lines listed in

Table 2. In some cases, it may be necessary to give multiple doses of the APC

chelates to elicit sufficient cancer cell kill. By using quantitative imaging coupled

with radiation dosimetry calculation, we can estimate the tumor absorbed dose

necessary to either kill the cancer cells (higher doses) or stimulate the immune

system, as disclosed herein (lower doses).

[00273] Coupling

[00273] dosimetry Coupling estimates dosimetry for a estimates variety for of cancer a variety cellcell of cancer lines (Table lines 1) 1) (Table

with their respective radiosensitivity indices (Table 2) supports the establishment of a

dose response landscape for NM600. By knowing the tumor targeting characteristics

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

and efficacy of NM600 within a series of cell lines, it is possible to estimate the

absorbed tumor dose and potential efficacy of cell lines with similar radiosensitivity

indices. Furthermore, treatment doses can be linearly scaled according to Table 1,

depending on the desired outcome of tumor eradication or immuno-stimulation (as

disclosed herein).

Table 2: Relative Radiosensitivity of Cancer Cells

Imaging uptake Refs.

and or growth Tumor Type Cell Line SF2 value inhibition with

APC chelates Breast MDA-MB- 0.82 Yes Yes 8

231

Pancreatic Mia-Paca 0.80 Yes Yes 6,7 Colorectal HCT-29 0.75 0.75 Yes Yes 7

Melanoma B-78 0.65 0.65 Yes 3, 4, 7 Yes Glioma (brain) U-87 0.63 Yes 1, 2, 7 Yes A-549 0.61 Yes 5, 7 Lung Yes 5,7 (NSCLC) Prostate PC-3 0.55 Yes 4 EL-4 0.30 Yes 3, 3,77 Lymphoma Yes

SF2=surviving fractionfollowing SF=surviving fraction followingexposure exposureto to22Gy Gyof ofin invitro vitroradiation radiationexposure exposure

* Several cell lines

1 'Taghian, "Taghian, Alphonse, et al. "In vivo radiation sensitivity of glioblastoma multiforme."

International Journal of Radiation Oncology* Biology* Physics 32.1 (1995): 99-104.

2Ramsay, 2Ramsay, J., J., R. R. Ward, Ward, and and N. M. Bleehen. N.M. Bleehen. "Radiosensitivity "Radiosensitivity testing testing of of human human malignant malignant

gliomas." International Journal of Radiation Oncology* Biology* Physics 24.4 (1992): 675-

680.

3 Fertil, Fertil, B., B., and and E. P. E.P. Malaise. Malaise. "Intrinsic "Intrinsic radiosensitivity radiosensitivity of of human human cell cell lines lines is is correlated correlated with with

radioresponsiveness of human radioresponsiveness tumors: of human analysis tumors: of 101 published analysis survival curves." of 101 published survival19 curves."

International Journal of Radiation Oncology* Biology* Physics 11.9 (1985): 1699-1707.

4Wollin,Michael, Wollin, Michael,et etal. al."Radio "Radiosensitivity sensitivityof ofhuman humanprostate prostatecancer cancerand andmalignant malignantmelanoma melanoma

cell lines." Radiotherapy and Oncology 15.3 (1989): 285-293.

Kodym, Elisabeth, et al. "The small-molecule CDK inhibitor, SNS-032, enhances cellular

radiosensitivity in quiescent and hypoxic non-small cell lung cancer cells." Lung Cancer 66.1

(2009): 37-47.

Unkel, Steffen, Claus Belka, and Kirsten Lauber. "On the analysis of clonogenic survival

data: Statistical alternatives to the linear-quadratic model." Radiation Oncology 11.1 (2016):

11.

EP Malaise, Patrick J. Deschavanne, and Bernard Fertil. "Intrinsic radiosensitivity of human

cells." Advances in radiation biology 15 (2016): 37-70.

Sules, Siles, E., et al. "Relationship between p53 status and radiosensitivity in human tumour cell

lines." British journal of cancer 73.5 (1996): 581-588.

References

[00274] References cited cited in in Example Example 13:13:

Bolch,W.E.,

[00275] Bolch, W. E., K.K.F.F.Eckerman, Eckerman, G. G. Sgouros, Sgouros,and S. S. and R. Thomas. 2009.2009. R. Thomas.

"MIRD Pamphlet No. 21: A Generalized Schema for Radiopharmaceutical

Dosimetry--Standardization of Nomenclature." Journal of Nuclear Medicine 50 (3):

477-84. doi: 477-84. :10.2967/jnumed.108.056036. doi:10.2967/jnumed.108.056036

[00276] Stabin, MM G,

[00276] Stabin, G, RRBB Sparks, Sparks, and and EECrowe. Crowe.2005. "OLINDA/EXM: 2005. The The "OLINDA/EXM:

Second-Generation Personal Computer Software for Internal Dose Assessment in

Nuclear Medicine." J Nucl Med 46 (6): 1023-27.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

Example 14:

Advantages of and differences when using alkylphosphocholine metal chelates in

place of radioiodinated compounds, such as those exemplified in Examples 1-4

and 7-10

[00277] In In thisexample, this example, we we discuss discussthe theadvantages of using advantages APC metal of using chelates APC metal chelates

instead of radioiodinated compounds (the compounds exemplified in Examples 1-4

and 7-10). We also discuss factors to be considered by the skilled artisan when

optimizing dosages of metal chelates to be used in the TRT step of the disclosed

methods.

[00278] Chelates permit the use of a wide variety of stable or radioactive metal ions

for imaging and therapy. They can be conjugated with a wide variety of alpha, beta,

Auger, gamma and positron emitters whereas iodine is limited to one positron (I-124),

one beta (I-131), one gamma (I-123) and 1 Auger (I-125) isotope.

[00279] Metal Isotopes are diapeutically more efficacious than I-131 and I-124.

[00280] Lu-177 has fewer high energy gammas which make it more favorable for

SPECT imaging and dosimetry. However, its beta energy is slightly less than I-131,

making it more ideal for treating smaller tumors.

[00281] I-131 and Lu-177 are comparable in therapeutic efficacy "horse power", but

there is significantly less contribution to the overall dose from gamma-emissions for

Lu-177. In the case of Y-90, there is negligible contribution to the radiation dose from

gamma-emissions.

[00282] Relative to I-131, Y-90 is more efficacious for killing cancer cells by

conventional TRT than I-131, as seen in Figure 52 and discussed further below.

[00283] The Committee on Medical Internal Radiation Dose (MIRD) develops

standard methods, models, assumptions, and mathematical schema for assessing

internal radiation doses from administered radiopharmaceuticals. The MIRD approach,

which simplifies the problem of assessing radiation dose for many different

radionuclides, has been implemented in the widely used 510(k) approved software,

OLINDA/EXM1. Along with its many standard anthropomorphic phantoms,

OLINDA/EXM has a Spheres Model which can be used to approximate tumor doses.

The Spheres Model assumes homogeneous distribution of a radiopharmaceutical within

unit-density spheres of a range of tumor masses (0.01 - 6,000 g).

[00284] Using this standard model, we compared Y-90 to I-131 in terms of radiation

dose normalized by administered radioactivity. The results of this comparison, for

81

WO wo 2019/094657 PCT/US2018/059927

tumor masses between 1 to 100 g, are displayed in Figure 52. Note that the Y-90-to-I-

131 ratio reaches 4 for a 4 g tumor, and remains between 4.0 and 4.2 up to a 100 g

tumor, strongly suggesting that on a mCi per mCi basis that Y-90 is between 3.6 and

4.1 times as cytotoxic as I-131 in tumors up to 10g in size, and about 4.1 times more

effective in tumors greater than 10 grams in size.

Different

[00285] Different Pharmacokinetic Pharmacokinetic Properties Properties

Unlike

[00286] Unlike iodinated iodinated analogs, analogs, APCAPC chelates chelates areare tootoo large large to to fitfit into into known known

albumin binding pockets in the plasma and therefore exhibit different in vivo

pharmacokinetic and biodistribution profiles (see Figure 53). Lower binding energies

lead to larger fractions of free molecule in the plasma which affords more rapid tumor

uptake. Some APC chelates are cleared via the renal system, whereas iodinated analogs

are eliminated through the hepatobiliary system. APC chelates also accumulate in

tumors and clear from the blood much quicker than iodinated analog. Faster blood

clearance is directly associated with lower bone marrow and off-target toxicity of

therapeutic radiopharmaceuticals.

[00287] These differences in PK and biodistribution profiles lead to differing dose

limiting organ toxicity and ultimate utility. Moving from hematological toxicity to renal

or liver for dose limiting toxicity would increase the utility of radiometal chelates for

TRT.

[00288] Moreover, the pharmacokinetic profile of the APC chelates can easily be

manipulated by minor changes in the structure of the chelate (e.g. chelate charge). The

choice of chelators is vast. Faster clearance from normal tissues improves imaging

contrast and therapeutic windows, resulting in higher maximum tolerable doses.

[00289] APC chelates possess different physico-chemical characteristics than

iodinated analogs. They are much more water-soluble, and therefore do not need

surfactants to render them suitable for intravenous injection. APC chelates are based on

ionic binding of the metal to the chelate, whereas iodinated compounds form covalent

bonds with their carrier molecules. In vivo de-iodination is quite common in alkyl

iodides whereas chelates tend to be extremely stable in vivo.

Once

[00290] Once de-iodination occurs, de-iodination occurs, free freeiodide rapidly iodide accumulates rapidly in thein accumulates thyroid the thyroid

with a very long subsequent excretion half-life, whereas free radiometals are in general

excreted from the body or detoxified much more quickly.

[00291] In vivo biodistribution of APC chelates can be quite different depending on

the metal ion SO so the metal and chelate also both contribute to the tumor targeting

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

characteristics of the APC. Not all chelates target tumors. Tumor targeting depends on

the cumulative properties of the APC carrier, the type of chelate (linear chelates

undergo rapid renal elimination whereas macrocyclic chelates undergo hepatobiliary

excretion), and the metal ion. Even slight changes in chelate structure result in

significant variations on the in vivo properties. Simple changes in isotope can result in

changes in tumor targeting larger than 50%.

[00292] Radioactive APC-metal chelates are easily radiolabeled in nearly quantitative (>98%) yields under facile conditions, whereas radioiodination yields of

iodinated analogs are much lower (typically about 50% for I-131 and 60% for I-124).

Moreover, high specific activities can be achieved with chelates. Synthesis can be done

using a radiolabeling kit in any nuclear pharmacy without the requirement of

sophisticated ventilation equipment or training. Radioiodination must be done in a fume

hood fitted with effluent monitoring equipment due to the volatility of radioactive

iodine during the labeling reaction.

Imaging

[00293] Imaging agents agents don't don't necessarily necessarily make make good good therapy therapy agents agents andand vice vice

versa.

[00294] It cannot be assumed that because there is good tumor uptake with an

imaging agent that it implies that therapy is obvious. In addition to having good tumor

uptake, a therapy agent needs to have prolonged tumor retention relative to normal

tissues and must be cleared from the blood quickly in order to lower bone marrow

exposure and associated toxicity. Iodinated analogs have prolonged blood residence

resulting in dose limiting bone marrow toxicity. In contrast, our APC chelates exhibit

much faster blood clearance kinetics most likely, as stated above, due to lower albumin

binding in the plasma.

[00295] Finally, due to the short path length and physical nature of metallic beta-

and alpha-emitters relative to Iodine-131, there are no exposure concerns for health care

workers or family members following injection. Patients undergoing I-131 therapy

often have to be held for some time (up to a week) in a lead shielded room prior to being

released from the hospital. Patients injected with radioactive alpha and beta-emitting

APC chelates will not be required to remain hospitalized, hospitalized.

wo 2019/094657 WO PCT/US2018/059927 PCT/US2018/059927

Example 15: TRT delivered by Y90-NM600 in combination with administering an anti-CLA4

immune checkpoint inhibitor synergistically inhibits cancer in an in vivo

melanoma model In this

[00296] In this

[00296] example, example, we demonstrate we demonstrate the the efficacy efficacy of the of the disclosed disclosed combination combination

method, where the in vivo immunization is performed by systemically administering

an immune checkpoint inhibitor (an anti-CTLA4 antibody), and the TRT is performed

by by systemically systemically administering the 90Y-NM600 administering chelate the Y-NM600 used in chelate previous used examples. in previous examples.

[00297] B78B78 melanoma melanoma subcutaneous subcutaneous flank flank xenografts xenografts were were implanted implanted in in male male

C57BL/6 mice, as described previously. Subsequently, the mice were randomized to

be be to to be betreated treatedwith varying with dosesdoses varying (25 uCi, (25 50 uCi,50orµCi, µCi, 100 uCi) of 90 or 100 Y-NM600 µCi) (Day of Y-NM600 (Day

1), both with and without anti-CTLA4 antibody (an immune-checkpoint inhibitor)

(200 ug µg on Days 4, 7, and 11) (n=6 for each experimental group). Both agents were

administered by via lateral tail vein injection (i.e., intravenously). Control groups of

PBS treatment alone and anti-CTLA4 alone were also included. Tumors were

measured with calipers twice a week, and animal survival was monitored for 60 days.

[00298] As As shownininFigure shown Figure 54, 54, the the three threecombination therapies combination (anti-CTLA4 therapies + (anti-CTLA4 +

Y-NM600 atat 90Y-NM600 three different three dosages) different showed dosages) substantial showed tumor substantial growth tumor inhibition, growth asas inhibition,

compared to compared toany anyofof thethe single therapies single (anti-CTLA4 therapies or Y-NM600 (anti-CTLA4 alone at or alone at three three

different dosages) or the PBS control. After Day 18, combination treatment with 50

or 100 uCi µCi of 90Y-NM600 with Y-NM600 with anti-CTLA4 anti-CTLA4 had had significantly significantly (p(p < < 0.05 0.05 byby ANOVA) ANOVA)

reduced tumor reduced tumorgrowth compared growth to PBS, compared Y-NM600 to PBS, alone, alone, or or anti-CTLA4 alone. anti-CTLA4 alone.

uCi Y-NM600 The 25 µCi 90Y-NM600 combination combination treatment treatment group group with with anti-CTLA-4 anti-CTLA-4 had had anan

intermediate growth delay response that showed a trend towards dose response.

[00299] As As

[00299] seen in in seen Figure 55,55, Figure mice treated mice with treated 50 50 with µCiuCi of of Y-NM600 combined 90Y-NM600 combined

with anti-CTLA4 exhibited significantly greater aggregate survival than mice treated

with TRT alone or PBS vehicle (p < 0.05). <0.05).The Thelog logrank rankwas waspp==0.06 0.06for forthe the

combination treatmnent, as compared to anti-CTLA4 alone.

As seen

[00300] As seen

[00300] in Figure in Figure 56, 56, all all three three combination combination treatments treatments significantly significantly

improved improved survival. survival. Significantly, Significantly, there there were were 6/12 6/12 (50%) (50%) complete complete responders responders in in the the

µCi doses of 90 combination TRT + CTLA4 arms at therapeutic 50 and 100 uCi Y-NM600, YON-NM600,

as compared to 0/24 complete responders in the non-combination control arms (PBS,

TRT alone at 50 uCi, µCi, TRT alone at 100 uCi, µCi, and anti-CTLA4 alone).

PCT/US2018/059927

[00301]

[00301]These results These illustrate results the the illustrate therapeutic potential therapeutic of combining potential the the of combining use use of aof a

molecular targeted radiotherapeutic agent with any agent that causes immune

checkpoint inhibition (ICI). The results show that a combination of molecularly

targeted TRT and an ICI affords a synergistic effect, relative to treatment with each

agent alone. In addition to demonstrating significant tumor regression, the combined

method also has the potential to generate immunologic memory and ultimately afford

a potent in situ cancer vaccine effect that prevents tumor recurrence.

Example 16: Utilization of Molecular Targeted Radiotherapy to Enhance the Efficacy of

Systemic Checkpoint Inhibition in Metastatic Cancer Models

[00302] In this follow-up to Example 15, we provide greatly expanded supporting

data demonstrating the efficacy of the disclosed method combining systemically

administering an immune checkpoint inhibitor and TRT performed by systemically

administering administeringthethe 90Y-NM600 Y-NM600chelate used chelate in previous used examples. in previous Efficacy examples. is Efficacy is

demonstrated in mouse melanoma, neuroblastoma and breast cancer models, as well

as in multiple tumor melanoma models having disseminated "cold" tumors.

[00303] Clinical studies demonstrate that a subset of patients treated with immune

checkpoint inhibitors (ICIs) experience durable and complete response (CR) at all

disease sites. However, ICIs are not typically effective in patients with

immunologically "cold" tumors characterized by low levels of T cell infiltrate and/or

few mutation-created neo-antigens. In this example, we demonstrate using the

disclosed combination method to stimulate an immune response in such tumors and to

enhance response in "hot" tumors. More specifically, we enhanced the efficacy of

systemic ICI by combining it with systemic molecular targeted radiotherapy (MTRT),

which can deliver immunostimulatory low dose radiation to all sites of disease

without causing resultant systemic lymphodepletion that would be counterproductive

in generating an anti-tumor immunotherapy response.

[00304] Methods:

[00305] ForFor tumoruptake tumor uptake studies studies of ofMTRT, MTRT,flank tumors flank (n =(n tumors 3 for = 3 each for of B78 of B78 each

melanoma melanomaand andPanc02) were Panc02) established were by injection established of 1-2x106 by injection cells incells of 1-2x10 100 uLin PBS 100 µL PBS

into C57BL/6 mice on an approved IACUC protocol. Both B78 and Panc02 tumors

are poor to moderately immunogenic, slow growing, radio-resistant tumor lines and

this profile makes them useful for studies of MTRT where slow growth permits time for MTRT decay and radio-resistance plus poor immunogenicity enables testing for cooperative improvements in efficacy with combined MTRT + ICI.

After

[00306] After

[00306] tumors tumors werewere wellwell established, established, approximately approximately 5 weeks 5 weeks after after injection, injection,

animals were treated with a dose of IV 6-Y-NM600 and Y-NM600 and serial serial PET/CT PET/CT images images were were

collected at 1,2, and 3 days after MTRT injection. PET uptake values were compared

to areas of background activity including the heart and liver. A paired t-test was

performed to test for significant differences in 86Y-NM600 uptake Y-NM600 uptake between between

background organs and tumor sites.

Y-NM600 and/or

[00307] To demonstrate the ability of 90Y-NM600 ICI and/or toto ICI decrease decrease

immunosuppressive Treg cell populations within B78 Melanoma flank tumors, we

generated flank tumor models (n = 4 for each group) of B78 melanoma. MTRT (50

uCi), µCi), anti-CTLA4 (200 ug µg Days 4,7,10), MTRT and CTLA4, and PBS placebo

control were our treatment groups. The effect of treatment on tumor immune cell

populations was examined at day 1, 7, and 14 after radiation or saline placebo

delivery by harvesting tumor tissue and freezing one portion for histology and saving

another portion for quantitative PCR. The rest of the tumor sample was prepped for

mRNA and RT-PCR analysis. Quantitative RT-PCR was used to evaluate changes in

tumor cell expression of immune susceptibility markers (e.g. Fas, MHC-I, and PD-

L1).

[00308] ForFor efficacystudies, efficacy studies, 22 bilateral bilateralflank tumors flank models tumors of B78 models ofmelanoma B78 melanoma

were generated in C57BL/6 mice. Once tumors grew to 80-120 mm³, they were

randomized into the following treatment groups: anti-CTLA-4 alone at 200 ug µg IP on

days 4, 7, 10, 90Y-NM600 Y-NM600 IVIV (50 (50 uCi) µCi) onon day day 1 1 and and anti-CTLA4, anti-CTLA4, 1212 GyGy whole whole body body

radiation (EBRT) and anti-CTLA4, 12 Gy EBRT + 50 uCi µCi 90Y-NM600, 12 Gy

EBRT + 50 uCi µCi 90Y-NM600 and anti-CTLA4. Tumor measurements were made

twice a week for 30 days, and survival was tracked to 60 days with a euthanasia

endpoint of 15 mm diameter for tumor burden.

[00309] Mice with complete response to therapy were re-challenged with 2x106 2x10

B78 or 1x106 Panc02cells 1x10 Panc02 cellsto tothe theopposite oppositeflank flank90 90days daysafter afterMTRT MTRTand andthen thenagain againat at

120 days with Panc02 (only for B78) and B16 melanoma to test for tumor specific

immune memory response.

[00310] Results:

[00311] TheThe

[00311] selective uptake selective of of uptake ourour MTRT agent, MTRT Y-NM600, agent, was confirmed 90Y-NM600, in in was confirmed

both B78 and Panc02 tumor models. For B78 melanoma, tumor uptake of 90Y-

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

NM600 demonstrated that after initial injection the majority of the agent was in the

blood pool as expected, however by 48 hours after injection the majority of the agent

was retained in the tumor or organs of elimination (liver, kidney). Gamma counts of

tissue sections taken at Day 48 confirms PET imaging uptake values with higher

radioactivity counts in tumor tissue that increase over time with lower values in

marrow spaces that decrease over time. Monte Carlo dosimetry performed as a

collaborative effort shows that approximately 2-3 Gy are delivered over the life of the

MTRT agent when our experimental dose of 50 uCi µCi is delivered. PET uptake and

tissue biodistribution studies in Panc02 pancreatic cancer also demonstrated increased

uptake uptake and andretention of 90Y-NM600 retention of Y-NM600in in tumor tissue tumor compared tissue to marrow compared tissue at to marrow 72 tissue at 72

hours.

[00312] To To studythe study the effect effect of of treatment treatmentonon tumor immune tumor cell cell immune populations, tumor tumor populations,

tissue samples were collected at various time points after radiation. At Day 14 after

µCi 90Y-NM600), MTRT treatment (50 uCi Y-NM600), wewe found that found combination that MTRT combination and MTRT anti- and anti-

CTLA4 significantly increases effector/suppressive immune t-cell ratios as

determined by the CD4/FoxP3 and CD8/FoxP3 infiltrates in tumor tissue.

Quantitative PCR (qPCR) studies of gene expression also showed increased

inflammatory gene expression including genes that are part of the stimulator of

interferon gene pathway (STING). Levels of Mx1, IFNa, IFNB, IFN, IFN, and and PDL1, PDL1, which which are are

all downstream of STING activation, were upregulated compared to PBS control.

[00313] We next established a single B78 R flank tumor in mice and once they

reached approximately 80 mm³, randomized them into 25, 50, and 100 uCi MTRT

dose treatment groups given on Day 1 with and without anti CTLA4 given on days

4,7,and 10, as well as PBS and anti CTLA4 alone as controls. We found that

combination MTRT at 50 and 100 uCi dose levels and anti-CTLA4 demonstrated

significantly improved tumor growth delay (Figure 57) and survival (Figure 58)

compared to other groups. At 25 uCi of MTRT there was an intermediate response.

Additionally, the only mice that had a complete response to therapy were in the

combination therapy groups with 66%, 33, and 16% of the animals in the 50 uCi, 100,

and 25 uCi MTRT dose groups. All mice that had a complete response at Day 60

after MTRT injection were challenged with B78 cells on the contralateral flank and

there was a 100% rejection rate compared to naive naïve controls, demonstrating that our

treatment was able to generate an immune memory response.

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

[00314] This study has since been replicated showing similar trends and survival

across both studies showed a significantly improved overall survival in mice treated

with combination MTRT (50, 100 uCi) and anti-CTLA4 compared to other groups by

log rank test.

[00315] We next extended this study to similar mouse models of neuroblastoma

(NXS2) and breast cancer (4T1). As seen in Figure 59 (NXS2) and in Figure 60

(4T1), the CTLA4 MTRT combination again was the only group demonstrating

significantly reduced tumor growth (in fact, tumor volume reduction).

[00316] Next, we extended the study to demonstrate improved response rates in

mice with multiple bulky tumors. We designed a study MTRT treatment in a two-

tumor mouse model, the goal being to treat mice with multiple bulky tumors, which

would correspond to a patient with bulky metastatic disease at multiple sites. For this

experiment, we examined if MTRT can improve response rates over the current

clinical paradigm of delivering EBRT to one site in combination with immune

checkpoint blockade.

[00317] Two-tumor models of Panc02 and B78 melanoma were established. First in

B78 melanoma, traditional immunosensitizing EBRT (12 Gy) to a primary site of

disease (secondary site shielded) was combined with anti-CTLA4 and compared to

radiation alone, MTRT and anti-CTLA4, or combination treatment with EBRT to a

primary site with MTRT to all sites and anti-CTLA4. Tumor growth curves show that

triple combination treatment results in improved tumor regression of both primary

(Figure 61) and secondary (Figure 62) tumors compared to other groups. Additionally,

survival was significantly improved with triple combination treatment (p < 0.01)

compared to dual combination treatment groups. Triple combination treatment resulted

in a 40% complete response rate (16% CR in MTRT + anti-CTLA4, 0% other groups)

with all responding animals having tumor specific immune memory to B78 or related

B16 melanoma. Finally,

[00318] Finally,

[00318] we simulated we simulated advanced advanced multi-site multi-site "cold" "cold" cancers cancers (i.e., (i.e., multi-site multi-site

tumors that do not provoke a strong immune system response, and thus are largely

resistant to checkpoint inhibition) using a mouse model having two distant

macrospcopic tumors and disseminated microscopic metastases.

[00319] To form the large primary tumor, mice were injected in one flank with 2 X

10 B78 106 B78melanoma melanomatumor tumorcells. cells.To Toform formthe thesmall smallsecondary secondarytumor, tumor,twelve twelvedays days

later, mice were injected in the opposite flank with 5 X 105 B78 melanoma 10 B78 melanoma tumor tumor wo 2019/094657 WO PCT/US2018/059927 cells. Seventeen days after this (Day 1), to create the disseminated metastases, mice

105B16 were injected intravenously with 2 X 10 B16melanoma melanomacells. cells.

Mice

[00320] Mice werethe were the exposed exposed to to various varioussingle or or single combination treatments: combination PBS treatments: PBS

control injection; MTRT, 50 uCi µCi IV on Day 1; ICI, Anti-CTLA4/PD1 on Days 4, 7

and 10; In Situ Vaccine (IS), 12 Gy local RT on Day 1 + intratumoral injection of

anti-GD2 mAb and IL2 on Days 6-10. Tested single and combination treatments

were PBS, MTRT, ICI, IS, MTRT + ICI, MTRT + IS, ICI + IS, and MTRT + IS +

ICI. Beginning on Day 60, mice were monitored for tumor growth and animal

survival, and tumor-free mice were rechallenged with B78 on Day 90.

On 90,

[00321] On Day

[00321] Day less 90, less thanthan 20%the 20% of of ICI the mice ICI mice survived, survived, while while about about halfhalf of the of the

MTRT + IS and the ICI + IS mice survived (the MTRT + IS had a somewhat higher

survival rate). Surprisingly, the MTRT + IS mice were 100 % alive (the survival rate

was zero for all the other groups). Notably, 83% of these mice were found to be

tumor-free, exhibiting complete remission (CR) with immune cell memory (i.e., were

cured), while the remainder retained an uncontrolled secondary tumor.

[00322] We also confirmed uptake and dose delivery in a variety of other cancers,

including neuroblastoma (NXS2, 9464D), rhabdomyosarcoma (M3-9-M), high grade

glioma, lewis lung carcinoma, and head and neck cancer (MOC-2). In addition to tumor

uptake and dosimetry, toxicity analysis was conducted, and no radiation-induced

marrow toxicity (as measured by seum white cells or lymphocytes) was observed at our

therapeutic radiation dose of 50 uCi µCi (2-3 Gy tumor dose). We have also irradiated mice

with both external beam and varying doses of 90Y-NM600 and collected histology

stained with IHC as well as tissue for mRNA analysis by PCR. Data from these studies

show upregulation of the interferon signaling pathway with 50 uCi µCi of 90Y-NM600 as

well as increased PDL1 expression. In addition, we have found that tumor infiltrating

regulatory T-cells are reduced wuth molecularly targeted radiotherapy.

[00323] In sum, our findings from this study suggest that low dose NM600 MTRT

can enhance abscopal response in tumors when combined with checkpoint blockade.

Notably, the NM600 MTRT radiotherapeutic delivery agent demonstrates the ability

to improve response in "cold" tumors that normally do not respond to immune

checkpoint blockade alone. In addition, a relatively low MTRT dose, 50 uCi µCi (2.5 Gy

tumor dose) is sufficient to achieve immunostimulatory effects to enhance ICI

efficacy without systemic lymphodepletion. MTRT can be added to single site EBRT

PCT/US2018/059927

and checkpoint blockade to achieve greater tumor response and cure rates at both

local and distant tumor sites. Our results show that MTRT has great potential to

improve therapeutic efficacy of immunotherapy treatments in patients.

Conclusion to the Examples

These

[00324] These

[00324] examples examples illustrate illustrate an anti-cancer an anti-cancer strategy strategy based based on the on the synergistic synergistic

and widely applicable combination of targeted systemic delivery of radiotherapy with

systemic delivery of an immunostimulatory agent, such as an immune checkpoint

inhibitor. As the disclosed metal chelated and radiohalogenated Ikylphosphocholine alkylphosphocholine

analogs can target cancers of virtually any histology, the systemic administration of

immune checkpoint-targeting mAbs or small molecules (immune checkpoint

inhibitors) finds use for virtually any cancer type (tumor reactive mAbs are approved

or in clinical testing for nearly all cancer histological types). Accordingly, the clinical

translation of the two different combined strategies have wide application for virtually

all high risk cancers.

Other

[00325] Other

[00325] embodiments embodiments and and usesuses of the of the invention invention willwill be apparent be apparent to those to those

skilled in the art from consideration from the specification and practice of the

invention disclosed herein. All references cited herein for any reason, including all

journal citations and U.S./foreign patents and patent applications, are specifically and

entirely incorporated herein by reference. It is understood that the invention is not

confined to the specific reagents, formulations, reaction conditions, etc., herein

illustrated and described, but embraces such modified forms thereof as come within

the scope of the following claims.

Claims

CLAIMS 1. A method of treating a metastatic cancer comprising a primary malignant solid tumor and one or more metastatic tumors capable of causing concomitant immune tolerance, comprising systemically administering to the subject: (a) an immunomodulatory dose of a targeted radiotherapy (TRT) agent that is differentially taken up by and retained within the malignant solid tumor tissue, wherein 2018366219

the TRT agent is a phospholipid ether metal chelate or a radiohalogenated phospholipid ether that has the formula:

(OCH2CHYCH2)m

or a salt thereof, wherein: R1 comprises (a) a chelating agent that is chelated to a metal atom, wherein the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater than 6 hours and less than 30 days; or (b) a radioactive halogen isotope; a is 0 or 1; Y is selected from the group consisting of –H, –OH, -COOH, -COOX, -OCOX, and –OX, wherein X is an alkyl or an arylalkyl; and wherein: (i) m is 0, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (ii) m is 1, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (iii) m is 0, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or

(iv) m is 1, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; and (b) one or more immunostimulatory agents capable of stimulating specific immune cells within the tumor microenvironment, wherein the one or more immunostimulatory agents are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules, wherein the one or more checkpoint molecules that the 2018366219

checkpoint inhibitors are capable of targeting are selected from the group consisting of A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associated protein 4), KIR (killer cell immunoglobulin-like receptor), LAG3 (Lymphocyte Activation Gene 3), PD-1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), CD40 (cluster of differentiation 40), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD137 (cluster of differentiation 137), OX40 (CD134; cluster of differentiation 134), OX40L (OX40 ligand; cluster of differentiation 252), GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T-cell costimulatory), ICOSL (inducible T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4 (VTCN1; V-set domain-containing T-cell activation inhibitor 1), IDO (Indoleamine 2,3-dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), Gal-9 (galectin-9), and VISTA (V-domain Ig suppressor of T cell activation); wherein the immunomodulatory dose delivers a tumor dose of 2-5 Gy, wherein the immunomodulatory dose depletes tumor infiltrating FoxP3+ Tregs without systemic lymphodepletion, and whereby the concomitant immune tolerance caused by the metastatic tumors is prevented and the metastatic cancer is treated in the subject.
2. The method of claim 1, wherein the one or more immune checkpoint inhibitors comprise one or more anti-immune checkpoint molecule antibodies selected

from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-LAG3 antibody, an anti-KIR antibody, an anti-A2AR antibody, and anti-BTLA antibody, an anti-CD40 antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-OX40L antibody, a GITR antibody, a GITRL antibody, an ICOS antibody, an ICOSL antibody, a B7H3 antibody, a B7H4 antibody, an IDO antibody, a TIM-3 antibody, a Gal-9 antibody, and a 2018366219

VISTA antibody; or one or more small molecule immune checkpoint inhibitors comprising a small molecule PD-L1 inhibitor that act to block one or more immune checkpoint molecules.
3. The method of claim 1, wherein (1) the metal isotope is selected from the group consisting of Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, and Th-227; or (2) the radioactive halogen isotope is selected from the group consisting of 123 I, 124I, 125I, 131I, 211At, 77Br, and 76Br; or (3) the chelating agent is selected from the group consisting of 1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid (DO3A); 1,4,7-triazacyclononane-1,4-diacetic acid (NODA); 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA); 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7-triazacyclononane,1- glutaric acid-4,7-diacetic acid (NODAGA); 1,4,7,10-tetraazacyclodecane,1-glutaric acid- 4,7,10-triacetic acid (DOTAGA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A); diethylene triamine pentaacetic acid (DTPA), its diester; 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A”-DTPA); deforoxamine (DFO); 1,2-[[6- carboxypyridin-2-yl]methylamino]ethane (H2dedpa); and DADA, wherein DADA comprises the structure:

︴ 2018366219

; or (4) each Z is independently –CH2CH3 or –CH3.
4. The method of any one of claims 1-3, wherein the chelating agent chelated to the metal atom is selected from the group consisting of:

︴ ,

︴

, 2018366219

︴

,

︴

,

︴ 2018366219

,

︴

,

︴ ,

︴

2018366219

, ︴ ,

︴ ,

︴︴ , 2018366219

︴ ,

︴ , and

︴ 2018366219

.
5. The method of any one of claims 1-4, wherein the radioactive phospholipid ether metal chelate has the formula selected from the group consisting of:

O OH N H O N (CH2)18OPOCH2CH2NMe3 N N O O O N OH HO O ,

O OH O N H N (CH2)18OPOCH2CH2NMe3 N N O O O N OH HO O ,

HO

O O N H N (CH2)18OPOCH2CH2NMe3 N N O O O

HO , 2018366219

HO

O O N H N (CH2)18OPOCH2CH2NMe3 N N O O O

HO ,

O OH OH

O N N O (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

O OH OH

O N N O (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

OH

O N N O HO N (CH2)18OPOCH2CH2NMe3 O O O OH , 2018366219

OH

O N N O HO N (CH2)18OPOCH2CH2NMe3 O O O OH ,

O OH OH O O N N HN (CH2)18OPOCH2CH2NMe3 O N N O

O HO O HO ,

O OH OH O O N N HN (CH2)18OPOCH2CH2NMe3 O N N O

O HO O HO ,

OH

O O N N HN (CH2)18OPOCH2CH2NMe3 HO N O O O O OH , 2018366219

OH

O O N N HN (CH2)18OPOCH2CH2NMe3 HO N O O O O OH ,

O OH OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

O OH OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O

HO O , 2018366219

OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O

HO O ,

O (CH2)18OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2 H , O (CH2)18 8OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H , O (CH2)18OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H ,

O (CH2)18 8OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H , 2018366219

O O O O Me3NCH2CH2OPO(CH2)18 O N N N O (CH2)18OPOCH2CH2NMe3 O HO2C CO2H O HO2C ,

O O HN N 5 HO O HO N O HN NH Me3NCH2CH2O P O(CH2)18 5 O O O N HO O , O O HN N 5 HO O HO N O HN NH Me3NCH2CH2O P O(CH2)18 5 O O O N HO O ,

O (CH2)18OPOCH2CH2NMe3 O NH HN

N N

OH HO O O , 2018366219

O (CH2)18 OPOCH2CH2NMe3 O NH HN

N N

OH HO O O ,

O O NH (CH2)18OPOCH2CH2NMe3 O

O NH HN O

HS SH , and

O O NH (CH2)18OPOCH2CH2NMe3 O

O NH HN O

HS SH ;

wherein the selected compound is chelated to the metal atom.
6. The method of claim 1, wherein a is 1, b is 1, m is 0, n is 18, and R2 is –N+(CH3)3.
7. The method of claim 6, wherein the radioactive phospholipid ether metal chelate is NM600 chelated to the metal atom, or wherein the radiohalogenated phospholipid ether is NM404. 2018366219
8. The method of claim 7, wherein the radioactive phospholipid ether metal chelate is 90Y-NM600 or 177Lu-NM600.
9. The method of any one of claims 1-8, wherein the TRT agent; the immune checkpoint inhibitor; or both; are administered intravenously.
10. The method of any one of claims 1-9, wherein the subject is a human.
11. The method of any one of claims 1-10, wherein the cancer that is treated is selected from the group consisting of melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell cancer of the skin or head or neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms’ tumor, and pediatric brain tumors.
12. Use of (a) an immunomodulatory dose of a targeted radiotherapy (TRT) agent and (b) one or more immunostimulatory agents capable of stimulating specific immune cells within the tumor microenvironment for treating a subject for a metastatic cancer comprising a primary malignant solid tumor and one or more metastatic tumors capable of causing concomitant immune tolerance,

wherein the TRT agent is a phospholipid ether metal chelate or a radiohalogenated phospholipid ether that is capable of being differentially taken up by and retained within a malignant solid tumor tissue and has the formula:

(OCH2CHYCH2)m 2018366219

or a salt thereof, wherein: R1 comprises (a) a chelating agent that is chelated to a metal atom, wherein the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater than 6 hours and less than 30 days; or (b) a radioactive halogen isotope; a is 0 or 1; Y is selected from the group consisting of –H, –OH, -COOH, -COOX, -OCOX, and –OX, wherein X is an alkyl or an arylalkyl; and wherein: (i) m is 0, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (ii) m is 1, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (iii) m is 0, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or iv) m is 1, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl, wherein the one or more immunostimulatory agents are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules, wherein the one or more checkpoint molecules that the checkpoint inhibitors are capable of targeting are selected from the group consisting of A2AR (adenosine A2a receptor), BTLA (B and T

lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associated protein 4), KIR (killer cell immunoglobulin-like receptor), LAG3 (Lymphocyte Activation Gene 3), PD- 1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), CD40 (cluster of differentiation 40), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD137 (cluster of differentiation 137), OX40 (CD134; cluster of differentiation 134), OX40L (OX40 ligand; cluster of differentiation 252), GITR (glucocorticoid- 2018366219

induced tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T-cell costimulatory), ICOSL (inducible T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4 (VTCN1; V-set domain-containing T-cell activation inhibitor 1), IDO (Indoleamine 2,3-dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), Gal-9 (galectin-9), and VISTA (V-domain Ig suppressor of T cell activation); wherein the TRT agent and the one or more immunostimulatory agents are both systemically administered to the subject; wherein the immunomodulatory dose delivers a tumor dose of 2-5 Gy, wherein the immunomodulatory dose depletes tumor infiltrating FoxP3+ Tregs without systemic lymphodepletion, and whereby the concomitant immune tolerance caused by the metastatic tumors is prevented and the metastatic cancer is treated in the subject.
13. Use of (a) an immunomodulatory dose of a targeted radiotherapy (TRT) agent and (b) one or more immunostimulatory agents capable of stimulating specific immune cells within the tumor microenvironment in the manufacture of a medicament for treating a metastatic cancer comprising a primary malignant solid tumor and one or more metastatic tumors capable of causing concomitant immune tolerance;

wherein the TRT agent is a phospholipid ether metal chelate or a radiohalogenated phospholipid ether that is capable of being differentially taken up by and retained within a malignant solid tumor tissue and has the formula:

(OCH2CHYCH2)m 2018366219

or a salt thereof, wherein: R1 comprises (a) a chelating agent that is chelated to a metal atom, wherein the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater than 6 hours and less than 30 days; or (b) a radioactive halogen isotope; a is 0 or 1; Y is selected from the group consisting of –H, –OH, -COOH, -COOX, -OCOX, and –OX, wherein X is an alkyl or an arylalkyl; and wherein: (i) m is 0, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (ii) m is 1, b is 1, n is an integer from 12 to 30, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or (iii) m is 0, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl; or iv) m is 1, b is 1, n is 18, and R2 is -N+Z3, wherein each Z is independently an alkyl or an aryl, wherein the one or more immunostimulatory agents are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules, wherein the one or more checkpoint molecules that the checkpoint inhibitors are capable of targeting are selected from the group consisting of A2AR (adenosine A2a receptor), BTLA (B and T

lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associated protein 4), KIR (killer cell immunoglobulin-like receptor), LAG3 (Lymphocyte Activation Gene 3), PD- 1 (programmed death receptor 1), PD-L1 (programmed death ligand 1), CD40 (cluster of differentiation 40), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD137 (cluster of differentiation 137), OX40 (CD134; cluster of differentiation 134), OX40L (OX40 ligand; cluster of differentiation 252), GITR (glucocorticoid- 2018366219

induced tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T-cell costimulatory), ICOSL (inducible T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4 (VTCN1; V-set domain-containing T-cell activation inhibitor 1), IDO (Indoleamine 2,3-dioxygenase), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), Gal-9 (galectin-9), and VISTA (V-domain Ig suppressor of T cell activation); wherein the medicament is to be systemically administered to the subject, wherein the immunomodulatory dose delivers a tumor dose of 2-5 Gy, and wherein the immunomodulatory dose depletes tumor infiltrating FoxP3+ Tregs without systemic lymphodepletion.
14. The use of claim 12 or 13, wherein the one or more immune checkpoint inhibitors comprise one or more anti-immune checkpoint molecule antibodies selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-LAG3 antibody, an anti-KIR antibody, an anti-A2AR antibody, and anti-BTLA antibody, an anti-CD40 antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-OX40L antibody, a GITR antibody, a GITRL antibody, an ICOS antibody, an ICOSL antibody, a B7H3 antibody, a B7H4 antibody, an IDO antibody, a TIM-3 antibody, a Gal-9 antibody, and a VISTA antibody; or one or more small molecule immune checkpoint inhibitors

comprising a small molecule PD-L1 inhibitor that act to block one or more immune checkpoint molecules.
15. The use of claim 12 or 13, wherein (1) the metal isotope is selected from the group consisting of Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, and 2018366219

Th-227; or (2) the radioactive halogen isotope is selected from the group consisting of 123 I, 124I, 125I, 131I, 211At, 77Br, and 76Br; or (3) the chelating agent is selected from the group consisting of 1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid (DO3A); 1,4,7-triazacyclononane-1,4-diacetic acid (NODA); 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA); 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7-triazacyclononane,1- glutaric acid-4,7-diacetic acid (NODAGA); 1,4,7,10-tetraazacyclodecane,1-glutaric acid- 4,7,10-triacetic acid (DOTAGA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A); diethylene triamine pentaacetic acid (DTPA), its diester; 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A”-DTPA); deforoxamine (DFO); 1,2-[[6- carboxypyridin-2-yl]methylamino]ethane (H2dedpa); and DADA, wherein DADA comprises the structure:

︴ ; or

(4) each Z is independently –CH2CH3 or –CH3.
16. The use of any one of claims 12-15, wherein the chelating agent chelated to the metal atom is selected from the group consisting of: 2018366219

︴ ,

︴

,

︴ , 2018366219

︴

,

︴ ,

︴,

︴ 2018366219

,

︴ , ︴ ,

︴ , 2018366219

︴︴ ,

︴ ,

︴ 2018366219

, and

︴ .
17. The use of any one of claims 12-16, wherein the radioactive phospholipid ether metal chelate has the formula selected from the group consisting of:

O OH N H O N (CH2)18OPOCH2CH2NMe3 N N O O O N OH HO , 2018366219

O

O OH O N H N (CH2)18OPOCH2CH2NMe3 N N O O O N OH HO O ,

HO

O O N H N (CH2)18OPOCH2CH2NMe3 N N O O O

HO ,

HO

O O N H N (CH2)18OPOCH2CH2NMe3 N N O O O

HO ,

O OH OH

O N N O (CH2)18OPOCH2CH2NMe3 N N O O HO O HO , 2018366219

O OH OH

O N N O (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

OH

O N N O HO N (CH2)18OPOCH2CH2NMe3 O O O OH ,

OH

O N N O HO N (CH2)18OPOCH2CH2NMe3 O O O OH ,

O OH OH O O N N HN (CH2)18OPOCH2CH2NMe3 O N N O

O HO O HO , 2018366219

O OH OH O O N N HN (CH2)18OPOCH2CH2NMe3 O N N O

O HO O HO ,

OH

O O N N HN (CH2)18OPOCH2CH2NMe3 HO N O O O O OH ,

OH

O O N N HN (CH2)18OPOCH2CH2NMe3 HO N O O O O OH ,

O OH OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O O HO O HO , 2018366219

O OH OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O O HO O HO ,

OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O

HO O ,

OH

O O N N (CH2)18OPOCH2CH2NMe3 N N O

HO O ,

O (CH2)18OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H ,

O (CH2)18 8OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO 2 H , 2018366219

O (CH2)18OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H ,

O (CH2)18 8OPOCH2CH2NMe3 O N N N HO2C CO2H HO2C CO2H CO2H ,

O O O O Me3NCH2CH2OPO(CH2)18 O N N N O (CH2)18OPOCH2CH2NMe3 O HO2C CO2H O HO2C ,

O O HN N 5 HO O HO N O HN NH Me3NCH2CH2O P O(CH2)18 5 O O O N 2018366219

HO O , O O HN N 5 HO O HO N O HN NH Me3NCH2CH2O P O(CH2)18 5 O O O N HO O ,

O (CH2)18OPOCH2CH2NMe3 O NH HN

N N

OH HO O O ,

O (CH2)18 OPOCH2CH2NMe3 O NH HN

N N

OH HO O O ,

O O NH (CH2)18OPOCH2CH2NMe3 O

O NH HN O

HS SH , and 2018366219

O O NH (CH ) 2 18 OPOCH2CH2NMe3 O

O NH HN O

HS SH ;

wherein the selected compound is chelated to the metal atom.

18. The use of claim 12 or 13, wherein a is 1, b is 1, m is 0, n is 18, and R2 is –N+(CH3)3.

19. The use of claim 16, wherein the radioactive phospholipid ether metal chelate is NM600 chelated to the metal atom, or wherein the radiohalogenated phospholipid ether is NM404.

20. The use of claim 16, wherein the radioactive phospholipid ether metal chelate is 90Y-NM600 or 177Lu-NM600.

21. The use of any one of claims 12-20, wherein the TRT agent; the immune checkpoint inhibitor; or both; are administered intravenously.

22. The use of any one of claims 12-21, wherein the subject is a human.

23. The use of any one of claims 12-22, wherein the cancer that is treated is selected from the group consisting of melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell cancer of the skin or head or neck, intestinal cancer, 2018366219

retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms’ tumor, and pediatric brain tumors.

24. The method of any one of claims 1-11 or use of any one of claims 12-23, wherein the immunomodulatory dose delivers a tumor dose of 2-3 Gy.

25. The method of any one of claims 1-11 or use of any one of claims 12-23, wherein the radioactive phospholipid metal chelate compound has the structure:

or a salt thereof; wherein the radioactive phospholipid metal chelate is chelated to a 90Y radioisotope.

26. The method or use of claim 25, wherein the radioisotope delivers a radiation dose of from 2 Gy to 3 Gy to tumors in the subject.

27. The method or use of claim 25, wherein the checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, or a LAG3 inhibitor. 2018366219

OF O I

(CH)OPOCHCHNMe O- 0 NM404 (unmodified)

Fig. 1

SUBSTITUTE SHEET (RULE 26)

12 Gy 12 Gy + + hu14.18-IL2 hu14.18-IL2

Mean Tumor Volume (mm³)

12 Gy + IgG

1200 1200 NS hu14.18-IL2 hu14.18-IL2 - Min

IgG * we " I = **

n 1000 1000

B78 Melanoma

800

353 - ** n = 5 600

400 %% *** CODE

the ** n=8

200 1-4

H n = 9 n=9 = HH 1 0

0 5 10 15 20 25 30

Days

Fig. 2A

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

3/78

n= n : 19 n = 19

1.0

Survival probability

0.8

nn=$ n3 :1515

0.6

B78 Melanoma it :9 n=9

Log rank rank pp <<0.001 0.001 0.4 0.4

***

12 Gy + hu14.18-IL2

12 Gy + IgG 0.2 NS * ***

*** n=9=n 99 n hu14.18-IL2 hu14. 18-IL2

/ IgG

0.0

- 0 10 20 30 40 50 60 60

Days

Fig. 2B

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

***

*** 100 ***

83% 100% 1009 0 10 70 80 50 30 90 40 60 20 90 100% 10% (n=6) (n=5) (n=5) not For

80 Disease-free Percent engraftment

70

60 aSurgically mSurgically resected

50

oT-cell depleted

40 disease-free

30 DNaive Naive

20 10% (n=20)

10

0

Fig. 2C

SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

5/78

Two Two tumors tumors ** 12 12 Gy Gy ++ IgG IgG

Two Two tumors tumors -* 12 12 Gy Gy ++ hu14.18-IL2 hu14.18-IL2

Mean Tumor Volume (mm³)

1400 Two Two tumors tumors - * 12 12 Gy Gy to to both both tumors tumors + + hu14.18-IL2 hu14.18-IL2

Single Single tumor- tumor ** 12 12 Gy Gy ++ hu14.18-IL2 hu14.18-IL2 1200

B78 melanoma 1000

800

600 600 **

400

200

0

0 5 10 15 20 25 30

Days

Fig. 3

SUBSTITUTE SHEET (RULE 26) wo 2019/094657 PCT/US2018/059927 6/78 6/78

1400 1400

Non-depleted control Non-depleted control

1200 1200 reg depleted ### Treg depleted

1000 1000

Mean Tumor Volume (mm³)

800 008 %

600 600 *

400 400 I-

###

- - - 200 200 I # - I- -

0-

0- 5 ot 10 15 ST 20 20 SZ 25 30 08

Days Days on

Fig. 4 Fig. 4

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

I Activity I Activity

[MBq/cc] 71h

0

B78 tumor

Fig. 5

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

Single Single tumor tumor *X 12 12 Gy Gy ++ hu14.18-IL2 hu14.18-IL2 ++ 33 uCil131-CLR1404 uCi I131-CLR1404

Mean Tumor Volume (mm³)

1200 Single tumor * -12 Gy ++ hu14.18-IL2 12 Gy hu14.18-IL2

1000 B78 melanoma

800 800

600

400

200

0 X

0 5 10 10 15 20 25 30

Days Days

Fig. 6

SUBSTITUTE SHEET (RULE 26)

T 1h Precontrast

T

24h 4h

Fig. 7

SUBSTITUTE SHEET (RULE 26)

Primary Tumor Response

22 tumors tumors << - IT-IgG IT-tgG(n (n :=4) 4)

2 tumors * IT-hu14.18-IL2 (n=5) 5) 2400

22 tumors tumors *-12-12Gy+IT-IgG(n=4) Gy + IT-lgG (n=4) 3

NS

2000 22 tumors - 12 Gy + IT-hu14.18-IL2 (n=5) (n =

Tumor volume (mm³)

1 1 tumor * 12 Gy +IT-hu14.18-IL2(n=6 =

B78 B78 Melanoma Melanoma primary primary and and secondary secondary tumors tumors 1600

#

1200 : #

#

800

T

400 400

X

- 0

0 $ 5 3 10 15 20 25 30

Day

Figure 8A Figure 8A

SUBSTITUTE SHEET (RULE 26)

2 tumors - 12 Gyto +-16- 1T-19G

NS NS

1 tumor 12 Gy +T-hu14.18-

80

n :: 13 n= 13

60

Survival Rate (%)

40

20 n= Il n=11 =

B78 B78 Melanoma Melanoma primary primary and and secondary secondary tumors tumors

Log-rank <0.0001 - - = *

10 20 40 50 3 8

Day

Figure 8B

SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

12/78

1600 Primary Tumor Response

Panc02-GD2+ primary Panc02-GD2+ primary andand Panc02-GD2- Panc02-GD2- secondary secondary (n=7) (n=7) 7)

Panc02-GD2+ primary Panc02-GD2+ primary only only (n := 7) (n=7) 7)

Tumor volume (mm³)

1200 All treated with 12 Gy + T-hu14.18-IL2 to the primary tumor All treated with 12 Gy + IT-hu14.18-IL2 to the primary tumor

800

400 the

0 I 0 I 5 10 15 20 25 30

Day

Figure 8C

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

13/78

Primary Tumor Response

B78 B78 primary primary and and B78 B78 secondary secondary (n (n 1=6) : 6) 2000

B78 B78 primary primary and and Panc02-GD2+secondary(n = 6 = Panc02-GD2+ secondary (n=6) for Tumor volume (mm³) NS B78 primaryalone B78 primary alone (n (n=7) : 7)

1600

All All treated treated with with 12 12 Gy Gy + + IT-hu14.18-IL21 IT-hu14. 18-IL2 to to the the primary primary tumor tumor

1200

800 800

400

0 =

0 - 5 10 15 of 20 25 30

Day Day

Figure 8D Figure 8D

SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

14/78

Primary Primary Tumor Tumor Response Response

1600 Panc02-GD2+ primary and Panc02-GD2- secondary (n 1=7) Panc02-GD2+ primary and Panc02-GD2- secondary (n : 7)

Panc02-GD2+ primary and B78 secondary (n=7) ///// (II) Panc02-GD2+ primary and B78 secondary (n=7) = 7)

NS Panc02-GD2+ primary Panc02-GD2+ primary only only (n (n (n=7) n=7) : Tumor volume (mm³)

1200 All treated with 12 Gy + IT-hu14.18-IL2 to the primary tumor All treated with 12 Gy + IT-hu14.18-IL2 to the primary tumor

800

*

400

0 =

0 5 10 15 20 25 30

Day

Figure 8E

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

B78 primary only Two B78 tumors

A1' A1 A3

12 12 Gy Gy

No RT NoRT Primary tumor Primary tumor

A2 A4

No RT 12 Gy 12 Gy 12 Gy Secondary lumor tumor

Figure 9A

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

16/78

60 ** field 200x per cells FoxP3+ 50

40

30

20 20 A1 A2 A3 A4 A3 A4 10 /

0 I No RT 12 Gy Primary Primary Secondary Secondary

Day Day 66 after after RT RT B78 primaryonly B78 primary only Two B78 B78 tumors tumors 12 12 Gy Gy to to primary primary tumor tumor ANOVA ANOVA pp << 0.001 0.001

Figure Figure 9A 9A - - Continued Continued

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

PCT/US2018/059927

17/78

Primary Tumor Response 1200

Non-depleted control Non-depleted control (n :(n=5) 5)

Treg depleted Treg depleted (n (n=5) : 5) you

Tumor Volume (mm³)

800 800

400

B78 B78 Melanoma Melanoma primary primary and and secondary secondary tumor tumor

"DEREG" transgenic mice

All All treated treated with with 12 12 Gy Gy + + IT-hu14.18-IL2 IT-hu14 18-IL2 to to the the primary primary tumor tumor

-

X 5 - 10 15 20 25 30 2 Day

Figure 9B

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

18/78

Non-depleted control Non-depleted (n :(n=5) control # W 5)

600 **

% : (Ifdepleted Treg depleted Treg SD (n=5)

All treated with 12 Gy + * IT-hu14.18-IL2 10 to the primary tumor Tumor Volume (mm³)

B78 Melanoma primary and secondary tumor

400 "DEREG" transgenic mice

200

0

= - 10 15 20 25 30

Day

Figure 9C

SURSTITUTE SHEETIRULE SUBSTITUTE SHEET 26) (RULE 26)

Primary Tumor Response

1600 1600

12 Gy to 12 Gy toprimary primary only only (n :(n =5) * 5)

** 12 Gy to 12 Gy toprimary primaryandand secondary secondary (n *= 5) (n : ** 5)

1200 1200 B78 Melanoma primary and secondary tumors

All treated with IT-hu14.18-IL21 IT-hu14. 18-IL2 to the primary tumor

Tumor Volume (mm³)

800

-

400

0

- 5 10 15 20 25 30

Day

Figure 10A

SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

20/78 20/78

100 100

n 11 == 21 21

80

Survival Rate (%)

60

40

$ 12 Gy to primary only

*** n ==- -21 n 21

12 Gy lo to primary and secondary 20

B78 Melanoma primary and secondary tumors

All treated with IT-hu14.18-IL2 IT-hu14. 18-IL2to tothe theprimary primarytumor tumor

Log-rank Log-rank- =w 0.0001 0.0001

I

0 = 10 20 30 40 50 60

Day

Figure 10B

SUBSTITUTE SHEET (RULE 26)

Primary Tumor Response

1200

IT-hu14.18-112 IT-hu14.18-IL2

2 2Gy $ IT-hu14.18-(L2 IT-hu14.18-IL2 ========= Gy+

12 12Gy + IT-hu14.18-IL2 #### Tumor volume (mm3)

900

B78 B78 Melanoma Melanoma primary primary tumor tumor only only

600

300

OF 0 5.5%

CD 0 5 10 10 15 20 25 30 35 5 Day

Figure 11A

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 PCT/US2018/059927

22/78

Primary Tumor Response

2000

IIIIIII 12 12 GyGytotoprimary primary +IT-hu14.18-1L2 IT-hu14.18-IL2 IIIIIII

\\\\\\\ 12 12 Gy Gy to to primary primary +2 +Gy2 to Gy secondary to secondary + iT-hu14.18-l2 + T-hu14.18-IL2 <<<<<<<

1600 12 Gy @@@@@@@@ 12 to Gy primary + 5 +5 to primary Gy Gy to to secondary+ IT-hu14.18-IL2 secondary + IT-hu14.18-I2 ####### Tumor volume (mm3)

IT-hu14.18-IL2 12 Gy to primary + 12 Gy to secondary + IT-hu14.18-1L2 30000000

12 12 /////// Gy Gy to to primary +12+12 primary Gyto Gy otoseconary seconary

1200

800

400 400

- 0 6.73 CHD

0 5 10 15 of 20 25 30 35 35 X Day

Figure 11B Figure 11B

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 PCT/US2018/059927

23/78 23/78

Secondary Tumor Response

3200

12Gy Gy to to primary primary+ IT-hu14.18-L2 IT-hu14.18-IL2

2800 12 12GyGytoto primary - 2 Gy primary +2 to Gysecondary + IT-hu14.18-112 to secondary + IT-hu14,18-IL2

<<<<<<<< 12 Gy to primary +5 Gy to secondary + IT-hu14,18-IL2 12Gytoprimary+SGytosecondary+ 1T-hu14.18-L2 Tumor volume (mm³) ammu 2400

12 Gy to primary + 1) 12 Gy to secondary & + IT-hu14.18-IL2

2000 IIIIIII 12 Gy to primary +12 Gy to secondary IIIIIIII

1600

1200

800

400

<<<

0

4.20%

of 0 5 10 15 20 25 30 26 $ Day Day

Figure 11C Figure 11C

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

24/78

124 1241 Activity I Activity

[MBq/cc]

[MBq/cc] 2.23 71h

0-

B78 tumor

Figure 12A

SUBSTITUTE SUBSTITUTESHEET SHEET(RULE : 26) (RULE 26)

AVG WBC from maxillary bleed samples

14

12

10

Average WBC 8

6 3

4

2

0

control T2 T1 T3 T4

Number of Half-Lives post injection

Figure 12B

SUBSTITUTE SHEET (RULE 26)

CD8+ Tumor Infiltrate

300

250

200

Count/HPF

150

100

50

0

C1 T1 T2 T3 T4

Figure 12C Figure 12C

SUBSTITUTE SHEET (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

27/78

FoxP3+ FoxP3+ Tumor Tumor Infiltrate Infiltrate 120

100

80

Count/HPF CHANGE

*

60

40

20

* = * = p<0.05

0 C T1 T2 T3 T4

Figure 12D

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 PCT/US2018/059927

28/78

Primary Tumor Response

NM404 NM404+ IC 2000 NM404 + RT NM404 NM404 / + RT is IC + IC

/ IC 12/0Gy +

1600 \\\\\\\\\\\\ 12/2G/ + IC 12/26y+1 mm 12/12Gy * IC 12/12Gy+1 Tumor Volume (mm³)

1200

800

400

0

0 5 $ 10 15 20 25 30 35

Day

Figure 13A

SUBSTITUTE SHEET (RULE 26)

Secondary Tumor Response

1600 NM404 NM404 of+ IC IC

NM404 + RT 18 NM404 is

NM404 NM404 in+ RT RT .+ ICIC

12/0Gy * + IC

12/2Gy of IC + IC

1200 12/12Gy + +10 12/12Gy IC Tumor Volume (mm³) 1200

800

400

0 CD 0 5 10 15 20 25 30 35 Day

Figure 13B

SUBSTITUTE SHEET (RULE 26)

O 0 -

N H O 0 II + N 64 (CH2 DPOCH2CH2NMe3 (CH)OPOCHCHNMe O 0 0 O N -

OH

0

Figure 14

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 31/78 31/78

2000 2000 110.0 110.0 mm mm

5.5 %ID/g 5.5 %ID/g

5.3 %ID/g 5.3 %ID/g 450 450

Figure 15 Figure 15

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 32/78 32/78

2000 2000 110.0 110.0 mm mm

0 400 400

Figure Figure 16 16

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

U87MG

10

H

L

(61019) (%ID/g)

T

0

3 h 24 h 48 h 3h

Figure 17

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

34/78 34/78

4T1

77

(%ID/g)

0

3 h 24 hh 48 h 3h

Figure Figure 18 18

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) wo 2019/094657 2019/094657 PCT/US2018/059927

35/78 35/78

HCT-116 HCT-116

7

(%ID/g)

0

3 h 24 h 48 h 3h

Figure Figure 19 19

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 36/78 36/78

A549 5

5E 367

(%ID/g)

0

Figure Figure 20 20

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

PC-3

5

(%ID/g)

0

Figure 21

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 38/78 38/78

HT-29 HT-29

5

(%ID/g)

0

Figure 22 Figure 22

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 39/78 39/78

MiaPaca MiaPaca

55

(%ID/g)

0

Figure 23 Figure 23

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 40/78 40/78 20

(%ID/g)

T

0

Figure Figure 24 24

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 41/78 41/78 15

(%ID/g)

T

0

Figure 25 Figure 25

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

(%ID/g)

HT-29

0

Figure 26

SUBSTITUTE SHEET (RULE 26)

<<<<<<<<

PC3

0 0

Figure 27

SUBSTITUTE SUBSTITUTESHEET SHEET(RULE 2 26) (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

44/78

2 2

(%ID/g)

Figure Figure 28 28

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

45/78

1 2 2

(%ID/g)

Figure 29

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927 46/78 46/78

86 15 15 Y 64 Uptake (%ID/g) Cu Cu 89 Zr 10 10 Zr I T

5 5

0 3h 14h 24h 24h 48h Time (h) Time (h)

Figure 30 Figure 30

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

Y 64 Uptake (%ID/g)

Cu Cu 89

10 + Zr Zr

T

T I 5 H

0

3h 14h 24h 48h

Time (h)

Figure 31

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927

48/78

86 30 30 Y 64 Uptake (%ID/g) request Cu Cu H J. 89 T Zr Zr 20 20 + I T 10 10 + F I

0 3h 14h 24h 48h

Time (h) Time (h)

Figure Figure 32 32

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

8Y Y 64 Uptake (%ID/g)

Cu Cu I 89 Zr Zr 4 4 T I T

F 2

0

3h 14h 24h 24h 48h

Time (h)

Figure 33

SUBSTITUTE SHEET (RULE 26)

Cu @ 48h

Tissue Uptake (%ID/g)

86y @ 48h

15

Zr@ 48h

¹Lu @ 48h

10

¹Lu @ 96h

5

0

BloodSkin Muscle Tumor HasLungLiver Bone Spleen //// Kidney Brain

Figure 34

SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927

51/78 51/78

O 0 -

N H H II + N N (CH2)18 H2CH2NMe (CH2)OPOCHCHNMe 0 O 0

OH

O

Figure Figure 35 35

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

Figure 36

SUBSTITUTE SHEET (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

53/78

Figure Figure 37 37

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

54/78

Figure 38

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

55/78

Figure 39

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

PCT/US2018/059927

56/78 56/78

MiaPaca Tumor

Figure 40

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

PCT/US2018/059927

57/78

4T1 Tumor

Figure 41

SUBSTITUTE SHEET (RULE 26)

Figure 42

SUBSTITUTE SHEET (RULE 26)

4T1 Tumor 4h

Figure 43

SUBSTITUTE SHEET (RULE 26)

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60/78

48 h

Figure 44

SUBSTITUTE SHEET (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

61/78

96 96hh

Figure 45

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26) wo 2019/094657 2019/094657 PCT/US2018/059927

62/78

3000 Control

90 90Y-NM600 (150Ci) Tumor Volume (mm³) Y-NM600 (150µCi) 90 Y-NM600 (300uCi) Y-NM600 (300µCi)

2000

1000

If

F

0 0 5 10 15 20 25 Days Days after after injection injection

Figure Figure 46 46

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO 2019/094657 PCT/US2018/059927

63/78

Control Control

3000 177 Lu-NM600 (500Ci) ¹Lu-NM600 (500µCi) Tumor Volume (mm³)

2000 2000

1000

0 0 10 20 30 40 40 Days after injection Days after injection

Figure Figure 47 47

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) wo 2019/094657 2019/094657 PCT/US2018/059927

64/78 64/78

Control 3000 3000 177 Lu-NM600 (400 ¹Lu-NM600 (400 µCi) uCi) Tumor Volume (mm³)

2000

1000 1000

0 0 10 20 30 40

Time Time (days) (days)

Figure Figure 48 48

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

65/78

177 5000 Lu-NM600 (500uCi) (500µCi) Control

Tumor Volume (mm³)

4000 4000 ***

*** 3000 3000

* * ** 2000

1000

0 0 5 10 10 15

Days Days after after injection injection

Figure 49

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

PCT/US2018/059927

66/78 66/78

177 177 Lu-NM600 (500 (500µCi+250µCi) Lu-NM600 uCi+250uCi) 2500

Tumor Volume (mm³) Control

2000 2000

1500

1000

500

T

0 0 5 10 15 20 25

Days Days after after injection injection

Figure 50

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

67/78

2500 Control

90 Y-NM600 Tumor Volume (mm³)

Y-NM600 (500uCi) (500uCi)

2000 2000 90 Y-NM600 Y-NM600 (250uCi) (250uCi)

1500

1000

500

0

0 5 10 15 20

Days Days after after injection injection

Figure 51

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

4.4

4.2- 4.2

Y-90/I-131

4.0 4.0

3.8

3.6

3.4

0 10 20 0 10 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100 100

Tumor Mass (g)

Figure 52

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

69/78 69/78

Average BindingEnergies Average Binding Energies

3.5 # APC-amine APC-amine 3.5

APC-DOTA # APC-DOTA (-kCal/moL) Energies Binding CD 3 APC-NOTA APC-NOTA

APC DOTA-M APC DOTA-M 2.5 2.5

lodo-PLE lodo-PLE - 2

1.5 1.5

-

0.5 0.5

0

Figure Figure 53 53

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

100 uCi 100 #C

100 100 uCi uCi of CTLA4 + CTLA4

1000 50 uCi 50 uO

uO +$CTLA4 50 uCi CTLA4

25 uCl 25 uC

25 u0 uCi* +CTLA4 CTLA4 800 Tumor volume mm3

PBS PBS

CTIA4 CTLA4 & + PBS

600

#

400

200

0 0 452

0 0 5 10 15 20 25 30 35 40 40

Time (Days)

Figure 54

SUBSTITUTE SHEET (RULE 26)

Percent survival

80

------ - ------------------------- 60

who ****** 100000 ****** 00000 ****

PBS 1 PBS 40 50 uCi + CTLA4 20 **** -

50 uCi + CTLA4

0 0 20 40 60 Time (Days)

Figure 55

SUBSTITUTE SHEET (RULE 26)

Percent survival

80 80

60 60

- 2525 uCi uCi + + CTLA4 CTLA4 40 40 50 uCi + CTLA4

- 100 uCi 1 100 uCi + + CTLA4 CTLA4 20

0

0 20 40 60 60

Time (Days)

Figure 56

SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

73/78

MTRT Dose Study 1000

100 uCi µCi

100 uCi µCi + CTLA4** Tumor Volume (mm³)

800 800 50 uCi µCi

50 uCi µCi + CTLA4**

25 uCi µCi 600 25 uCi µCi + CTLA4

PBS

400 CTLA4

89

200

0

0 5 10 15 20 25 30

Days

Figure 57

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 PCT/US2018/059927 PCT/US2018/059927

74/78 74/78

1/

0.8 0.8

inconclusive Survival

0.6 0.6

100 uCi 100 µCi

100 pC µCi+ +CTLA4** CTLA4**

0.4 50 µCi uCi

50 uCi µCi + CTLA4**

25 µCi uCi

0.2 0.2 25 uCi µCi + CTLA4

PBS CTLA4 Alone

0 0 10 20 30 40 50 60

Days

Figure 58

SUBSTITUTE SHEET (RULE 26)

NXS2 4500

4000 PBS

Tumor Volume (mm³) 3500 50 uCi µCi

3000 CTLA4

2500 µCi + CTLA4** 50 uCi

2000

1500 -

1000

500

0

0 5 10 15 20 25 30

Days

Figure 59

SUBSTITUTE SHEET (RULE 26)

WO wo 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927

76/78 76/78

4T1 2500

PBS

CTLA4 2000 Tumor Volume (mm³)

50 uCi µCi

50 50 uCi µCi + + CTLA4** CTLA4** 1500

1000

500 I -

0 0 = 5 10 15 20 25 30

Days

Figure Figure 60 60

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

WO WO 2019/094657 2019/094657 PCT/US2018/059927 PCT/US2018/059927 77/78

B78 B78 Primary Primary Tumor Tumor 1000 1000

12 12 Gy Gy ++ 50 50 uCi µCi

12 12 Gy Gy + + CTLA4 CTLA4

50 50 uCi µCi ++ CTLA4 CTLA4 800 800 Tumor Volume (mm³)

12 Gy + 50 uCi + CTLA4** 12 Gy + 50 µCi + CTLA4**

600 * -

- 400 *

200 **

0 C

0 5 10 15 15 20 25 30

Days Days

Figure Figure 61 61

SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26)

PCT/US2018/059927

78/78

B78 B78 Distant Distant Tumor Tumor (No (No EBRT) EBRT) 1000 1000

12 12 Gy Gy + + 50 50 uCi µCi

12 Gy + CTLA4

800 800 50 uCi µCi + CTLA4 Tumor Volume (mm³)

12 12 Gy Gy + + 50 50 uCi µCi + + CTLA4** CTLA4**

600

400 1888

200

0 0 0 5 10 15 15 20 25 30

Days Days

Figure 62

SUBSTITUTE SUBSTITUTE SHEET SHEET (RULE (RULE 26) 26)