Use of targeted radiation therapy (TRT) to drive anti-tumor immune responses to immunotherapy

Abstract

The disclosed method of treating a malignant solid tumor in a subject comprises the steps of administering to the subject an immunomodulatory dose of a radiophosphatide ether metal chelate, a radiohalogenated phosphatide ether, or other targeted radiotherapy (TRT) agent differentially retained in the malignant solid tumor, and treating the malignant solid tumor by systemically administering an immunostimulatory agent to the subject. Immunotherapy is performed in a subject, such as an immune checkpoint inhibitor. In a non-limiting example, a radioactive phospholipid ether metal chelate or radioactive halogenated phospholipid ether has the formula: # imgabs0 # wherein R1 comprises a chelating agent that chelates with a metal atom wherein the metal atom is an alpha, beta or Auger emitting metal isotope having a half-life of greater than 6 hours and less than 30 days, or wherein R1 comprises a radioactive halogen isotope. In one such embodiment, a is 1, n is 18, m is 0, b is 1, and R2 is-N + (CH3) 3.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 15 / 809,427, filed November 10, 2017, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federal Funding

[0004] Research or development

[0005] This invention was made with government support from the National Institutes of Health under Grants OD024576 and CA197078. The government has certain rights in this invention. Technical Field

[0006] The present disclosure generally relates to methods for treating cancer. Specifically, the present disclosure relates to methods for treating cancer, including 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 radioactive halogenated compound, a radiolabeled antibody, or a radioisotope that is differentially taken up by and retained in solid tumor tissue; and (2) systemically administering to the subject one or more immunostimulants, such as one or more immune checkpoint inhibitors. Background Art

[0007] Current cancer treatments typically involve systemic chemotherapy, in which non-targeted small molecules or antibody-mediated cytotoxic agents preferentially enter or bind (in the case of antibody-mediated agents) to cancer cells and kill them through a variety of mechanisms. External beam radiation therapy (xRT) is often combined with chemotherapy to kill cancer cells by inducing nuclear DNA double-strand breaks that lead to cell cycle death. Unlike systemic chemotherapy, xRT depends on the ability to accurately determine the anatomical location of the tumor. Surgical resection of the tumor also depends on the ability to visualize the tumor and completely resect it, as residual tumor cells will quickly repopulate the tumor after surgery. Surgery and xRT are typically limited to local treatment of malignancies and are therefore limited in treating disseminated or metastatic disease, which is why chemotherapy is often used in combination with these treatment modalities. Although systemic chemotherapy is able to reach many distant metastatic sites (with the possible exception of brain metastases), for all so many patients, responses are often short-lived (months to years) and ultimately lead to tumor recurrence.

[0008] Because the body's innate immune system is also capable of destroying cancer cells upon recognition, immune approaches are becoming increasingly popular in cancer treatment models. However, some cancer cells, and to a greater extent cancer stem cells, manage to initially evade immune surveillance and actually gain the ability to evolve and ultimately survive by remaining relatively immune invisible [Gaipi et al., Immunotherapy 6:597-610, 2014].

[0009] One specific immunological approach that is being increasingly investigated is "in situ vaccination," a strategy that seeks to enhance tumor immunogenicity, generate tumor-infiltrating lymphocytes (TILs), and drive systemic antitumor immune responses against "unvaccinated" disseminated tumors. In in situ vaccination, malignant solid tumors are injected (or treated) with one or more substances that promote the release of tumor antigens while simultaneously providing proinflammatory signals to reverse the tumor's immune-tolerant microenvironment [Pierce et al., Human Vaccines & Immunotherapoeutics 11(8):1901-1909, 2015; Marabelle et al., Clin. Cancer Res. 20(7):1747-56, 2014; Morris et al., Cancer Res; 76(13);3929–41, 2016].

[0010] A second, entirely different approach is systemically administered immunotherapy. In systemically administered immunotherapy, an immune stimulatory agent (such as an immune checkpoint inhibitor) is administered to circulate throughout the body (e.g., intravenously) rather than being injected locally into a tumor. Such agents can be used to treat tumors in which an anti-tumor immune response exists but has been “exhausted” or rendered ineffective. In the case of checkpoint inhibitors, tumor cells express “checkpoint ligands” or other checkpoint molecules that interact with “checkpoint receptors” on existing anti-tumor immune cells, triggering the inactivation of those cells. By blocking this interaction, systemically administered checkpoint inhibitors can turn on a cancer patient’s exhausted, pre-existing immune response, thereby promoting a more effective attack on cancer cells by the patient’s own immune system.

[0011] While recent data from clinical trials and preclinical models illustrate the potential of these approaches, there is a pressing need in the art for systemically administered immunotherapeutic approaches with improved systemic efficacy.

[0012] Irradiation 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]. When stimulated, it is hypothesized that the backup repair mechanisms are sufficiently effective not only to eliminate the deleterious effects of ionizing RT but also to suppress disease unrelated to RT exposure. Perhaps related, abscopal effect is a phenomenon reported in the 1950s in which xRT treatment of one tumor actually results in the shrinkage of another tumor outside the RT treatment area. Although rare, this phenomenon is thought to depend on the activation of the immune system. Together, hormesis and abscopal effect support the potential interaction and stimulation of the immune system by low dose (immunostimulatory but non-cytotoxic) RT, which can then be combined with other immunological approaches (e.g., systemically administered immunotherapy).

[0013] We have previously reported that the combination of local xRT plus orthotopic vaccination and / or systemic checkpoint inhibitor immunotherapy is synergistically effective in treating large tumors in mice when a single tumor is present [Morris et al., Cancer Res; 76(13); 3929–41, 2016]. However, we were surprised to find that the combination of orthotopic vaccination and xRT did not result in suppressed tumor growth in the presence of a second non-irradiated tumor. Apparently, the non-irradiated tumor exhibited a suppressive effect on the immunomodulatory effects of xRT and orthotopic vaccination on the irradiated tumor (which we termed “concomitant immune tolerance”).

[0014] When xRT is administered to all regions of the tumor, this concomitant immune tolerance can be overcome, thereby achieving the efficacy of in situ vaccination. However, in the presence of multiple tumors, xRT cannot be effectively combined with an in situ vaccination approach, particularly if the number of tumors is not small, if the location of one or more tumors is not precisely known, or if delivering xRT to all tumor sites is not feasible. Furthermore, administering xRT to all tumor sites in patients with metastatic disease may result in systemic immunosuppression, thereby defeating the primary purpose of systemically administered immunotherapy.

[0015] Therefore, there is a need for improved methods of delivering immunomodulatory doses of RT to all tumors in a subject, regardless of their number and anatomical location, in conjunction with systemically administered immunotherapy. Summary of the Invention

[0016] 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 (the entire contents of which are incorporated herein by reference), Weichert et al. disclose the use of the basic compound 18-(p-iodophenyl)octadecylphosphocholine (NM404; see Figure 1 ) analogs to detect, locate, and treat various solid malignancies. If the iodine moiety is an imaging-optimized radionuclide, such as iodine-124 ([ 124 I]-NM404), the analog can be used for positron emission tomography-computed tomography (PET / CT) or single photon emission computed tomography (SPECT) imaging of solid tumors. Alternatively, if the iodine moiety is a radionuclide optimized for delivering therapeutic doses of RT to solid tumor cells that take up the analog, such as iodine-125 or iodine-131 ([ 125 I]-NM404 or [ 131 I]-NM404), which can be used to treat solid tumors.

[0017] This analog not only targets multiple solid tumor types in vivo but also experiences prolonged selective retention in tumor cells, thus offering high potential as a RT agent. Furthermore, tumor uptake is limited to malignant cancers, rather than premalignant or benign lesions.

[0018] However, there are metal isotopes that have better properties for optimized imaging and / or RT than the radioiodine isotopes used in previously disclosed alkylphosphocholine analogs. For example, I-124 as an imaging isotope suffers from poor positron output (only about 24% of the emissions are positrons), and it is also subject to confounding gamma emissions (600 KeV) that actually interfere with normal 511 KeV PET detection. Certain positron-emitting metals have better imaging properties. As another example, I-131 as an RT isotope produces other non-therapeutic emissions at other energies, which increase the undesirable radiation dose to adjacent normal tissues (including bone marrow). The beta particle range of I-131 is also very long, which leads to off-target toxicity. Several metallic radiotherapy isotopes can provide sharper emission curves and shorter optical paths, and therefore are less potentially toxic.

[0019] We have developed improved alkylphosphocholine analogs that include chelated radioactive metal isotopes rather than radioactive iodine isotopes (see, e.g., U.S. Patent Publication No. 2018 / 0022768, the entire contents of which are incorporated herein by reference). The analogs include the same backbone as previously disclosed radioiodinated compounds, so they are still selectively taken up and retained in tumor cells. However, the chelated radiometal isotope provides improved emission for imaging and / or radiotherapy applications. This class of materials is well suited for delivering subcytotoxic but immunomodulatory doses of ionized RT to all malignancies present in a subject, regardless of whether their quantity and location are known.

[0020] Thus, in a first aspect, the present invention encompasses a method of treating cancer comprising one or more solid malignant tumors in a subject. The method comprises the steps of systemically administering to the subject (a) an immunomodulatory dose of a targeted radiation therapy (TRT) agent that is differentially taken up by and retained in the solid malignant tumor tissue; and (b) one or more immunostimulatory agents.

[0021] In some embodiments, the one or more immunostimulatory agents are immune checkpoint inhibitors that can target one or more checkpoint molecules.

[0022] Non-limiting examples of one or more immune checkpoint inhibitors include agents that can target one or more of the following checkpoint 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 molecule), ICOSL (inducible T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4 (VTCN1; V-set domain-containing inhibitor of T-cell activation 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 inhibitor of T-cell activation).

[0023] In some embodiments, the one or more immune checkpoint inhibitors comprise one or more anti-immune checkpoint molecule antibodies. In some such embodiments, the one or more anti-immune checkpoint molecule antibodies comprise at least one monoclonal antibody.

[0024] In some embodiments, the one or more immune checkpoint inhibitors include one or more small molecules that can inhibit or block one or more immune checkpoint molecules. Non-limiting examples of such small molecule checkpoint inhibitors include CA-170 and CA-327, which target PD-L1.

[0025] In some embodiments, the one or more anti-immune checkpoint molecule antibodies include: anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG3 antibody, anti-KIR antibody, anti-A2AR antibody and anti-BTLA antibody, anti-CD40 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-OX40L antibody, anti-GITR antibody, anti-GITRL antibody, anti-ICOS antibody, anti-ICOSL antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-IDO antibody, anti-TIM-3 antibody, anti-Gal-9 antibody, or anti-VISTA antibody.

[0026] In some embodiments, the TRT agent is meta-iodobenzylguanidine (MIBG), wherein the iodine atom in MIBG is a radioactive iodine isotope.

[0027] In some embodiments, the TRT agent is a radiolabeled tumor-targeting antibody.

[0028] In some embodiments, the TRT agent is a radioactive isotope of radium, such as Ra-223.

[0029] In some embodiments, the TRT agent is a radioactive phospholipid ether metal chelate or a salt thereof having the formula:

[0030]

[0031] R1 comprises a chelating agent chelated to a metal atom, wherein the metal atom is an α, β or Auger emitting metal isotope with a half-life 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, wherein X is an alkyl or aryl group; R2 is -N + H3, -N + H2Z,-N + HZ2 or -N +Z3, wherein each Z is independently alkyl or aroalkyl; and b is 1 or 2. Non-limiting examples of metal isotopes that can 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.

[0032] In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one of its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or one of its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or one of its derivatives; 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-diacetic acid (NODAGA) or one of its derivatives; 1,4,7,10-tetraazacyclodecane, 1-pentanedioic acid-4,7,10-triacetic acid (DOTAG A) or one of its derivatives; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or one of its derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) or one of its derivatives; diethylenetriaminepentaacetic acid (DTPA), its diester, or one of its derivatives; 2-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A"-DTPA) or one of its derivatives; deferoxamine (DFO) or one of its derivatives; 1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H2dedpa) or one of its derivatives; and DADA or one of its derivatives, wherein DADA comprises the structure:

[0033]

[0034] In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In some embodiments, a is 0 (aliphatic alkyl chain).

[0035] 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 -OX.

[0036] In some embodiments, X is -CH2CH3 or -CH3.

[0037] In some embodiments, m is 0 (alkyl phospholipid series).

[0038] In some embodiments, b is 1.

[0039] In some embodiments, n is 18.

[0040] In some embodiments, R2 is -N + Z3. In some embodiments, each Z is independently -CH2CH3 or -CH3. In some such embodiments, each Z is -CH3.

[0041] In some embodiments, the chelating agent that chelates metal atoms is:

[0042]

[0043]

[0044]

[0045]

[0046] In some embodiments, the radioactive phospholipid ether metal chelate is one of the following compounds, wherein the selected compound chelates the metal atom:

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] 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 + (CH3)3. In some such embodiments, the phospholipid ether metal chelate is NM600 chelated to a metal atom, such as (but not limited to) 90 Y-NM600.

[0054] In some embodiments, the TRT agent is a radioactive halogenated phospholipid ether or a salt thereof having the formula:

[0055]

[0056] R1 includes a radioactive halogen isotope; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is selected from -H, -OH, -COOH, -COOX, -OX and -OCOX, wherein X is an alkyl group or an arylalkyl group; R2 is selected from -N + H3, -N + H2Z, -N + HZ2 and -N + Z3, wherein each Z is independently alkyl or aryl.

[0057] In some embodiments, the radiohalogen isotope is 123 I. 124 I. 125 I. 131 I. 211 At 76 Br or 77 Br.

[0058] In some embodiments, a is 1 and m is 0.

[0059] In some embodiments, n is 18.

[0060] In some embodiments, R2 is -N + (CH3)3. In some such embodiments, a is 1, m is 0, and n is 18. In some such embodiments, the radiohalogen isotope is 123 I. 124 I. 125 I, or 131 I (radioactive halogenated phospholipid ether is [ 123 I]-NM404, [ 124 I]-NM404, [ 125 I]-NM404, [ 131 I]-NM404, [ 211 At]-NM404, [ 76 Br]-NM404, or [ 77 Br]-NM404).

[0061] In some embodiments, the TRT agent, the immunostimulatory agent, or both are administered intravenously.

[0062] In some embodiments, the subject is a human.

[0063] Non-limiting examples of cancers presenting as malignant solid tumors that can be treated using the methods include melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma of the skin or head or neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms' tumor, or a pediatric brain tumor.

[0064] In some embodiments, cancer is treated without administering to the subject an antibody against a tumor antigen that is not a checkpoint molecule.

[0065] In some embodiments, the subject is not administered an anti-GD2 antibody.

[0066] In a second aspect, the present disclosure includes the use of a TRT agent and one or more immunostimulatory agents for treating cancer, including one or more malignant solid tumors, in a subject.

[0067] In a third aspect, the present disclosure includes the use of a TRT agent and / or one or more immunostimulants in the preparation of a medicament for treating cancer, including one or more malignant solid tumors, in a subject, as described above.

[0068] Other objects, features and advantages of the present invention will become apparent from the following detailed description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] This 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.

[0070] Figure 1 The chemical structure of the base compound 18-(p-iodophenyl)octadecylphosphocholine (NM404) is shown.

[0071] Figure 2A is a graph showing that xRT+IT-IC triggers in situ tumor vaccination. More specifically, Figure 2A Tumor growth curves are shown, demonstrating synergy between xRT and IT-hu14.18-IL2. 71% (22 / 31) of mice treated with xRT+IT-IC became disease-free.

[0072] Figure 2B is another image showing xRT+IT-IC triggering orthotopic tumor inoculation. More specifically, Figure 2BKaplan-Meier survival curves are shown, demonstrating synergy between xRT and IT-hu14.18-IL2. 71% (22 / 31) of mice treated with xRT+IT-IC became disease-free.

[0073] Figure 2C is another image showing xRT+IT-IC triggering orthotopic tumor inoculation. More specifically, Figure 2C showed that 90% of the treated mice rejected subsequently implanted B78 melanoma tumors.

[0074] Figure 3 Figure 2 is a graph showing concomitant immune tolerance. Figure 2 shows primary tumor response. Distant untreated tumors in a dual-tumor B78 melanoma model suppress responses to xRT+IT-IC, and this suppression can be overcome by irradiating the second tumor.

[0075] Figure 4 Figure 2 shows that concomitant immune tolerance is induced by Tregs. Primary tumor responses are shown. Distant untreated tumors in a dual-tumor B78 melanoma model suppressed responses to xRT + IT-IC, and this suppression could be overcome by depletion of Tregs (using transgenic DEREG mice expressing diphtheria toxin receptors on their Tregs, thereby depleting Tregs by administering diphtheria toxin).

[0076] Figure 5 B78 is shown to be selectively taken up by melanoma 124 Image of I-NM404. Carrying about 200mm 3 Mice with B78 tumors received IV 124 I-NM404 and serial PET / CT scans were performed. This image at 71 hours shows selective uptake in the tumor with some residual background uptake in the heart and liver.

[0077] Figure 6 Figure 1 demonstrates that in situ vaccination can be initiated in the presence of residual levels of molecularly targeted radiotherapy (TRT). 131 In the case of I-NM404, treatment with the combined xRT+IT-IC was equally effective. This approximates the residual activity of TRT that would be present if we delivered xRT (d0) followed by IT-IC (d6-10), as described in Example 4.

[0078] Figure 7 Shown is a time course of MRI images of tumor-bearing mice after injection of Gd-NM600, showing tumor enlargement at 24 hours (T).

[0079] Figure 8A Figure 3: Tumor-specific inhibition of primary tumor response to a combination of local RT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models. C57BL / 6 mice bearing syngeneic primary ipsilateral tumors expressing disialoganglioside (GD2+) + / - contralateral secondary tumors were treated with primary tumors only, as indicated, with xRT on day 1 and intratumoral (IT) injections of 50 mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of an anti-GD2 mAb and IL2), on days 6-10. Mean primary tumor volumes are shown in Table 3. Figure 8A More specifically, Figure 8A We show that in mice bearing primary B78 melanoma tumors, the presence of untreated secondary B78 tumors antagonizes the primary tumor's response to RT+IT-IC. We describe this effect as "concomitant immune tolerance"—antagonism of the local response of treated tumors to xRT+IT-IC by untreated distant tumors.

[0080] Figure 8B Another figure showing tumor-specific inhibition of primary tumor response to a combination of local RT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models. C57BL / 6 mice bearing syngeneic primary ipsilateral tumors expressing disialoganglioside (GD2+) + / - contralateral secondary tumors were treated with only the primary tumor, as indicated, with xRT on day 1 and intratumoral (IT) injections of 50mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of an anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8B Kaplan-Meier survival curves of mice plus replicates are shown. Almost all mice were euthanized due to primary tumor progression.

[0081] Figure 8CAnother figure shows tumor-specific inhibition of primary tumor response to a combination of local RT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models. C57BL / 6 mice bearing a homologous primary ipsilateral tumor expressing disialoganglioside (GD2+) + / - contralateral secondary tumor were treated with only the primary tumor, as indicated, with xRT on day 1 and intratumoral (IT) injection of 50mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of an anti-GD2 mAb and IL2), on days 6-10. More specifically, in mice bearing primary Panc02-GD2+ pancreatic tumors with or without secondary Panc02-GD2- tumors on the contralateral flank, the presence of untreated Panc02 secondary tumors inhibited the response of the primary Panc02-GD2+ tumor to RT + IT-IC.

[0082] Figure 8D Another figure showing tumor-specific inhibition of primary tumor response to a combination of local RT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models. C57BL / 6 mice bearing syngeneic primary ipsilateral tumors expressing disialoganglioside (GD2+) + / - contralateral secondary tumors were treated with only the primary tumor, as indicated, with xRT on day 1 and intratumoral (IT) injections of 50mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of an anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8D We show that in mice bearing primary B78 melanoma tumors, secondary B78 tumors, but not secondary Panc02-GD2+ pancreatic tumors, inhibited the response of the primary tumor to RT+IT-IC.

[0083] Figure 8E Another figure showing tumor-specific inhibition of primary tumor response to a combination of local RT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models. C57BL / 6 mice bearing syngeneic primary ipsilateral tumors expressing disialoganglioside (GD2+) + / - contralateral secondary tumors were treated with only the primary tumor, as indicated, with xRT on day 1 and intratumoral (IT) injections of 50mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of an anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8EFigure 3. In mice bearing primary Panc02-GD2+ tumors, secondary Panc02-GD2- tumors suppressed the primary tumor's response to combined xRT and IT-hu14.18-IL2, whereas B78 secondary tumors did not. n = number of mice per group. NS = not significant, ***p < 0.001.

[0084] Figure 9A Included are immunohistochemical images (left and center) and diagrams (right) showing that concomitant immune tolerance is circumvented by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9A Shown in a Figure 9A , the leftmost A1 and A2) or two ( Figure 9A Immunohistochemistry of Treg marker FoxP3 in tumors assessed on day 6 after xRT in mice bearing tumors (A3 and A4 in the middle) (representative 400x images are shown). Mice did not receive xRT or received xRT to the primary tumor only. Primary tumors are shown in Figure 9A In A1-A3, secondary tumors are shown in Figure 9A Small arrows point out some FoxP3+ cells (brown nuclei = FoxP3+, blue = hematoxylin counterstain). The graph shown on the right is a blind quantification of FoxP3+ cells per 200x field, corresponding to Figure 9A The conditions shown in A1, A2, A3 and A4.

[0085] Figure 9B Another figure showing that concomitant immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9B We show that DEREG mice express the diphtheria toxin receptor under the control of the Treg-specific FoxP3 promoter, enabling specific Treg depletion upon IP injection of diphtheria toxin. DEREG mice bearing primary and secondary B78 melanoma tumors were treated with xRT+IT-IC for the primary tumor and IP injected with diphtheria toxin or PBS (first replicate shown). Depletion of Tregs in these mice abolished concomitant immune tolerance, resulting in improved ( Figure 9B ) Primary tumor response. n = number of mice per group. **p < 0.01, ***p < 0.001.

[0086] Figure 9C Another diagram showing the avoidance of concomitant immune tolerance by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9CWe show that DEREG mice express the diphtheria toxin receptor under the 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 diphtheria toxin or PBS (first replicate shown). Depletion of Tregs in these mice abolished concomitant immune tolerance, resulting in improved ( Figure 9C ) Secondary tumor response. n = number of mice per group. **p < 0.01, ***p < 0.001.

[0087] Figure 10A Graph showing overcoming of concomitant immune tolerance by delivering xRT to two tumor sites. In mice bearing primary and secondary B78 tumors, the secondary tumor suppressed the primary tumor's response to treatment with xRT + IT-IC. This could be overcome by delivering 12 Gy xRT to both the primary and secondary tumors, along with IT-IC to the primary tumor, resulting in improved ( Figure 10A ) Primary tumor response (first replicate shown). n = number of mice per group. **p<0.01, ***p<0.001.

[0088] Figure 10B Another figure showing overcoming concomitant immune tolerance by delivering xRT to two tumor sites. In mice bearing primary and secondary B78 tumors, the secondary tumor suppressed the primary tumor's response to treatment with xRT + IT-IC. This could be overcome by delivering 12 Gy xRT to both the primary and secondary tumors, along with IT-IC to the primary tumor, resulting in improved ( Figure 10B ) enhances animal survival. n = number of mice per group. **p < 0.01, ***p < 0.001.

[0089] Figure 11A showed that low-dose xRT alone did not elicit in situ vaccination, but did overcome concomitant immune tolerance when delivered to distant tumor sites along with 12 Gy+IT-IC treatment at the in situ vaccine site. More specifically, Figure 11A It was shown that in mice bearing only primary B78 tumors, 12 Gy + IT-IC triggered in situ vaccination (as previously shown) and resulted in complete tumor regression and memory immune responses in the majority of mice (4 / 6 in this experiment) (Morris, Cancer Res, 2016). On the other hand, after IT-IC alone or low-dose (2 Gy) xRT + IT-IC, no animals showed complete tumor regression (0 / 6 in both groups) (p < 0.05).

[0090] Figure 11B Another picture shows that low-dose xRT alone does not elicit in situ vaccination, but does overcome concomitant immune tolerance when delivered to distant tumor sites along with 12Gy+IT-IC treatment at the in situ vaccine site. More specifically, Figure 11B showed that in mice bearing primary and secondary B78 melanoma tumors, low-dose xRT (2 Gy or 5 Gy) delivered to the secondary tumors was equivalent to 12 Gy in its ability to overcome concomitant immune tolerance in the primary tumor.

[0091] Figure 11C Another picture shows that low-dose xRT alone does not elicit in situ vaccination, but does overcome concomitant immune tolerance when delivered to distant tumor sites along with 12Gy+IT-IC treatment at the in situ vaccine site. More specifically, Figure 11C They showed that in these same animals, systemic responses to IT-IC immunotherapy were apparently rescued by overcoming concomitant immune tolerance through the delivery of low-dose xRT to secondary tumors. In this case, when xRT was delivered to all tumor sites, subsequent IT-IC injection of the primary tumor elicited a systemic antitumor effect, such that the response of secondary tumors to 2 Gy or 5 Gy was greater than the response to 12 Gy xRT in the absence of IT-IC injection of the primary tumor.

[0092] Figure 12A It is a PET image showing the 131 Low-dose TRT of I-NM404 effectively depletes tumor-infiltrating FoxP3+ Tregs without systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical situations, it is not feasible to deliver an external beam (even a low dose) to all tumor sites without causing significant bone marrow depletion and leukopenia leading to immunosuppression. Here, we tested whether TRT can be given systemically to specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without triggering systemic immune cell depletion and leukopenia. More specifically, Figure 12A Display using positron emission 124 I-NM404 was used in a dosimetry study in a B78 melanoma tumor model to demonstrate tumor-selective uptake of NM404. C57BL / 6 mice bearing B78 tumors were treated with 60 μCi 131 I-NM404 treatment. This activity is similar to that required to deliver approximately 2 Gy TRT to B78 tumors 131 Amount of I-NM404. Peripheral blood and tumor samples were collected from untreated control mice (labeled "C") and thereafter at 8-day intervals (T1 = d8, T2 = d16, T3 = d24, T4 = d32).

[0093] Figure 12B Is a bar chart, showing the 131 Low-dose TRT of I-NM404 effectively depletes tumor-infiltrating FoxP3+ Tregs without systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical situations, it is not feasible to deliver an external beam (even a low dose) to all tumor sites without causing significant bone marrow depletion and leukopenia leading to immunosuppression. Here, we tested whether TRT can be given systemically to specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without triggering systemic immune cell depletion and leukopenia. More specifically, Figure 12B This dose of TRT was shown not to cause any significant systemic leukopenia.

[0094] Figure 12C is another bar chart showing the 131 Low-dose TRT of I-NM404 effectively depletes tumor-infiltrating FoxP3+ Tregs without systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical situations, it is not feasible to deliver an external beam (even a low dose) to all tumor sites without causing significant bone marrow depletion and leukopenia leading to immunosuppression. Here, we tested whether TRT can be given systemically to specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without triggering systemic immune cell depletion and leukopenia. More specifically, Figure 12C This dose of TRT was shown to not significantly affect the levels of tumor-infiltrating CD8+ effector T cells (ANOVA p=0.25).

[0095] Figure 12D is another bar chart showing the 131 Low-dose TRT of I-NM404 effectively depletes tumor-infiltrating FoxP3+ Tregs without systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical situations, it is not feasible to deliver an external beam (even a low dose) to all tumor sites without causing significant bone marrow depletion and leukopenia leading to immunosuppression. Here, we tested whether TRT can be given systemically to specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without triggering systemic immune cell depletion and leukopenia. More specifically, Figure 12D This dose of TRT was shown to significantly deplete tumor-infiltrating FoxP3+ Tregs (ANOVA p=0.03; *p<0.05).

[0096] Figure 13A Display using 131Low-dose TRT of I-NM404 effectively overcame concomitant immune tolerance and rescued the systemic antitumor effects of in situ vaccination. 131 We tested the ability of I-NM404 TRT to deplete tumor-infiltrating Tregs without leukopenia in mice. 131 Can I-NM404 effectively overcome concomitant immune tolerance? As shown, 60-mcCi 131 C57BL / 6 mice bearing two B78 tumors were treated with I-NM404 (NM404). After one half-life (day 8), animals received 12 Gy xRT to the primary tumor (orthotopic vaccine site) or no xRT. 131 Secondary tumors in control mice treated with I-NM404 (0, 2, or 12 Gy). Mice received daily IT injections of IC to the primary tumor (in situ vaccine site) on days 13-17 as indicated. More specifically, Figure 13A Primary tumor responses were demonstrated, demonstrating that administration of low-dose TRT effectively overcomes concomitant immune tolerance and rescues the systemic antitumor effects of in situ vaccination.

[0097] Figure 13B Display using 131 Another picture is that low-dose TRT of I-NM404 effectively overcomes concomitant immune tolerance and rescues the systemic antitumor effect of in situ vaccination. 131 We tested the ability of I-NM404 TRT to deplete tumor-infiltrating Tregs without leukopenia in mice. 131 Can I-NM404 effectively overcome concomitant immune tolerance? Considering the low dose 131 We tested the ability of I-NM404 TRT to deplete tumor-infiltrating Tregs without leukopenia in mice. 131 Can I-NM404 effectively overcome concomitant immune tolerance? After one half-life (day 8), animals received 12 Gy xRT to the primary tumor (in situ vaccine site) or no xRT. 131 Secondary tumors in control mice treated with I-NM404 (0, 2, or 12 Gy). Mice received daily IT injections of IC to the primary tumor (in situ vaccine site) on days 13-17 as indicated. More specifically, Figure 13B Secondary tumor responses were demonstrated, demonstrating that administration of low-dose TRT effectively overcame concomitant immune tolerance and rescued the systemic antitumor effects of in situ vaccination.

[0098] Figure 14 Exemplary alkylphosphocholine metal chelates are shown ( 64 Chemical structure of Cu-NM600). Other metals can be used instead 64 Cu.

[0099] Figure 15 It is for personal use 86 PET / CT images of two single-tumor B78 mice scanned 48 hours after Y-NM600 injection.

[0100] Figure 16 It is for personal use 86 PET / CT images of two B78 mice with two tumors scanned 48 hours after Y-NM600 injection.

[0101] Figure 17 Including from 64 PET / CT images of U87MG mice scanned 3 hours (left), 24 hours (middle), and 48 hours (right) after Cu-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (% ID / g, with the ratio shown on the far right).

[0102] Figure 18 Including from 64 PET / CT images of 4T1 mice scanned 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure) after Cu-NM600 injection. These images show the tissue activity calculated as the percentage of injected dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0103] Figure 19 Including from 64 PET / CT images of HCT-116 mice scanned after Cu-NM600 injection 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure). These images show the tissue activity calculated as the percentage of injected dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0104] Figure 20 Including from 64 PET / CT images of A549 mice scanned after Cu-NM600 injection 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure). These images show the tissue activity calculated as the percentage of injected dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0105] Figure 21 Including from 64PET / CT images of PC-3 mice scanned 3 hours (left panel), 24 hours (middle panel) and 48 hours (right panel) after Cu-NM600 injection. These images show the tissue activity calculated as the percentage of injected dose / g tissue (%ID / g, ratio is presented on the far right).

[0106] Figure 22 Including from 64 Cu-NM600 is injected after 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure) scanning HT-29 mice PET / CT image.These images show the tissue activity calculated with the percentage of injection dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0107] Figure 23 Including from 64 Cu-NM600 is injected after 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure) scanning MiaPaca mouse PET / CT image.These images show the tissue activity calculated with the percentage of injection dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0108] Figure 24 Including from 86 PET / CT images of 4T1 mice scanned 3 hours (left), 24 hours (middle) and 48 hours (right) after Y-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (% ID / g, ratio is shown on the far right).

[0109] Figure 25 Including from 89 PET / CT images of 4T1 mice scanned 3 hours (left figure), 24 hours (middle figure) and 48 hours (right figure) after Zr-NM600 injection.These images show the tissue activity calculated with the percentage of injected dose / g tissue (%ID / g, ratio is presented at the rightmost).

[0110] Figure 26 Including from 52 PET / CT images of HT-29 mice scanned 4 hours (left) and 1 day (right) after Mn-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (% ID / g, with the ratio shown on the far right).

[0111] Figure 27 Including from 52PET / CT images of PC-3 mice scanned 4 hours (left panel) and 1 day (right panel) after Mn-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (% ID / g, with the ratio shown on the right side of each image).

[0112] Figure 28 Including from 52 PET / CT images of HT-29 mice scanned 2 days (left panel), 3 days (second panel on the left), 5 days (second panel on the right) and 7 days (right panel) after Mn-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (% ID / g, ratio is shown on the right side of the image).

[0113] Figure 29 Including from 52 PET / CT images of PC-3 mice scanned 2 days (left panel), 3 days (second panel on the left), 5 days (second panel on the right), and 7 days (right panel) after Mn-NM600 injection. These images show tissue activity calculated as a percentage of injected dose / g tissue (%ID / g, ratio displayed on the right side of the image).

[0114] Figure 30 It is used 86 Y-NM600, 64 Cu-NM600 and 89 Graph of PET quantification region of interest data (chelate uptake versus time) for 4T1 tumor tissue in Zr-NM-600 injected 4T1 mice.

[0115] Figure 31 It is used 86 Y-NM600, 64 Cu-NM600 and 89 Graph of data (chelate uptake versus time) for PET quantification of region of interest in cardiac tissue from Zr-NM-600 injected 4T1 mice.

[0116] Figure 32 It is used 86 Y-NM600, 64 Cu-NM600 and 89 Graph of data (chelate uptake versus time) for PET quantification of region of interest in liver tissue from Zr-NM-600 injected 4T1 mice.

[0117] Figure 33 It is used 86 Y-NM600, 64 Cu-NM600 and 89Graph of whole body region of interest PET quantitative data (chelate uptake versus time) in Zr-NM-600 injected 4T1 mice.

[0118] Figure 34 is to describe the 48 hours after metal chelate injection ( 86 Y-NM600, 64 Cu-NM600, 89 Zr-NM-600 and 177 Lu-NM600) and 96 hours ( 177 Bar graph of ex vivo chelate biodistribution in healthy and tumor tissues in 4T1 mice harboring Lu-NM600).

[0119] Figure 35 Exemplary alkylphosphocholine metal chelates are shown ( 177 Chemical structure of Lu-NM600). Other metals can be used instead 177 Lu.

[0120] Figure 36 It is used 90 Autoradiographic images of three B78 mice acquired 48 hours after Y-NM600 injection. Xenografted B78 tumors are shown as large dark spots located in the lower right portion of each mouse image.

[0121] Figure 37 It is used 90 Autoradiographic images of three B78 mice acquired 96 hours after Y-NM600 injection. Xenografted B78 tumors are shown as large dark spots located in the lower right portion of each mouse image.

[0122] Figure 38 It is used 177 Autoradiographic images of a B78 mouse taken 5 days after Lu-NM600 injection. Xenografted B78 tumors are shown as two dark spots at the bottom of the mouse image.

[0123] Figure 39 It is used 177 Autoradiographic images of a B78 mouse taken 13 days after Lu-NM600 injection. Xenografted B78 tumors are shown as two dark spots at the bottom of the mouse image.

[0124] Figure 39 It is used 177 Autoradiographic images of a B78 mouse taken 13 days after Lu-NM600 injection. Xenografted B78 tumors are shown as two dark spots at the bottom of the mouse image.

[0125] Figure 40 It is used 177Autoradiograph of a MiaPaca mouse acquired 10 days after Lu-NM600 injection. The location of the xenografted MiaPaca tumor is indicated by the arrow and dashed circle.

[0126] Figure 41 It is used 177 Autoradiographic images of three 4T1 mice acquired 48 hours after Lu-NM600 injection. The locations of xenografted 4T1 tumors are indicated by arrows and dashed circles.

[0127] Figure 42 It is used 177 Autoradiographic images of three 4T1 mice acquired 96 hours after Lu-NM600 injection. The locations of xenografted 4T1 tumors are indicated by dashed circles.

[0128] Figure 43 It is used 90 Autoradiographic images of three 4T1 mice acquired 4 hours after Y-NM600 injection. The locations of xenografted 4T1 tumors are indicated by arrows and dashed circles.

[0129] Figure 44 It is used 90 Autoradiographic images of three 4T1 mice acquired 48 hours after Y-NM600 injection. Xenografted 4T1 tumors are shown as large dark spots located in the lower right portion of each mouse image.

[0130] Figure 45 It is used 90 Autoradiographic images of three 4T1 mice acquired 96 hours after Y-NM600 injection. Xenografted 4T1 tumors are shown as large dark spots located in the lower right portion of each mouse image.

[0131] Figure 46 The present study illustrates the effect of two different doses (150 μCi and 300 μCi) of dapoxetine on the B78 xenograft mouse model compared to the control (vehicle only). 90 Graph showing the radiotherapy effect of Y-NM600. Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection.

[0132] Figure 47 The present study demonstrates the efficacy of a single 500 μCi dose of 1% β-catenin in a B78 xenograft mouse model compared to a control (vehicle only) 177 Graph showing the radiotherapy effect of Lu-NM600. Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection.

[0133] Figure 48The present study demonstrates the efficacy of a single 400 μCi dose of 1% β-lactamase in a MiaPaca xenograft mouse model compared to a control (vehicle only) 177 Graph showing the radiotherapy effect of Lu-NM600. Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection.

[0134] Figure 49 The present study demonstrates the efficacy of a single 500 μCi dose of 4T1 xenograft in a mouse model compared to a control (vehicle only). 177 Graph showing the radiotherapy effect of Lu-NM600. Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection. *P<0.05; **P<0.01; ***P<0.001.

[0135] Figure 50 The present study demonstrates that two dose series of 1,2-dimethylthiazide (DHT) and 1,2-dimethylthiazide (DHT) compared to control (vehicle only) in a 4T1 xenograft mouse model. 177 Graph showing the radiotherapy effect of Lu-NM600 (500 μCi and 250 μCi). Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection.

[0136] Figure 51 The present study demonstrates the effects of two different doses (500 μCi and 250 μCi) of 4T1 xenograft mice compared to controls (vehicle only). 177 Graph showing the radiotherapy effect of Lu-NM600. Data are expressed as measured tumor volume in mm 3 Relationship with the number of days after injection.

[0137] Figure 52 The effect of tumor mass on conventional TRT is 90 Y-NM600 and 131 Graph showing the comparative therapeutic efficacy of I-NM404.

[0138] Figure 53 This is a bar graph comparing the average albumin binding energies of three different metal chelate analogs and an amine analog of NM404. The binding energy of I-NM404 is shown as a dotted line for comparison.

[0139] Figure 54 Figure 2 shows the tumor volume (mm) of mice with B78 melanoma flank tumors. 3) over time (days) in mice treated with the anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or different doses (25μCi, 50μCi, or 100μCi) of the targeted radiotherapy (TRT) agent Y90-NM600. Control mice were given a vehicle (PBS) without anti-CTLA4 or TRT agents. After 18 days, treatment with anti-CTLA4 combined with 50 or 100μCi Y90-NM600 significantly reduced tumor growth compared to PBS, Y90-NM600 alone, or anti-CTLA4 alone (ANOVA test p<0.05). The group treated with 25μCi Y90-NM600 combined with anti-CTLA-4 showed an intermediate growth delay response, showing a dose-response trend.

[0140] Figure 55 Shown are the overall animal survival rates of mice given a combination of TRT (50 μCi Y90-NM600) and a checkpoint blocker (anti-CTLA4) compared to mice given TRT alone, the checkpoint blocker (anti-CTLA4) alone, or PBS vehicle.

[0141] Figure 56 Shown are the overall animal survival rates of mice given three different TRTs (25 μCi, 50 μCi, and 100 μCi Y90-NM600) in combination with a checkpoint blocker (anti-CTLA4).

[0142] Figure 57 Tumor volume (mm) of mice with B78 melanoma flank tumors 3 ) over time (days) in mice treated with an anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or different doses (25 μCi, 50 μCi, or 100 μCi) of a molecularly targeted radiotherapy (MTRT) agent. 90 The control group of mice was treated with vehicle (PBS) without anti-CTLA4 or MTRT agents. 90 Compared with Y-NM600 or anti-CTLA4 alone, anti-CTLA4 combined with 50 or 100 μCi 90 Y-NM600 treatment significantly reduced tumor growth (ANOVA test p < 0.05).

[0143] Figure 58 Patients who received anti-CTLA4 immune checkpoint inhibitors (CTLA4) and / or different doses (25 μCi, 50 μCi, or 100 μCi) of molecularly targeted radiotherapy (MTRT) agents 90 The overall survival rate of mice bearing B78 melanoma flank tumors treated with Y-NM600 was significantly improved compared with the other groups. 90Y-NM600 or 100μCi 90 The survival rate of mice treated with the combination of anti-CTLA4 (anti-CTLA4) and anti-Y-NM600 was significantly improved.

[0144] Figure 59 Tumor volume (mm) of NXS2 neuroblastoma mice is shown. 3 ) over time (days) in mice treated with an anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi of molecularly targeted radiotherapy (MTRT) 90 Treatment with Y-NM600. The control group of mice was given vehicle (PBS) without anti-CTLA4 or TRT reagents. 90 Compared with Y-NM600 or anti-CTLA4 alone, the combination 90 Y-NM600 MTRT and anti-CTLA4 significantly reduced tumor growth.

[0145] Figure 60 Tumor volume (mm) of mice with 4T1 breast cancer tumors 3 ) over time (days) in mice treated with an anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi of molecularly targeted radiotherapy (MTRT) 90 Treatment with Y-NM600. The control group of mice was given vehicle (PBS) without anti-CTLA4 or TRT reagents. 90 Compared with Y-NM600 or anti-CTLA4 alone, the combination 90 Y-NM600 MTRT and anti-CTLA4 significantly reduced tumor growth.

[0146] Figure 61 Figure 2 shows the change in tumor volume (mm3) as a function of time (days) after irradiation of the primary B78 tumor in mice bearing both primary and secondary (distant) tumors of B78 melanoma flank tumors. Irradiated mice were treated with EBRT to the primary tumor only (12 Gy, with secondary tumors shielded), an anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi of a molecularly targeted radiotherapy (MTRT) agent. 90 The mice were treated with various combinations of Y-NM600. Compared with other groups, the combination of 12Gy EBRT, 90 Y-NM600 MTRT and anti-CTLA4 treatment significantly reduced primary tumor growth.

[0147] Figure 62Figure 3 shows tumor volume (mm) of shielded secondary (distant) B78 tumors in mice bearing both primary and secondary B78 melanoma flank tumors. 3 ) versus time (days). Patients were treated with EBRT (12 Gy, shielded from secondary tumors) directed only at the primary tumor, an anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi of molecularly targeted radiotherapy (MTRT) agents. 90 Mice were treated with various combinations of Y-NM600. Compared with other groups, EBRT to primary tumors, 90 Combination therapy of Y-NM600 MTRT and anti-CTLA4 significantly reduced the growth of secondary tumors. DETAILED DESCRIPTION

[0148] I. Overview

[0149] It should be understood that this disclosure is not limited to the particular methodology, protocols, materials and reagents described, as these may vary. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will be limited only by any later filed non-provisional application.

[0150] As used herein and in the appended claims, the singular "a," "an," and "the" include the plural unless the context clearly indicates otherwise. Likewise, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. The term "including" and its variations do not have a limiting meaning where these terms appear in this specification and claims. Thus, the terms "including," "comprising," and "comprising" are used interchangeably.

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, preferred methods and materials are described herein. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including describing and disclosing the chemical substances, equipment, statistical analyses and methods reported in the publications that can be used in conjunction with the present invention. All references cited in this specification should be considered as an indication of the level of technology in the art.

[0152] The terms herein are only used to describe the embodiments and are not intended to limit the invention as a whole. Unless otherwise specified, "a", "an", "the" and "at least one" are used interchangeably to mean one or more than one.

[0153] The present invention includes any pharmaceutically acceptable form of the compounds described herein (including intermediates), including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, etc. In particular, if the compound is optically active, the present invention specifically includes each enantiomer of the compound and the racemic mixture of such enantiomers. It should be understood that the term "compound" includes any or all such forms, whether or not explicitly stated (although sometimes explicitly stated as a "salt").

[0154] As used herein, "pharmaceutically acceptable" means that the compound or composition or carrier is suitable for administration to a subject to achieve the treatment described herein without undue adverse side effects as required for therapeutic purposes.

[0155] As used herein, the term "effective amount" refers to that amount or dosage of a compound that elicits the biological or medical response of a subject, tissue or cell that is desired by the researcher, veterinarian, medical doctor or other clinician.

[0156] As used herein, the term "pharmaceutically acceptable carrier" includes any and all dry powders, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. A pharmaceutically acceptable carrier is a substance useful for the purpose of administering the compound of the present invention, which is preferably non-toxic and can be a solid, liquid, or gaseous substance that is otherwise inert and pharmaceutically acceptable and compatible with the compound of the present invention. Examples of such carriers include, but are not limited to, various lactoses, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, and detergents such as Tween 80, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrins, and starch.

[0157] As used herein, the term "administering" or "administering" refers to providing a compound or pharmaceutical composition of the present invention to a subject having or at risk for the disease or condition to be treated or prevented.

[0158] In pharmacology, the route of administration is the route by which a drug enters the body. The route of administration can generally be categorized based on where the substance is administered. Common examples may include oral administration and intravenous administration. Routes can also be categorized based on the location of the target of action. The action can be local (topical), enteral (a systemic effect, but delivered via the gastrointestinal tract), or parenteral (a systemic effect, but delivered via a route other than the gastrointestinal tract), such as through the lungs via inhalation.

[0159] In enteral administration, the desired effect is systemic (not local), and the substance is administered through the digestive tract. In parenteral administration, the desired effect is systemic, and the substance is administered by a route other than the digestive tract.

[0160] Enteral administration can be administration involving any part of the gastrointestinal tract and has a systemic effect. Examples include those given orally (orally), many medications in tablets, capsules, or drops, those given through a gastric feeding tube, duodenal feeding tube, or abdominal hole, many medications and enteral nutrition, and those given rectally, various medications in suppositories.

[0161] Examples of parenteral administration may include intravenous (into a vein), for example, many drugs, total parenteral nutrition intraarterial (into an artery), for example, vasodilators for treating vasospasm and thrombolytics for treating embolism, intraosseous infusion (into the bone marrow), intramuscular, intracerebral (into the brain parenchyma), intraventricular (into the ventricular system), intrathecal (injection into the spinal canal), and subcutaneous (under the skin). Among them, intraosseous infusion is effective in indirect venous access because the bone marrow drains directly into the venous system. Intraosseous infusion can occasionally be used for drugs and fluids in emergency medicine and pediatrics when venous access is difficult.

[0162] The following abbreviations are used in this disclosure: ADCC, antibody-dependent cell-mediated cytotoxicity; anti-CTL4, an antibody targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA4), which is found on cytotoxic T lymphocytes (CTLs); B16, a melanoma syngeneic to C57Bl / 6 mice; B78, a B16 variant that expresses GD2 due to transfection with GD2 synthase; D, day; Hu14.18-IL2, a primary immunocytokine (reactive with GD2) used in the studies disclosed in the Examples; IC, immunocytokine (fusion protein of a tumor-reactive mAb linked to IL2); ICI, immune checkpoint inhibitor; IL2, interleukin 2; IT, intratumoral; IV, intravenous; mAb, monoclonal antibody; MAHA, mouse anti-human antibody; NM404, is used to denote Figure 1 The phospholipid ether shown in , which is selectively taken up by most tumors and used for TRT in the studies disclosed in the Examples; NM600, used to represent Figure 14 The phospholipid ethers shown in , which can chelate with any metal and are also selectively taken up by most tumors and TRT used in the studies disclosed in the Examples; NXS2, neuroblastoma syngeneic to AJ mice; Panc02-GD2, pancreatic cancer syngeneic to C57Bl / 6 mice, expressing GD2 due to transfection with GD2 synthase; PLE, phospholipid ether; RT, radiation therapy; TRT, targeted radiation therapy; W, week; 9464D-GD2, neuroblastoma syngeneic to C57Bl / 6 mice, expressing GD2 due to transfection with GD2 synthase.

[0163] II. The present invention

[0164] The present disclosure relates to methods for treating any cancer that presents as one or more malignant solid tumors. The disclosed method combines two processing steps with an unexpected synergistic effect, resulting in greatly improved effects on malignant solid tumors. Specifically, an immunomodulatory dose of a radioactive phospholipid metal chelate compound or a radioactive halogenated phospholipid compound that is differentially taken up by and retained in the malignant solid tumor tissue is administered to the patient, and further immunomodulation is performed by systemic administration (e.g., by IV injection) of a composition comprising one or more agents capable of stimulating specific immune cells, regardless of whether additional xRT treatment is performed on at least one malignant solid tumor that is being treated with an immunostimulatory agent.

[0165] Immunomodulatory doses of radioactive phospholipid metal chelate compounds or radiohalogenated compounds may reduce Treg levels (and other immunosuppressive elements) and prevent the immune system suppression (concomitant immune tolerance) that occurs when xRT is used against tumors and one or more additional tumors are not irradiated, although an understanding of this mechanism is not necessary to practice the invention and the invention is not limited to any particular mechanism of action.

[0166] A. Systemically administered immunotherapy: immune checkpoint inhibitors as exemplary immunostimulants.

[0167] In contrast to immunostimulatory approaches that involve direct administration of immunomodulatory agents into a tumor (e.g., intratumoral immunization via in situ vaccination as shown in some of the examples below), systemic immunotherapy involves systemic administration of immunostimulatory agents. The immunostimulatory agents circulate throughout the subject's body, thereby stimulating the body's natural immune response.

[0168] Immune checkpoint inhibitors are non-limiting examples of such immunostimulatory agents. Activated T cells express a variety of 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 regulate T cell responses to tumor antigens in the tumor microenvironment through unique and non-redundant pathways.

[0169] More specifically, cancer growth is mediated in part by cancer-induced immunosuppression. Tumors can activate inhibitory immune checkpoint pathways to reduce the general immune response to the tumor. Therefore, blockade of key immune checkpoint pathways can induce anti-tumor immunity that is promoted by the patient's own immune system.

[0170] CTLA4 was the first immune checkpoint molecule to be clinically targeted by administering a CTLA4-targeting (anti-CLA4) mAb. To date, the most promising immune checkpoint inhibitor strategies for cancer treatment involve administering mAbs targeting CTLA-4 and / or PD-1 / PD-L1. Other immune checkpoint inhibitor strategies are currently under development, and the disclosed combination approaches are not limited to targeting any specific immune checkpoint pathway.

[0171] A series of reviews on checkpoint inhibitors and cancer immunotherapy were recently published in Immunology Reviews, Volume 276. These reviews, including the introductory overview, Sharpe, AH, “Introduction to checkpoint inhibitors and cancer immunotherapy,” Immunol Rev. 276 (March 4, 2017): 5-8, are hereby incorporated by reference in their entirety.

[0172] B. Immunomodulatory Dosages of Radioactive Phospholipid Metal Chelate Compounds

[0173] The radioactive phospholipid metal chelate compound used should selectively target a wide range of solid tumor cell types, so that the RT emitted by the metal isotope chelated with the metal chelate compound is directed to the malignant solid tumor tissue, while substantially not exposing other tissue types to the emitted RT. The radioactive metal isotope contained in the radioactive phospholipid metal chelate compound can be any radioactive metal isotope that is known to emit ionized RT in the form of immune stimulation of cells that ingest the compound. Non-limiting examples of radioactive metal isotopes that can 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.

[0174] The immunomodulatory RT dose of radioactive phospholipid metal chelate compounds (as opposed to the injected dose) is much smaller than that used for conventional RT therapy for malignant solid tumors. Specifically, the dose should be sufficient to stimulate responses in immune cells within the tumor microenvironment (possibly by reducing immunosuppressive Treg levels and other immunosuppressive cells or molecules) without ablating the desired immune cells responsible for the immunostimulatory effect.

[0175] An appropriate immunomodulatory dose can be calculated from imaging data obtained after administration of a "detection-facilitating" dose of a radioactive metal chelate compound. The detection-facilitating dose can be completely different from the immunomodulatory dose, and the radioactive metal isotope chelated into the radioactive metal chelate compound can be different (although the rest of the compound structure should be the same). The radioactive metal isotope used in the detection step and the radioactivity determination calculation can be any radioactive halogen isotope known to emit RT in a form that is easily detected by conventional imaging means. Non-limiting examples of "conventional imaging means" include: gamma ray detection, PET scanning and SPECT scanning. Non-limiting examples of radioactive metal isotopes that can be 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.

[0176] C. Metal Chelates of PLE Analogs

[0177] The disclosed structure utilizes an alkylphosphocholine (APC) carrier backbone. Once synthesized, the material should have formulation properties that make it suitable for injection while maintaining tumor selectivity, as previously demonstrated with related radiohalogenated compounds. The disclosed structure includes a chelating moiety to which the radiometal isotope will be chelated to produce the final imaging or therapeutic agent.

[0178] D. Methods for Synthesizing Exemplary M-PLE Analogs

[0179] The proposed synthesis of compound 1 is shown below. The first step of the synthesis is similar to that described in Org Synth, 2008, 85, 10-14. The synthesis begins with the conversion of cyclen to a DO3A tris-Bn ester. This intermediate is then coupled with NM404 in the presence of a base and a Pd catalyst.

[0180] Finally, the benzyl protecting group is removed by catalytic hydrogenation.

[0181]

[0182] The synthesis of compound 2 is shown below. It begins with the alkylation of DO3A tris-Bn ester with 3-(bromo-prop-1-ynyl)-trimethylsilane. After alkylation, the trimethylsilyl group is removed, and the intermediate acetylene is coupled with NM404 via a Sonogashira reaction. The benzyl group is removed, and the triple bond is simultaneously hydrogenated in the final step of the synthesis.

[0183]

[0184] Compounds 5 and 6 can be synthesized from the same precursors, DTPA dianhydride and 18-p-(3-hydroxyethyl-phenyl)-octadecylphosphocholine, using the following scheme.

[0185]

[0186] NOTA-NM404 conjugates can be synthesized in a similar manner. An example of NOTA-NM404 conjugate 7:

[0187]

[0188] E. Dosage Form and Method of Administration

[0189] For co-targeted RT, any route of administration may be suitable. In one embodiment, the disclosed alkylphosphocholine analogs can be administered to a subject by intravenous injection. In another embodiment, the disclosed alkylphosphocholine analogs can be administered to a subject by any other suitable systemic delivery method, such as parenteral, intranasal, sublingual, rectal, or transdermal administration.

[0190] In another embodiment, the disclosed alkylphosphocholine analogs can be administered to a subject via the nasal system or by, for example, inhalation into the mouth.

[0191] In another embodiment, the disclosed alkylphosphocholine analogs can be administered to a subject by intraperitoneal injection or IP injection.

[0192] In certain embodiments, the disclosed alkylphosphocholine analogs can be provided as pharmaceutically acceptable salts. However, other salts can also be used to prepare the alkylphosphocholine analogs or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include, but are not limited to, acid addition salts, which can be formed, for example, 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.

[0193] When the alkylphosphocholine analogs have at least one asymmetric center, they may therefore exist as enantiomers. When the disclosed alkylphosphocholine analogs have two or more asymmetric centers, they may additionally exist as diastereomers. It should be understood that all such isomers and mixtures thereof in any proportion are included within the scope of the present invention.

[0194] The present invention also includes methods of using pharmaceutical compositions comprising one or more of the disclosed alkylphosphocholine analogs and a pharmaceutically acceptable carrier. Preferably, these compositions are in unit dosage form, such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosols or liquid sprays, drops, ampoules, automatic injector devices, or suppositories; for parenteral, intranasal, sublingual, or rectal administration, or administration by inhalation or insufflation.

[0195] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutically acceptable carrier, such as conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum, and other pharmaceutical diluents such as water to form a solid preformulated composition comprising a homogeneous mixture of the compound of the invention or a pharmaceutically acceptable salt thereof. When these preformulated compositions are described as homogeneous, it is meant that the active ingredient is evenly dispersed throughout the composition, allowing the composition to be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. The solid preformulated composition is then further subdivided into unit dosage forms of the above-mentioned type, containing approximately 0.1-500 mg of the active ingredient of the invention. Typical unit dosage forms contain 1-100 mg, for example, 1, 2, 5, 10, 25, 50, or 100 mg of active ingredient. The tablets or pills of the novel compositions can be coated or otherwise compounded to provide a dosage having the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage component and an outer dosage component, the latter encapsulating the former. The two components may be separated by an enteric layer, which resists disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be released after a delay. A variety of materials can be used for such enteric layers or coatings, including various polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0196] Liquid forms for oral or parenteral administration that can incorporate alkylphosphocholine analogs include: aqueous solutions appropriately flavored with syrups; aqueous or oily suspensions; and emulsions flavored with edible oils, including cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical carriers. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginates, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, or gelatin.

[0197] The disclosed alkylphosphocholine analogs are particularly useful when formulated in the form of a pharmaceutical injectable dosage form, including in combination with an injectable carrier system. As used herein, injectable and infusible dosage forms (i.e., parenteral dosage forms) include, but are not limited to, liposome injections or lipid bilayer carriers having phospholipids encapsulating the active drug. Injectables include sterile preparations for parenteral use.

[0198] The UPS defines five different categories of injectables: emulsions, lipids, powders, solutions, and suspensions. Emulsion injections include emulsions that contain sterile, pyrogen-free preparations for parenteral administration. Liposomes and powders for solution injection are sterile preparations intended to be reconstituted into solutions for parenteral use. Powders for suspension injection are sterile preparations intended to be reconstituted into suspensions for parenteral use. Lyophilized powders for liposomal suspension injection are sterile, freeze-dried preparations intended for reconstitution for parenteral use that are prepared in a manner that allows for the incorporation of liposomes, such as lipid bilayer vehicles with phospholipids, with which the active drug is encapsulated within the lipid bilayer or in the aqueous space, thereby forming a formulation upon reconstitution. Lyophilized powders for solution injection are dosage forms for solutions prepared by freeze-drying ("freeze-drying"), a process that involves removing water from the product in a frozen state at very low pressure, followed by the addition of a liquid to produce a solution that meets all requirements for injection. Lyophilized powders for suspension injection are liquid preparations intended for parenteral use that contain a solid suspended in a suitable liquid medium and that meet all requirements for a sterile suspension, thereby preparing a pharmaceutical agent for suspension by lyophilization. Solution injections involve liquid preparations that contain one or more drug substances dissolved in a suitable solvent or mutually miscible solvent mixture suitable for injection.

[0199] Solution concentrate injections refer to sterile preparations for parenteral use that, when a suitable solvent is added, produce a solution that meets the requirements for injection in all aspects. Suspension injections refer to liquid preparations (suitable for injection) that comprise solid particles dispersed in a liquid phase, the particles being insoluble, with an oil phase dispersed in an aqueous phase or vice versa. Suspension liposome injections are liquid preparations (suitable for injection) that have an oil phase dispersed throughout an aqueous phase in a manner that forms liposomes (a lipid bilayer carrier, typically containing phospholipids for encapsulating active drugs within the lipid bilayer or in the aqueous space). Suspension ultrasonic injections are liquid preparations (suitable for injection) that comprise solid particles dispersed in an aqueous phase, the particles being therefore insoluble. In addition, when gas is bubbled from the suspension, the product can be ultrasonically treated so that the solid particles form microspheres.

[0200] Parenteral delivery systems include one or more pharmaceutically suitable excipients, such as solvents or co-solvents, solubilizers, wetting agents, suspending agents, thickening agents, emulsifiers, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents, protective agents, tonicity adjusters and special additives.

[0201] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, various variations beyond the present invention shown and described will be apparent to those skilled in the art through the above description and the following examples, and said variations are also included within the scope of the appended claims.

[0202] III. Examples

[0203] Example Introduction

[0204] These examples demonstrate the potential to integrate two distinct frontiers in cancer therapy research with unexpected and very effective synergies. These two disciplines are: 1) systemically administered TRT; and 2) locally directed antibody-mediated cancer immunotherapy or systemically administered cancer immunotherapy. The data presented herein demonstrate that powerful synergies can be generated by combining these approaches. In summary, these two strategies can be used to destroy visible macroscopic tumors, allowing the destroyed cancer cells to act as a potent immunostimulatory agent, generating tumor-specific T cell immunity that can eradicate persistent residual metastatic disease in any type of solid tumor at any site.

[0205] Our ongoing preclinical studies have shown that the combination of tumor-specific mAbs with IL2 (which activates innate immune cells) leads to enhanced antibody-dependent cell-mediated cytotoxicity (ADCC)[1,2]; this process has translated into clinical benefit in children with neuroblastoma[3]. Recent preclinical data show that the antitumor effect of mAb-IL2 fusion protein is even greater when it is injected intratumorally (IT)[4,5]. Notably, large tumors that did not respond to these mAb / IL2 injections and continued to grow if treated with local xRT alone can be completely eradicated if xRT is combined with mAb / IL2 therapy. The majority of mice were cured and developed T cell memory that resisted rechallenge with similar tumor cells[6]; demonstrating that the combined xRT+mAb / IL2 acts as an effective “in situ” anticancer vaccine.

[0206] A key limitation is that when these animals receive xRT+mAb / IL2 to treat the primary (first) tumor, if another macroscopic tumor is present, the second tumor will continue to grow and, to our surprise, suppress the immune response, preventing any shrinkage of the first-treated tumor. This “concomitant immune tolerance” arises in part from suppressive regulatory T cells (Tregs) in the second tumor. RT alone to both tumors has minimal antitumor effect but depletes these Tregs. Therefore, when the first tumor is treated with xRT+mAb / IL2, adding RT to the second tumor can circumvent this immune tolerance, allowing eradication of both tumors [7]. These observations highlight the limitations of in situ tumor vaccination in the metastatic setting but also suggest that RT has a powerful ability to overcome this limitation.

[0207] It is generally not possible to deliver xRT to all metastatic sites without excessive normal tissue toxicity and immunosuppression. However, not delivering xRT to all macroscopic disease sites allows the suppressive immune repertoire to remain intact, capable of suppressing the immune response to our local xRT+mAb / IL2 immunotherapy. Therefore, what is needed is a means to deliver RT to all tumor sites in a targeted manner in cancer patients.

[0208] We have developed TRT carriers that enable targeted systemic delivery of RT to primary and metastatic cancers. One such TRT agent 131 I-NM404 is an intravenously (IV) administered phospholipid ether (PLE) analog that has demonstrated nearly universal tumor targeting properties in more than 60 in vivo cancer and cancer stem cell models. This agent is currently in clinical trials for multiple imaging and therapeutic applications [8,9]. 131 I-NM404 localizes to all tumors regardless of anatomical location and delivers sufficient RT internally to abrogate intratumoral immunosuppressive pathways that can prevent the development of an immune response that is effective for tumor eradication. The unique attributes of this approach are the near-universal tumor targeting capability of NM404, and the ability to deliver immunomodulatory sublethal doses of RT to all tumor sites, which is generally not feasible with xRT. What is new about this is that our TRT agent can immunomodulate all tumors regardless of anatomical location, overcoming the concomitant tolerance, which will result in a long-term orthotopic tumor vaccination effect of local xRT followed by injection of tumor-specific mAb+IL2. As more and more tumor-specific mAbs are 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, this approach can be readily generalized to all orthotopic tumor vaccination strategies.

[0209] Recently, we have discovered 131 The iodine in I-NM404 can be replaced by chelators that enable a variety of metal imaging (MRI and PET) and TRT radiotherapy components. In these examples, we describe how to evaluate 131 I-NM404 (and therefore, related metal chelate analogs) have the ability to initiate a systemic immunomodulatory response, which is required to enable combined xRT + immunotherapy treatment to induce an effective radioimmunostimulatory response against cancerous solid tumors. A similar approach can be used for TRT delivered by PLE analogs in combination with other immunotherapies against cancerous solid tumors. For example, we illustrate below that this combined approach can use an immunomodulatory step that is completely different from local in situ tumor vaccination: systemic administration of an immunostimulatory agent, such as an immune checkpoint inhibitor.

[0210] In summary, we have disclosed here a therapeutic and research process that combines two distinct approaches from seemingly unrelated cancer treatment disciplines into a single unified therapy. The data presented in these examples demonstrate that these two approaches can be synergistically combined to effectively eliminate malignant solid tumors and prevent tumor recurrence.

[0211] In Example 1, we present background data from our B78 GD2+ model to support the approach.

[0212] In Example 2, we provide guidance for determining the xRT dose required for optimal in situ vaccine efficacy in primary tumors and the minimum xRT dose required to prevent concomitant immune tolerance in distant tumors.

[0213] In Example 3, we provide a method for determining 131 Guidance on I-NM404 dosing that approximates the required dose of xRT for metastasis, as determined in Example 2, and subsequent evaluation of the 131 Effect of I-NM404 Dosage on Immune Function in Vivo Such guidance can similarly be applied when using the disclosed radioactive phospholipid metal chelate compounds as TRT agents.

[0214] In Example 4, we provide guidance for designing / testing / developing a novel gene therapy in mice bearing two or more tumors using data from Examples 2 and 3. 131 The regimen of I-NM404 + local xRT + IT-mAb / IL2 was used to destroy the locally treated tumor and induce T cell-mediated eradication of all distant tumors. The key issues of TRT and xRT dose and timing were optimized for antitumor efficacy. Similarly, such guidance can be applied similarly when using the disclosed radioactive phospholipid metal chelate compounds as TRT agents.

[0215] In Example 5, we provide an exemplary synthesis of similar compounds that have also been found to be useful for synthesizing chelated radioactive metal isotopes.

[0216] In Example 6, we demonstrate that a chelated metal analog (Gd-NM600) with a chelator and substituted iodine moiety for NM404 is taken up by (and can be imaged in) solid tumor tissue, thus providing proof of concept for the use of the disclosed metal chelates as TRT agents.

[0217] In Examples 7, 8, 9 and 10, we provide information and specific data from experimental studies conducted according to the guidance of Examples 1-4.

[0218] In Examples 11 and 12, we demonstrate that other analogs of chelated metals having chelators and replacing the iodine moiety of NM404 are taken up in a range of in vivo models of solid tumors and can be imaged therein and can be used therapeutically for TRT, thereby further providing proof of concept for the use of the disclosed metal chelates as TRT agents in the disclosed methods.

[0219] In Example 13, we discuss how one skilled in the art can use dosimetry in combination with known radiosensitivity to optimize therapeutic doses for any solid tumor.

[0220] In Example 14, we discuss the differences and advantages of using alkylphosphocholine metal chelates in the disclosed methods rather than the iodinated compounds shown in Examples 1-4 and 7-10.

[0221] In Examples 15 and 16, we demonstrate that TRT combined with systemically administered immunotherapy, rather than in situ vaccination, is also effective in treating solid tumors. The systemically administered immunostimulatory agent can be an immune checkpoint blocker or inhibitor (in these examples, anti-CTLA4).

[0222] Example 1: Background Support Data

[0223] The Sondel laboratory has demonstrated that tumor-specific mAb + IL2 activates innate immune cells to mediate ADCC in mice [2], with clinical benefit in children with neuroblastoma [3]. In mice, intravenous administration of hu14.18-IL2 IC is more effective than intravenous administration of anti-GD2 mAb + IL2 [2,10]. This can provide significant antitumor effects against very small, recently established GD2+ tumors or very small micrometastases, potentially explaining the clinical application of this approach in patients who have achieved remission but are at high risk of relapse [3]. When IC is injected intratumorally (IT-IC) rather than IV (intravenously), the efficacy of hu14.18-IL2 against large, measurable tumors (i.e., ∼50 mm) is significantly improved. 3 GD2+ tumors] have a stronger anti-tumor effect [4,5].

[0224] We are now focusing on approaches that may provide benefit to larger macroscopic tumors. 3 ) Mice bearing B78 melanoma tumors did not respond to IV-IC, and growth was slowed by IT-IC, but the tumors continued to grow. After 12 Gy xRT, these 200 mm 3 In contrast, when IT-IC and xRT were combined, 73% of the animals were tumor-free and appeared to be cured of disease ( Figure 2A and 2BThese mice then displayed T cell-mediated rejection of rechallenge with the same tumor ( Figure 2C ). Therefore, IT-IC+xRT act synergistically to induce tumors to become “in situ tumor vaccines”[6].

[0225] To simulate clinical metastasis, we inoculated one side of the mice with B78 on day 1 and the other side on week 2. At week 5, the first tumor was 200 mm 3 , the second tumor was 50 mm 3 We expected that xRT + IT-IC would destroy the first tumor, and then the resulting T cell response would destroy the second tumor. However, adding IT-IC to xRT did not 3 Tumor or 200mm 3 Tumors had little effect ( Figure 3 This demonstrates a key limitation of the treatment we provide; namely, if a second tumor is present when these mice receive xRT+IT-IC to the first tumor, the second tumor will induce a systemic tumor-specific concomitant immune tolerance effect, preventing any tumor shrinkage. Importantly, we found that simultaneous local xRT (12 Gy) to the first and second tumors abolished this tolerance effect, allowing IT-IC to induce an immune response against the first tumor, eradicating both tumors in the majority of mice ( Figure 4 )[7]. Recent data, using Treg-depleting mAbs (not shown) or transgenic mice that allow selective Treg depletion ( Figure 4 )[7], demonstrating that this immune tolerance is mediated in part by regulatory T cells (Tregs); RT of the first and second tumors partially depletes these Tregs, potentially explaining how irradiating both tumors circumvents the tolerance effect[7].

[0226] While local xRT to the primary and secondary tumors circumvents tolerance, clinical metastatic disease is often present in several locations. All macroscopic metastatic disease must receive RT to break immune tolerance and enable xRT+IT-IC to effectively eradicate all tumor sites. However, delivering 12 Gy xRT to all disease sites may resemble "whole-body RT" with major dose-dependent (potentially lethal) toxicities and profound systemic immunosuppression.

[0227] Previously, the Weichert laboratory pioneered the development of TRT to deliver RT to all systemic tumor sites while minimizing "off-target" RT to normal tissues, particularly bone marrow and immune tissues.

[0228] Based on the finding that tumor cells contain an excess of phospholipid ethers (PLEs)

[11] , we synthesized over 30 radioiodinated PLE analogs in the hope of identifying analogs that could selectively target tumors

[12] . One of these, NM404, showed near-universal tumor uptake in all but three of the over 70 in vivo models tested, regardless of anatomical location, including brain metastases and cancer stem cells, and once inside tumor cells, they also experienced prolonged selective retention [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 entry portals for PLEs, including NM404, to enter cancer and cancer stem cells [8]. Radioiodinated NM404 (I-124 and I-131) have now been evaluated in five phase 1 and phase 2 PET imaging trials and three phase 1 TRT radiotherapy trials, respectively, providing similar tumor uptake and retention properties in over a dozen human cancer types [8]. 124 I-NM404 PET imaging demonstrated excellent tumor uptake in the cancer model relevant to these examples (B78 GD2+ murine melanoma) ( Figure 5 ).

[0229] Example 2: Determining the dose of xRT

[0230] Our data suggest four hypotheses: (1) the xRT doses we used to treat a single tumor induced modest direct in vivo tumor death and increased susceptibility to immune-mediated death (via ADCC and T cells); (2) the robust T-cell responses afforded by the addition of IT-IC, but not IT mAb, suggest that mAb binding to irradiated tumor cells in the presence of IL2 promotes antigen presentation and enhanced adaptive immunity induction; (3) the presence of a second tumor results in xRT+IT-IC exerting little antitumor effect on the first tumor, primarily due to tolerance induced by the systemic effects of immunosuppressive cells present in the second tumor [e.g., Tregs and possibly myeloid-derived suppressor cells (MDSCs)]; and (4) the depletion of Tregs ( Figure 4 ) or irradiate a second tumor ( Figure 3 ) can avoid this tolerance; (4) the RT dose required for the second tumor to avoid tolerance can be much lower than the xRT dose required for the first tumor to become an "in situ vaccine"

[14] .

[0231] Optimizing xRT doses at the primary ("in situ vaccine") tumor site.

[0232] Our in vivo studies of xRT+IT-IC focused on a single dose of 12 Gy to the first tumor. This was based on our data showing that in vitro RT induced a dose-dependent functional upregulation of Fas on B78 tumor cells (near peak at >12 Gy), combined with our in vivo data demonstrating that mice with functional Fas-L were required for the in situ vaccine effect of our xRT+IT-IC (6). Prior to selecting the 12 Gy dose, we performed in vivo pilot studies showing that higher doses (16 Gy) or increased fractionation of flank RT were toxic (dermatitis, ulceration, and late limb edema) and did not improve tumor response. Although we selected a single 12 Gy fraction of xRT for our in vivo studies, as we progress toward clinical translation, it would be beneficial to better understand the mechanisms of local xRT effects and their dose requirements to safely and effectively induce an in situ vaccine effect.

[0233] Our mouse data ( Figure 2A , 2B and 2C) showed that we could induce a potent vaccine effect with 12 Gy xRT + IT-IC, even though 12 Gy xRT alone did not result in tumor shrinkage; it only slowed progressive growth. We considered the possibility that we might see an equally potent in situ vaccine effect with lower doses of RT. To test this possibility, we performed a 200 mm 3 A range of xRT doses (4-16 Gy) as single components will be evaluated in mice bearing B78 tumors, followed by our standard IT-IC regimen (50 mcg / d on days 6-10). We will determine which xRT dose provides optimal tumor eradication and T cell memory when combined with IT-IC. If doses below 12 Gy are less toxic and show comparable efficacy, these lower doses will be better targets for our xRT doses at the "in situ vaccine" site in Examples 3 and 4. Similar approaches can be used to optimize doses for specific targets or subjects.

[0234] Optimizing xRT doses to distant tumors to prevent resistance to blocking “in situ vaccination”.

[0235] The first and second tumors were treated with 12 Gy ( Figure 3) enables IT-IC to generate an effective response against the first tumor thereby eradicating both tumors. Our goal is to achieve the same in situ vaccine effect by delivering xRT + IT-IC to a single tumor while circumventing tolerance using the minimum RT dose required at the metastatic site. We recognize that xRT itself (especially if ubiquitous) can be a myelosuppressant / immunosuppressant. This is why we pursued TRT in Examples 3 and 4. Even though it is targeted, TRT does have some systemic delivery of RT. To minimize the systemic immunosuppression of TRT, we want to give the lowest possible TRT dose to effectively suppress tumor-induced immune tolerance while not causing global immunosuppression induced by systemic RT. Therefore, it is best to select the lowest dose of xRT that needs to be delivered to the distant tumor so that when combined with IT-IC to the first tumor, a higher xRT dose to the first tumor can be used as an in situ vaccine.

[0236] As an exemplary optimization experiment, a 200mm 3 The first B78 tumor is about 50 mm 3 Mice with a secondary B78 tumor will receive 12 Gy of xRT to the primary tumor on day 0 (approximately 5 weeks after implantation of the primary B78 tumor). This will be followed by our standard IT-IC protocol on days 6-10. Different groups of mice will receive different doses of xRT to the secondary tumor. Based on data from the B. Johnson laboratory demonstrating that 3 Gy of whole-body xRT can prevent immunosuppression in a myeloma model (15), we will evaluate doses of 0, 1, 5, and 8 Gy (in addition to the 12 Gy dose, which is known to be effective). We will see whether doses significantly less than 12 Gy are as effective against the secondary tumor as the full 12 Gy dose in eliminating immune tolerance.

[0237] Once we select a critical dose of xRT at which beneficial effects are lost, 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 will compare 2, 3, and 4 Gy to determine the critical minimum effective RT dose required to eliminate tolerance and achieve efficacy in these two tumor models receiving 12 Gy plus IT-IC to the first tumor.

[0238] The study was then repeated to confirm whether an effective in situ vaccine could still be achieved when the dose to the first tumor was the lowest effective dose in the single tumor model (tested in Example 2 above) instead of the 12 Gy dose. In summary, the study of Example 2 optimized what the lowest xRT dose to the first and second tumors was without losing the efficacy we demonstrated with 12 Gy for both.

[0239] A study of the xRT doses required for the first and second tumors in mice bearing tumors other than B78 was initiated.

[0240] To provide more clinical generalizability to our mouse studies, we will initiate RT+IT-IC analysis in other GD2+ tumor models. We have published IT-IC with hu14.18-IL2 IC in AJ mice bearing GD2+NXS2 neuroblastomas [5]. We are also evaluating IT-IC with this same IC in C57BL / 6 mice bearing GD2+9464D-GD2 neuroblastomas and Panc02-GD2 pancreatic cancers expressing GD2 by insertion of the GD2 synthase gene. For Example 2, for each model, we will determine the minimum effective xRT dose required to maintain orthotopic vaccine efficacy in primary and secondary tumors.

[0241] Example 3:

[0242] Determined with TRT and immunosuppression from TRT in C57BL / 6 mice 131 Dosage of I-NM404 and Assessment of the Effect on Immune Function by Radiometric Measurement

[0243] 131 I-NM404 demonstrated selective uptake in >95% of tumor lines (human and mouse) in vitro, with poor uptake by non-malignant cells, and similar tumor specificity was observed in vivo. This included selective uptake in B78 tumors ( Figure 5 In our preliminary dosimetry studies, we administered 124 I-NM404, and characterize the time course of TRT exposure by serial PET / CT imaging (e.g. Figure 5 Monte Carlo dosimetry calculations based on this study [16-18] indicate that approximately 60 μCi of 131 I-NM404 will deliver approximately 3 Gy to established B78 tumors. After these 4 weeks, the remaining TRT dose to B78 tumors will be less than 0.25 Gy. We will use xRT to replicate our data obtained in the dual tumor model ( Figure 3 ), but using the lowest possible dose of targeted 131 I-NM404 TRT was shown to effectively eliminate tumor-induced tolerance at all sites of distant disease. However, unlike xRT, which delivers the entire dose within a few minutes and then completes, TRT deposits the dose over time, depending on the biological and physical half-life of the target isotope ( 131We hope to induce an initial TRT effect at distant tumor sites to eliminate immune tolerance; however, when we administer IT-IC to induce ADCC and in situ vaccine antitumor effects, we hope to minimize the immunosuppressive TRT effect. This is critical for complete tumor destruction at all sites.

[0244] Using dosimetry calculations from preliminary data, we estimated a dose of 3 μCi 131 I-NM404 should deliver a dose equivalent to approximately 0.2 Gy to the tumor site, a dose that we hypothesized would not be immunosuppressive and would not prevent lymphocyte-mediated tumor destruction. As described above, this is our estimate for an initial dose of 60 μCi. 131 The dose still needed to be delivered 28 days after the I-NM404 dose. Therefore, we evaluated the 3 On day 0, all mice received 12 Gy xRT to their tumors, and on days 6-10, all mice received 50 mcg / d IT-IC. One group also received 3 μCi of 131 I-NM404 (~0.2Gy). Figure 6 Display Acceptance 131 I-NM404 group and no 131 The I-NM404 group had the same degree of tumor eradication, demonstrating that this low dose of "residual" TRT in the tumor does not prevent the immune-mediated destruction of the RT+IT-IC in situ vaccine. Therefore, we hypothesized that if we used an initial dose of 60 μCi of 131 I-NM404 TRT, which will effectively block the tolerogenic effects of distant tumors, but xRT on day 0 and IT-IC on days 6-10 (28 days after TRT) enable the first tumor to be used as an in situ vaccine, inducing an adaptive response and then eradicating all tumors.

[0245] Experimental Optimization Outlined in This Example Figure 6 In our single tumor B78 model, we will test a range of doses of 131I-NM404 TRT to select the optimal TRT dose that results in sufficient undesirable systemic immunosuppression to interfere with the desired in situ vaccine effect (thus slowing or preventing eradication of the first tumor). This is important for Example 4 because it allows us to ensure that the residual radioactivity from TRT decays to less than this value in mice with distant disease by the time we initiate IT-IC of the first tumor. We will also evaluate the kinetics of the TRT response after different TRT doses to select the optimal time period we should wait after giving an animal with multiple tumors a "tolerance-preventing TRT dose" to allow RT+IT-IC treatment of the first tumor to still induce an in situ vaccine effect and eradicate both the primary and distant tumors.

[0246] Related studies will also explore what dose of TRT, given as monotherapy, is most effective in causing slowing, shrinkage, or eradication of individual B78 tumors. The TRT dose that is most effective in eliminating tumor-induced immune tolerance will be significantly smaller than the TRT dose required to actually induce complete tumor destruction (from TRT alone).

[0247] Finally, once the effects of various optimized doses of TRT are determined in the single tumor model, we will evaluate the subtle immunosuppressive effects of TRT by assessing the immune response of sera from these subjects to the human IgG component of IC. We have shown that immunocompetent mice produce readily quantifiable levels of mouse anti-human antibodies (MAHA) after treatment with these humanized ICs (19). We will use this to determine at which dose TRT results in a detectable, dose-dependent decrease in the magnitude of the murine immune response, in order to assess the overall immunosuppressive effect of the systemic dose of RT that these mice will receive from this TRT. We will need a low TRT dose that prevents tumor-induced immune tolerance and results in minimal systemic immunosuppression.

[0248] Example 4: Development of a genomic DNA library in mice bearing two or more tumors 131 The best regimen of I-NM404+local xRT+IT-mAb / IL2

[0249] The efficacy of TRT+RT+IT-IC was tested in the dual-tumor B78 model.

[0250] The dose and timing information obtained from the studies outlined in Examples 2 and 3 will provide us with the information needed to optimize the dose and timing of TRT for efficacy in the dual tumor model. C57BL / 6 mice will be inoculated with B78 simultaneously on the left (L) and right (R) flanks. After 2 weeks, each tumor should be approximately 50 mm 3 , about 200mm after 5 weeks 3If we assume that our dosimetry calculations in Example 3 indicate that we need to deliver 60 μCi of TRT to the second tumor to achieve close to 3 Gy of RT (to break immune tolerance), our external beam xRT studies predict that this dose should have minimal effect on slowing tumor growth. We plan to deliver 60 μCi of TRT to the second tumor at the 2-week time point (when the tumor is approximately 50 mm 3 ) Treat different groups of mice with 30, 60, or 90 μCi. After 3 weeks, tumors should be approximately 200 mm 3 At that time, we will administer xRT (at the dose determined as described in Example 2) 6 days later (approximately 28 days after TRT), followed by five daily injections of IT-IC to the tumor on the L flank to induce an in situ vaccine effect. Control mice will not receive TRT and will only receive xRT and IT-IC to the L flank, anticipating no in situ vaccine due to tolerance of distant tumors. A separate group will receive local xRT to both tumors and IT-IC to the L flank, anticipating eradication of both tumors through an in situ vaccine effect. Another group will receive TRT + IT-IC, but no local xRT, anticipating an incomplete vaccine effect.

[0251] Subsequent experiments will further evaluate different doses of TRT to the primary tumor (L side) and various timings between TRT and local xRT + IT-IC. Readouts will be: (A) eradication of the primary tumor; (B) eradication of secondary tumors; and (C) systemic immunosuppression as measured by ELISA analysis of MAHA responses. Our goal is to determine the optimal TRT dose and timing for the specific subject and disease model to add to the local xRT + IT-IC regimen, which will eradicate both tumors in the majority of subjects while minimizing systemic immunosuppression (as measured by MAHA responses).

[0252] Optimization of TRT+xRT+IT-IC in mice bearing more than two B78 tumors.

[0253] This portion of Example 4 is most similar to the relevant clinical setting; that is, patients with injectable tumors that can be used as in situ vaccine sites, but who also have multiple distant metastases that can each induce tumor-induced immune tolerance. These studies will replicate the conditions found most effective in the first portion of Example 4 (above). The important difference is that these subjects will each have 4 separate tumors, on the L and R sides, and the L and R subscapular regions. TRT will be given at the dose and time found most effective in the studies outlined in the first portion of Example 4, with xRT+IT-IC then given only to the L-side lesion. The goal here is to test the TRT dose and timing issues to make the most effective in situ vaccine possible, as TRT will effectively eliminate the tumor-induced immune tolerance caused by the 3 sites that did not receive xRT. The measure of efficacy will be the elimination of all 4 tumors in the majority of subjects. Modifications to TRT dose and timing will be tested to produce the most effective optimized regimen. This type of regimen is used clinically in patients with multiple distant metastases who cannot all be irradiated by external beam irradiation but can be irradiated by TRT when combined with local xRT+IT-IC for an “in situ vaccine”.

[0254] Example 5: Synthesis of metal-chelated NM600.

[0255] In this example, we show the synthetic scheme for the synthesis of an exemplary phospholipid chelate, Gd-NM600. Analogs incorporating various radioisotopes can be synthesized in a similar manner, wherein the radioisotope in question is substituted for Gd.

[0256]

[0257] Example 6: In vivo imaging proof of concept

[0258] In this example, we demonstrate successful in vivo MRI imaging of tumors using Gd-NM600 as an MRI contrast agent. The data demonstrate that the backbone phospholipid and chelating agent are taken up and retained by solid tumors, suggesting that such chelates incorporating various radiometals as disclosed herein will exhibit similar properties.

[0259] In order to carry out concept verification in vivo imaging to the tumor uptake of Gd-NM404 material, athymic nude mice with A549 tumor (non-small cell lung cancer) xenografts on one side were scanned. Gd-NM600 material (2.7 mg) was delivered via tail vein injection. Mice were anesthetized and scanned before contrast agent was given and after 1, 4, 24, 48 and 72 hours of delivery of contrast agent. Imaging was performed on a 4.7T Varian preclinical MRI scanner with a volume quadrature coil. T1-weighted images were obtained at all imaging time points using fast spin back scanning with the following pulse sequence parameters: repetition time (TR) = 206ms, echo interval = 9ms, echo train length = 2, effective echo practice (TE) = 9ms, 10 average values, with a 40x40mm 2 Field of view, 192x192 matrix, 10 slices, each 1 mm thick.

[0260] like Figure 7 As shown, MRI imaging of the tumor was significantly enhanced 24 hours after injection.

[0261] These results demonstrate that the metal chelated analogs disclosed in the present disclosure maintain the differential uptake and retention of alkylphosphocholine analogs. Therefore, the disclosed metal chelates can be readily applied in clinical therapeutic and imaging applications.

[0262] Example 7: Experiments to determine the xRT dose required for optimal in situ vaccine efficacy against primary tumors and the minimum xRT dose required to prevent concomitant immune tolerance in distant tumors

[0263] As a follow-up to Examples 1-4, dose titration experiments have been performed in mice with 1 or 2 tumors, evaluating various xRT doses. The first goal was to test the xRT dose required in mice with 1 tumor to promote synergy with IT-IC (IL2-linked tumor-reactive mAb) and "in situ vaccine". The initial experiments confirmed our previous observations that in mice carrying a single tumor, 12Gy RT alone did not eradicate or even regress the growth of established B78 melanoma tumors (0% complete regression), while 12Gy+IT-IC resulted in complete regression of the majority of B78 tumors (66%). On the other hand, 2Gy+IT-IC slowed tumor progression compared to IT-IC alone (mean tumor size at day 32 = 472 mm 3 Compare to 1214mm 3 ), but did not render any mice disease-free (0% complete regression).

[0264] In our "dual tumor model," we previously demonstrated that treating one "primary" tumor with xRT plus IT-IC had no effect, regardless of whether the treated primary tumor or the untreated "secondary" tumor was treated. Indeed, in this dual tumor model, we observed that the presence of a secondary tumor abolished the efficacy of xRT followed by IT-IC injection. We have termed this phenomenon "concomitant immune tolerance" (CIT) and demonstrated that it arises, at least in part, from T regulatory cells (Tregs) in the distant (non-irradiated) secondary tumor, which circulate throughout the body and repopulate the xRT-treated / IT-IC-injected primary tumor. These Tregs homing to the primary tumor appear to interfere with the desired "in situ vaccine" effect.

[0265] We have now confirmed our previous observations that CIT can be overcome by delivering 12Gy xRT to both primary and secondary tumors. Importantly, given that Tregs are very sensitive to RT, we hypothesized that lower doses of RT could be delivered to secondary tumors to overcome CIT and rescue the response to in situ vaccination at the primary tumor (treatment of the primary tumor with 12Gy+IT-IC). We have now tested this and observed that, for secondary tumors, xRT doses of 2Gy or 5Gy were comparable to 12Gy in blunting CIT and rescuing the response to treatment of the primary tumor with 12Gy+IT-IC. These important experiments were repeated in duplicate and suggest (as hypothesized) that the dose of xRT that must be given to distant tumors to prevent CIT is much smaller than the dose required to IT-IC inject the primary tumor site for the purpose of producing an in situ vaccine effect.

[0266] This supports our overall hypothesis in this disclosure and suggests that in animals bearing multiple tumors, we will be able to use targeted radiotherapy (TRT) NM404 to deliver relatively low doses of RT to all sites of disease, thereby overcoming CIT, when combined with local xRT and IT-IC injection of a single tumor site (orthotopic vaccine site).

[0267] Example 8: Determination of the dose required for xRT to metastasize (as determined above) 131 Experiments to evaluate the effects of 131I-NM404 doses on immune function in vivo

[0268] Based on the preliminary data described in Examples 1-4 above, studies have been conducted to translate these concepts into in vivo testing using TRT. Dosimetry studies have been performed on mice bearing one or two B78 tumors (the tumor model we used to demonstrate our orthotopic vaccine approach and CIT barrier). This was done to estimate the dose required for approximately 2 Gy of xRT. 131 I-Amount of NM404.

[0269] Then, to determine the equivalent dose of approximately 2 Gy 131 Whether I-NM404 has the desired effect on intratumoral lymphoid cells (particularly Tregs) has been investigated in two different ways. 131 I-NM404 was administered to mice bearing radiosensitive lymphoma tumors, which exhibited NM404 uptake comparable to that of B78 tumors. Following this, we have documented effective lymphoid tumor shrinkage / dose-dependent inhibition without causing significant shrinkage / slowdown of B78 tumors or any apparent depletion of circulating lymphocytes (as measured by peripheral complete blood counts). These data are consistent with the fact that lymphocytes are more sensitive to low-dose RT than typical solid tumor cells and indicate that selective uptake of TRT in tumors can deplete intratumoral lymphocytes without systemic lymphopenia. These studies also indicate that this lymphoid tumor can be used as an in vivo biological "radiometry" to identify and monitor the effects of TRT on intratumoral lymphocytes.

[0270] The second approach involves using these same doses of 131 I-NM404 was used to treat mice with B78 tumors. These animals were then sacrificed at half-life intervals (8 days) and, after sufficient delay in radioactive decay, the tumors were stained for effector T cells and Tregs by immunohistochemistry. Interestingly, in this initial experiment, 131 Animals receiving I-NM404 did not show systemic lymphopenia (by peripheral complete blood count) at any time point, but did show a decrease in intratumoral FoxP3+ Tregs 2 half-lives after TRT administration. At this 2 half-life time point, we also observed a decrease in intratumoral effector CD8+ T cells. However, importantly, at the subsequent 3 and 4 half-life time points, we observed an increase in intratumoral CD8+ effector T cells, but a further decrease in intratumoral Treg levels, compared to the untreated baseline and 2 half-life levels. This observation again supports our hypothesis that it is feasible to use TRT to overcome Treg-mediated CIT in order to rescue in situ vaccine effects in animals bearing multiple tumors.

[0271] Finally, to characterize the immune effects of TRT on intratumoral immune cells, we used 131I-NM404 processes mice carrying B78, and collects tumor tissue at intervals of pre-treatment and half-life (8d) thereafter.Then analyze the gene expression of a group of immune features of these tissues by RT-PCR.The results show that single TRT treatment causes the expression of tumor cell markers of immune susceptibility and the significant changes of genes usually expressed only by immune cells, the latter showing a time course of obvious expression reduction, which rebounds to overexpression.

[0272] Example 9: Using the data from Examples 5 and 6 to develop 131 An experiment in which the regimen of I-NM404+local xRT+IT-mAb / IL2 induced T cell-mediated eradication of all distant tumors

[0273] This example demonstrates the treatment of tumor-bearing animals at at least two sites. Our strategy involves the use of xRT and localized IT-IC at the in situ vaccine site, combined with systemic TRT to inhibit CIT, to achieve enhanced antitumor immune activity at all tumor sites. The key issues of TRT and xRT dose and timing will be optimization for antitumor efficacy.

[0274] Using the data summarized in Examples 7 and 8, studies were performed in mice bearing two separate B78 tumors. Mice received the estimated required systemic 131 I-NM404 dose followed by local immunotherapy and xRT to the in situ vaccine site. 131 I-NM404 appears to attenuate CIT, as expected in mice with two tumors. Furthermore, in mice with one tumor, this TRT dose does not appear to interfere with the local in situ vaccine effect (as hypothesized and expected). Further testing and modification of some experimental variables are underway to attempt to maximize the desired effect of blocking CIT without inhibiting the in situ vaccine effect. More details about these experiments are disclosed in Example 10 below.

[0275] Example 10: Data from mice bearing two or more tumors Tumor-specific inhibition of the primary tumor's response to the combination of local xRT + IT-IC in distant untreated tumors in murine melanoma and pancreatic tumor models.

[0276] Primary tumor only C57BL / 6 mice bearing syngeneic GD2+ primary flank tumors + / - contralateral secondary tumors were treated with xRT on day 1 and 50 mcg of anti-GD2 IC, hu14.18-IL2 IT on days 6-10 as indicated.

[0277] In mice bearing primary B78 melanoma tumors, the presence of untreated secondary B78 tumors antagonized the primary tumor response to xRT+IT-IC ( Figure 8A We describe this effect as “concomitant immune tolerance”—antagonism of the local response of treated tumors to xRT+IT-IC by untreated distant tumors. Kaplan-Meier survival curves were obtained for these mice plus replicates ( Figure 8B ). Almost all mice were euthanized due to primary tumor progression.

[0278] In mice bearing primary Panc02-GD2+ pancreatic tumors with or without secondary Panc02-GD2- tumors on the opposite flank, the presence of untreated Panc02 secondary tumors suppressed the response of the primary Panc02-GD2+ tumors to xRT+IT-IC ( Figure 8C In mice bearing primary B78 melanoma tumors, secondary B78 tumors suppressed the primary tumor's response to xRT+IT-IC, but secondary Panc02-GD2+ pancreatic tumors did not ( Figure 8D In mice bearing primary Panc02-GD2+ tumors, secondary Panc02-GD2- tumors suppressed the primary tumor response to combined xRT and IT-hu14.18-IL2, whereas B78 secondary tumors did not ( Figure 8E ).

[0279] Avoiding concomitant immune tolerance by specifically depleting regulatory T cells (Tregs).

[0280] Immunohistochemistry images of Treg markers were obtained, and tumors were assessed for FoxP3 ( Figure 9A Mice received no xRT or xRT directed only to the primary tumor. DEREG mice express the diphtheria toxin receptor under the control of the Treg-specific FoxP3 promoter, enabling specific depletion of Tregs upon IP injection of diphtheria toxin ( Figure 9B and 9C DEREG mice bearing primary and secondary B78 melanoma tumors were treated with xRT+IT-IC for the primary tumor and IP injection of diphtheria toxin or PBS. Depletion of Tregs in these mice abolished concomitant immune tolerance, resulting in improved primary ( Figure 9B ) and secondary ( Figure 9C ) tumor response.

[0281] Overcoming concomitant immune tolerance by delivering xRT to two tumor sites.

[0282] In mice bearing primary and secondary B78 tumors, the secondary tumors suppressed the primary tumor response to primary tumor treatment with xRT+IT-IC. This could be 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 responses in replicated experiments ( Figure 10A ) and enhanced animal survival ( Figure 10B ).

[0283] Low-dose xRT alone did not elicit in situ vaccination but did overcome concomitant immune tolerance when delivered to distant tumor sites together with 12 Gy+IT-IC treatment at the in situ vaccine site.

[0284] In mice bearing only primary B78 tumors, 12 Gy + IT-IC triggered in situ vaccination (as previously shown) and resulted in complete tumor regression in most mice ( Figure 11A ) and memory immune response (Morris, Cancer Res, 2016). On the other hand, no animal showed complete tumor regression after IT-IC alone or low-dose (2 Gy) xRT + IT-IC (0 / 6 in both groups) p < 0.05.

[0285] In mice bearing primary and secondary B78 melanoma tumors, low-dose xRT (2 Gy or 5 Gy) delivered to the secondary tumors was equivalent to 12 Gy ( Figure 11B In these same animals, it was apparent that overcoming concomitant immune tolerance by delivering low-dose xRT to the secondary tumors rescued the systemic response to IT-IC immunotherapy ( Figure 11C In this case, when RT is delivered to all tumor sites, then IT-IC injection of the primary tumor elicits a systemic antitumor effect, making the response of secondary tumors to 2 Gy or 5 Gy greater than the response to 12 Gy RT in the absence of IT-IC injection of the primary tumor.

[0286] have 131 Low-dose TRT with I-NM404 effectively depleted tumor-infiltrating FoxP3+ Tregs without systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells.

[0287] In most clinical settings, it is not feasible to deliver an external beam (even a low dose) to all tumor sites without causing significant bone marrow depletion and leukopenia leading to immunosuppression. Here, we tested whether TRT can be given systemically to specifically deplete tumor-infiltrating suppressive immune cells (Tregs) without triggering systemic immune cell depletion and leukopenia.124 I-NM404 was studied in a radiometric manner in a B78 melanoma tumor model, demonstrating tumor-selective uptake of NM404. Figure 12A ). C57BL / 6 mice bearing B78 tumors were treated with 60 μCi 131 I-NM404 treatment. This activity is similar to that required to deliver approximately 2 Gy TRT to B78 tumors 131 This activity approximates the amount of I-NM404 required to deliver approximately 2 Gy TRT to B78 tumors. 131 This dose of TRT did not result in any significant systemic leukopenia ( Figure 12B ) and did not significantly affect the levels of tumor-infiltrating CD8+ effector T cells ( Figure 12C However, this dose of TRT significantly depleted tumor-infiltrating FoxP3+ Tregs ( Figure 12D ).

[0288] have 131 Low-dose TRT of I-NM404 effectively overcame concomitant immune tolerance and rescued the systemic antitumor effects of in situ vaccination.

[0289] Considering the low dose 131 We tested the ability of I-NM404 TRT to deplete tumor-infiltrating Tregs without leukopenia in mice. 131 Can I-NM404 effectively overcome concomitant immune tolerance? C57BL / 6 mice bearing two B78 tumors were treated with 60-μCi 131I-NM404 (NM404) on day 1 as shown. After one half-life (day 8), the animals received 12 Gy xRT to the primary tumor (in situ vaccine site) or no xRT. 131 Secondary tumors in control mice treated with I-NM404 (0, 2, or 12 Gy). Mice that received daily IT injections of IC to primary tumors (orthotopic vaccine sites) on days 13-17 were treated as indicated. Primary tumors ( Figure 13A ) and secondary tumors ( Figure 13B ) responses, indicating that administration of low-dose TRT effectively overcomes concomitant immune tolerance and rescues the systemic antitumor effects of in situ vaccination.

[0290] References cited in Examples 1-4 and 7-10

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[19] Imboden M, Murphy KR, Rakhmilevich AL, Neal ZC, Xiang R, Reisfeld RA, Gillies SD, and Sondel PM. The level of MHC Class I expression on murine adenocarcinoma can change the antitumor effector mechanism of immunocytokine therapy. Cancer Res. 61:1500-7.2001.

[0310] Example 11:

[0311] In vivo uptake of multiple NM600 metal chelates in mice xenografted with eight different solid tumor types as demonstrated by PET imaging

[0312] In this example, we demonstrate differential uptake of NM600 chelated with four different metals within the context of solid tumors in vivo, as demonstrated by PET / CT imaging of such tumors. This further supports 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 14 , as with 64 Exemplary substances for Cu chelation ( 64 Cu-NM600); however, any metal can be easily chelated with NM600.

[0313] Specifically, mice were xenotransplanted with any of eight different solid tumor cell lines (B78 (melanoma), U87MG (glioblastoma), 4T1 (breast cancer), HCT-116 (colon cancer), A549 (lung cancer), PC-3 (prostate cancer), HT-29 (colorectal cancer) or MiaPaca (pancreatic cancer). For each xenotransplanted mouse, a cell suspension containing tumor cells was inoculated into the subcutaneous tissue on one or both sides of the mouse. Once the xenograft tumor reached the minimum size, 150-300 μCi of the drug was injected via the tail vein. 64 Cu, 89 Zr, 86 Y or 52 Each mouse was injected with Mn radiolabeled NM600. After the uptake phase, PET imaging was performed in an Inveon microPET / CT. Before each scan, mice were anesthetized with isoflurane (2%) and placed in a recumbent position in the scanner. Longitudinal PET scans with 40-80 million coincident events were acquired 3, 12, 24, and 48 hours after radiotracer injection, and images were reconstructed using the OSEM3D / MAP reconstruction algorithm.

[0314] Figure 15 Shows the injection 86 Image of a single tumor in a B78 mouse with Y-NM600 at 48 hours post-injection. Figure 16 Shows the injection 86 Results of two B78 mice with Y-NM600 tumors 48 hours after injection; Figure 17 Shows the injection 64 Results of Cu-NM600 in U87MG mice at 3, 24, and 48 hours after injection; Figure 18 Shows the injection 64 Results of Cu-NM600 injection in 4T1 mice at 3, 24, and 48 hours post-injection. Figure 19 Shows the injection 64 Results of Cu-NM600 injection in HCT-116 mice at 3, 24, and 48 hours post-injection. Figure 20 Shows the injection 64 Results of Cu-NM600 injection in A549 mice at 3, 24, and 48 hours post-injection; Figure 21 Shows the injection 64 Results of Cu-NM600 injection in PC-3 mice at 3, 24, and 48 hours post-injection; Figure 22 Shows the injection 64 Results of Cu-NM600 injection in HT-29 mice at 3, 24, and 48 hours post-injection; Figure 23 Shows the injection 64Results of Cu-NM600 injection in MiaPaca mice at 3, 24, and 48 hours post-injection. Figure 24 Shows the injection 86 Results of Y-NM600 injection in 4T1 mice at 3, 24, and 48 hours post-injection. Figure 25 Shows the injection 89 Results of Zr-NM600 injection in 4T1 mice at 3, 24, and 48 hours post-injection.

[0315] For injection 52 Mn-NM600 in HT-29 and PC3 mice at 4 hours and 1 day after injection ( Figure 26 It is HT-29; Figure 27 is PC3), and PET images were obtained 2, 3, 5, and 7 days after injection ( Figure 28 It is HT-29; Figure 29 is PC-3).

[0316] like Figure 15-29 As shown, the scanned mice produced PET / CT three-dimensional volumes that were able to display the cumulative absorbed dose distribution concentrated in the xenografted tumor. This result confirms the differential uptake of metal-chelated NM600 into xenografted solid tumor tissue and demonstrates the potential use of NM600 analogs incorporating radiometal isotopes in the disclosed therapeutic methods.

[0317] Quantitative region of interest analysis of the images was performed by manually outlining the tumor and other organs of interest. Quantitative data are expressed as percent injected dose per gram of tissue (% ID / g). Exemplary data show that 4T1 tumor tissue increases its uptake over time and effectively retains the three tested NM600 chelates ( 86 Y-NM600, 64 Cu-NM600 and 89 Zr-NM600, see Figure 30 ), while a healthy heart ( Figure 31 ),liver( Figure 32 ) and whole body tissues ( Figure 33 ) all exhibited significantly decreased uptake / retention over time.

[0318] Ex vivo biodistribution analysis was performed after the final longitudinal PET scan. Mice were euthanized and tissues were harvested, wet weight was measured, and counted in an automated gamma particle counter (Wizard 2480, Perkin Elmer). Exemplary biodistribution data show the biodistribution of different NM-600 chelates ( 86 Y-NM600, 64 Cu-NM600, 89Zr-NM600 and 177 Lu-NM600, see Figure 34 ) Significant uptake and retention.

[0319] Together, these results demonstrate that the disclosed metal chelates can be readily used in the TRT step of the disclosed therapeutic methods.

[0320] Example 12:

[0321] Demonstration of antitumor activity and tumor autoradiography with two different NM600 metal chelates against various solid tumor types in xenografted mice

[0322] In this example, using three different solid tumor models, we show that alkylphosphocholine metal chelate analogs can be effectively used to promote conventional TRT. These results further demonstrate the feasibility of using metal chelates in the TRT step of the currently disclosed treatment methods.

[0323] B78, MiaPaca, and 4T subcutaneous flank xenografts were induced in mice as described previously. Subsequently, mice were given a therapeutic dose (250-500 μCi) of 90 Y-NM600, 177 Lu-NM600 or control solution.

[0324] Planar 2D phosphorimagers of the substance biodistribution were acquired using a Cyclone Phosphorimager (PerkinElmer). Mice were anesthetized and placed in a supine position in direct contact with the phosphor plate, which they maintained for 15-30 minutes; the plate was then read in the phosphorimager. Images were recorded between 4 and 96 hours after the radioactive dose was injected. The autoradiographic images obtained showed rapid and selective uptake and long-term retention of the chelate in all solid tumor tissue types tested (see Figure 40 、 41 , 42, 43, 44 and 45).

[0325] Tumor response was assessed by comparing tumor growth in treated mice relative to control mice. Tumor volume was determined by measuring the length and width of the tumor with calipers and calculating the volume using the formula for ellipsoid volume. The body weight of the mice was also recorded. The humanized endpoint was defined as: tumor volume > 2500 m 3 or significant weight loss less than 13g.

[0326] like Figure 46 、 47, 48, 49, 50 and 51, the results demonstrated that the two tested NM600 chelates had statistically significant in vivo therapeutic effects when compared to controls, resulting in a double dose of 4T1 xenografts. 177 Lu-NM600 reduced the mean tumor volume (see Figure 50 ) and reduce growth to near zero or to the following rate: a single dose of 177 MiaPaca, 4T1, or B78 xenografts of Lu-NM600 (see Figure 47 、 48 and 49) or given a single dose of 90 B78 or 4T1 xenografts of Y-NM600 (see 46 and 51).

[0327] These results further demonstrate the efficacy of the disclosed alkylphosphocholine metal chelates for delivering TRT to effectively treat various types of solid tumors.

[0328] Example 13:

[0329] Combining radiation dosimetry with radiosensitivity index to predict TRT response in various solid tumor types

[0330] In this example, we discuss factors used to determine the appropriate chelate dosage for the TRT step of the disclosed methods in various solid tumor types.

[0331] Estimation of tumor absorbed dose

[0332] give 177 Lu / 90 Whether the amount of Y-NM600 is immunostimulatory or cytotoxic depends on the dose absorbed by the tumor. 64 Cu / 86 Y-NM600 can be used as therapeutic metal 177 Lu / 90 An imaging alternative to the Y-NM600 facilitates the estimation of tumor dosimetry. 64 Cu / 86 Y-NM600 PET / CT is used to quantitatively measure biodistribution in the body and estimate radiation dose, which can help identify dose-limiting organs and 177 Lu / 90 Potential tumor efficacy of Y-NM600 TRT.

[0333] The general concepts are as follows: (1) Use longitudinal PET / CT imaging to analyze tumor internal structure over time. 64 Cu / 86 The concentration of Y-NM600 was quantified, (2) decay correction64 Cu / 86 The concentration of Y-NM600 is calculated 64 Cu / 86 Y-NM600 and 177 Lu / 90 Differences in decay rates between Y-NM600, (3) 177 Lu / 90 The concentration of Y-NM600 was integrated over time to calculate the cumulative activity, or the total amount of decay, and (4) the deposition of radioactive nucleic acid decay within the tumor was modeled and quantified.

[0334] Steps (1) to (3) can be performed in any medical image processing software package, whereas step (4) requires sophisticated radiation dosimetry software. OLINDA / EXM (Stabin, Sparks, and Crowe 2005) is a 510(k) approved radiation dosimetry estimation software that uses a system developed by the Medical Internal Radiation Dose (MIRD) Committee of the Society of Nuclear Medicine (Bolch et al., 2009). The MIRD method estimates the mean absorbed dose received by a tissue or organ due to radiation emitted into the organ from within the organ itself or from another source. The simplest form of the MIRD equation is

[0335]

[0336] The absorbed dose D [mGy] from the radionuclide activity in the source region s to the target region t is given by It is the total number of radionuclide decays given in MBq-s. The S factor S(t←s)[mGy / MBq-s] is the fraction of the energy released by the decay of a radionuclide in the source region s that is deposited in the target region t, given by the mass m of the target region t. t Standardization. The S factor is a tabulated value calculated using Monte Carlo on a set of standard phantoms and organs. Usually, we are interested in the dose of each unit of injected activity. [mGy / MBq]. Equation relative to retention time τ h [MBq-s / MBq inj ]write,

[0337]

[0338] τ h It is the cumulative activity and injection activity A inj The ratio of [MBq] is

[0339]

[0340] In the case of tumor radioactivity calculations, OLINDA / EXM models the tumor as a discrete unit density sphere whose volume is estimated from the tumor region of interest (ROI) generated as part of step (1). The concentration of NM600 (% ID / g) within the tumor is determined at each time point and corrected for decay. Cumulative activity is then calculated by integrating the concentration over time using trapezoidal piecewise integration.

[0341] Radiation dosimetry results for a number of cell lines are shown in Table 1. This information can be used to estimate absorbed doses for radiotherapy studies aimed at eradicating tumors or stimulating the immune system.

[0342] Table 1: Usage 64 Cu-NM600 or 86 Y-NM600 PET imaging as an alternative 177 Lu-NM600 and 90 Radiation dosimetry estimates (Gy / MBq) for Y-NM600 inj )

[0343] PC3 A549 HT-29 MiaPaca U87MG 4T1 B78 Lu-177 0.39 0.30 0.49 0.24 0.58 1.50 0.92 Y-90 0.69 0.53 0.84 0.45 1.01 4.68 2.86

[0344] Radiosensitivity index to predict dose response

[0345] Intrinsic radiosensitivity is a potential important factor in radiotherapy response, and for a type of cancer, knowing in advance that it can help predict how it can respond to radiation from TRT. However, because there is no method for its conventional measurement in tumors, after irradiation with 2Gy (SF2), radiosensitivity is measured as a survival fraction (between 0 and 1) by clonogenic assay. The relative radiosensitivity of cancer cell phenotypes varies from those with low radiosensitivity (pancreas, colorectal, glioma and breast) to those with high radiosensitivity (lymphoma). Cancer (Table 2) can be classified or arranged according to the radiosensitivity index of cancer.

[0346] If we can demonstrate good tumor uptake and growth inhibition of APC metal chelates in highly radiosensitive tumors such as lymphomas and in highly radioresistant tumors such as gliomas, breast, pancreatic or colorectal, then this would suggest that if these substances could be targeted to tumors in vivo, then they would be effective against any tumor with an SF2 value between those of lymphomas and gliomas (0.3-0.82). Then, it would also be expected that the radiation dose required to eradicate glioma tumor cells would be higher than the radiation dose required to treat the more radiosensitive lymphocytes.

[0347] We currently use in vivo imaging to confirm tumor selectivity and therapy response (tumor growth inhibition) data in all tumor cell lines listed in Table 2. In some cases, multiple doses of APC chelates may need to be administered to induce sufficient cancer cell killing. By using quantitative imaging combined with radiation dosimetry calculations, we can estimate the dose absorbed by the tumor necessary to kill cancer cells (higher doses) or stimulate tumor cells (lower doses) as disclosed herein.

[0348] Combining dose measurement estimates for various cancer cell lines (Table 1) with their respective radiosensitivity indices (Table 2) supports the establishment of a dose response landscape for NM600. By knowing the efficacy and tumor targeting characteristics of NM600 across a range of cell lines, it is possible to estimate the potential cellular efficacy and tumor dose absorbed by cell lines with similar radiosensitivity indices. Furthermore, depending on the desired outcome of tumor clearance or immune stimulation (as disclosed herein), the therapeutic dose can be linearly scaled according to Table 1.

[0349] Table 2: Relative radiosensitivity of cancer cells

[0350]

[0351] 1 Taghian, Alphonse, et al. "In vivo radiation sensitivity of glioblastoma multiforme." International Journal of Radiation Oncology*Biology*Physics 32.1 (1995): 99-104.

[0352] 2 Ramsay, J., R. Ward, and NM Leehen. "Radiosensitivity testing of human malignant gliomas." International Journal of Radiation Oncology*Biology*Physics 24.4 (1992): 675-680.

[0353] 3Fertil, B. and EP Malaise. "Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves." International Journal of Radiation Oncology*Biology*Physics 11.9(1985):1699-1707.

[0354] 4 Wollin, Michael, et al. "Radiosensitivity of human prostate cancer and malignant melanoma cell lines." Radiotherapy and Oncology 15.3 (1989): 285-293.

[0355] 5 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.

[0356] 6 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.

[0357] 7 EP Malaise, Patrick J. Deschavanne, and Bernard Fertil. "Intrinsic radiosensitivity of human cells." Advances in radiation biology 15(2016):37-70.

[0358] 8 Siles, E., et al. "Relationship between p53 status and radiosensitivity in human tumor cell lines." British journal of cancer 73.5 (1996): 581-588.

[0359] References cited in Example 13:

[0360] Bolch, WE, KFEckerman, G. Sgouros, and SR Thomas. 2009. “MIRD Pamphlet No. 21: A Generalized Schema for Radiopharmaceutical Dosimetry—Standardization of Nomenclature.” Journal of Nuclear Medicine 50(3):477–84. doi:10.2967 / jnumed.108.056036.

[0361] Stabin, MG, RB Sparks, and E Crowe. 2005. “OLINDA / EXM: The Second-Generation Personal Computer Software for Internal Dose Assessment in Nuclear Medicine.” J Nucl Med 46(6):1023–27.

[0362] Example 14:

[0363] Advantages and differences when using alkylphosphocholine metal chelates instead of radioiodinated compounds such as those exemplified in Examples 1-4 and 7-10

[0364] In this example, we discuss the advantages of using APC metal chelates rather than radioiodinated compounds (such as the compounds exemplified in Examples 1-4 and 7-10). We also discuss the factors that one skilled in the art would consider when optimizing the dose of the metal chelate to be used in the TRT step of the disclosed methods.

[0365] Chelates allow the use of a variety of stable or radioactive metal ions for imaging and therapy. They can be coupled to a variety of α, β, helical, γ, and positron emitters, with iodine being limited to one positron (I-124), one β (I-131), one γ (I-123), and one helical (I-125) isotope.

[0366] Metal isotopes are more effective than I-131 and I-124 in diagnostic imaging and therapy.

[0367] Lu-177 has less high-energy gamma radiation, making it more suitable for SPECT imaging and dosimetry. However, its beta energy is slightly lower than that of I-131, making it more ideal for treating smaller tumors.

[0368] I-131 and Lu-177 are comparable in therapeutic efficacy "horse power," but Lu-177 results in significantly less total dose from gamma emission. In the case of Y-90, there is virtually no radiation dose from gamma emission.

[0369] Compared to I-131, Y-90 is more effective than I-131 in killing cancer cells through conventional TRT. Figure 52 and further discussion below.

[0370] The Medical Internal Radiation Dose Committee (MIRD) has developed standard methods, models, assumptions, and mathematical models for estimating internal radiation doses from administered radiopharmaceuticals. The MIRD approach simplifies the problem of estimating radiation doses from a variety of different radionuclides and has been implemented in the widely used 510(k)-approved software OLINDA / EXM1. Along with its many standard anthropomorphic phantoms, OLINDA / EXM has a sphere model that can be used to roughly estimate tumor doses. The sphere model assumes that the radiopharmaceutical is uniformly distributed within a sphere of unit density over a range of tumor masses (0.01-6,000 g).

[0371] Using this standard model, we compared Y-90 versus I-131 in terms of radiation dose normalized by the administered radioactivity. The results of this comparison are shown for tumor masses of 1-100 g. Figure 52 Of note, the Y-90 to I-131 ratio reached 4 for 4 g of tumor and remained above 4.0 and 4.2 for 100 g of tumor, strongly suggesting that Y-90 was 3.6-4.1 times more cytotoxic than I-131 on an mCi / mCi basis in up to 10 g of tumor and approximately 4.1 times more effective in tumors greater than 10 g.

[0372] Different pharmacokinetic properties

[0373] Unlike the iodinated analogs, the APC chelate is too large to fit into the known albumin binding pocket in plasma and the factors display different in vivo pharmacokinetic and biodistribution profiles (see Figure 53 Lower binding energy results in a larger fraction of free molecules in the plasma, which leads to faster tumor uptake. Some APC chelates are cleared via the urinary system, whereas iodinated analogs are eliminated via the hepatobiliary system. APC chelates also accumulate in tumors and are cleared from the blood more rapidly than iodinated analogs. Faster blood clearance is associated with lower bone marrow and off-target toxicity of therapeutic radiopharmaceuticals.

[0374] These differences in PK and biodistribution profiles result in varying dose-limiting organ toxicity and ultimate efficacy. Dose-limiting toxicity that shifts hematologic toxicity to the kidney or liver would increase the utility of radiometal chelates for TRT.

[0375] Furthermore, the pharmacokinetic profile of APC chelates can be easily manipulated by minor changes in the chelate structure (e.g., chelate charge). The choice of chelators is broad. Faster clearance from normal tissues improves imaging contrast and therapeutic window, leading to a higher maximum tolerable dose.

[0376] APC chelates have different physicochemical characteristics than their iodinated analogs. They are more water-soluble and therefore do not require surfactants to make them suitable for intravenous injection. APC chelates are based on ionic binding of the metal to the chelate, whereas the iodinated compound forms a covalent bond with its carrier molecule. Deiodination reactions in vivo are very common with iodinated alkanes, however, chelates tend to be extremely stable in vivo.

[0377] Once deiodination occurs, free iodide rapidly accumulates in the thyroid gland and has a very long subsequent excretion half-life, whereas the free radiometal is generally excreted from the body or detoxified more rapidly.

[0378] Depending on the metal ion, the in vivo biodistribution of APC chelates can be quite different, and therefore, both the metal and the chelate contribute to the tumor targeting characteristics of APCs. Not all chelates target tumors. Tumor targeting depends on the accumulation properties of the APC carrier, the type of chelate (linear chelates undergo rapid renal elimination, while macrocyclic chelates undergo hepatobiliary excretion), and the metal ion. Even slight changes in the chelate structure can lead to significant changes in in vivo properties. A simple change in the isotope can result in a greater than 50% change in tumor targeting.

[0379] Radioactive APC metal chelates are readily radiolabeled in quantitative (>98%) yields under mild conditions; however, the radioiodination yields of the iodinated analogs are very low (typically about 50% for I-131 and about 60% for I-124). Furthermore, the chelates achieve highly specific activity. Synthesis can be accomplished using radiolabeling kits in any nuclear pharmacy, requiring no sophisticated ventilation equipment or training. Due to the volatility of radioiodine during the labeling reaction, radioiodination must be performed in a fume hood equipped with effluent monitoring equipment.

[0380] Imaging agents are not necessarily good therapeutic agents, and vice versa.

[0381] It cannot be assumed that because an imaging agent exhibits good tumor uptake, that treatment is readily apparent. In addition to having good tumor uptake, therapeutic agents need to exhibit prolonged tumor retention relative to normal tissues and must be rapidly cleared from the blood to reduce bone marrow exposure and associated toxicity. Iodinated analogs have prolonged blood retention, leading to dose-limiting bone marrow toxicity. In contrast, our APC chelates exhibit faster blood clearance kinetics, likely due to reduced albumin binding in plasma, as shown above.

[0382] Finally, due to the short path lengths and physical properties of metallic beta- and alpha-emitters relative to iodine-131, there are no exposure concerns for healthcare workers or family members following injection. Patients undergoing I-131 treatment typically must wait in a lead-shielded room for a period of time (up to a week) before being discharged. Patients receiving injections of radioactive alpha- and beta-emitting APC chelates do not require hospitalization.

[0383] Example 15:

[0384] Combination of TRT delivered by Y90-NM600 and anti-CLA4 immune checkpoint inhibitor synergistically inhibits cancer in an in vivo melanoma model

[0385] In this example, we demonstrate the efficacy of the disclosed combination approach, where in vivo immunization is performed by systemic administration of an immune checkpoint inhibitor (anti-CTLA4 antibody), and TRT is performed by systemic administration of the same combination as used in the previous examples. 90 Y-NM600 chelate.

[0386] As previously described, B78 melanoma xenografts were implanted subcutaneously in the flank of male C57BL / 6 mice. Mice were then randomized to receive either 25 μCi, 50 μCi, or 100 μCi of dapoxetine. 90 Y-NM600 (day 1), with and without anti-CTLA4 antibody (immune checkpoint inhibitor) (200 μg on days 4, 7, and 11) (n=6 per experimental group), was treated. Both agents were administered by lateral tail vein injection (i.e., intravenous). A control group treated with PBS alone and anti-CTLA4 alone was also included. Tumors were measured with a caliper twice a week, and animal survival was monitored for 60 days.

[0387] like Figure 54 As shown, compared with any of the monotherapy (anti-CTLA4 or three different doses of 90 Compared with Y-NM600 alone) or PBS control, three combination therapies (anti-CTLA4 + three different doses of 90 Y-NM600) showed significant tumor growth inhibition. 90 Compared with Y-NM600 or anti-CTLA4 alone, anti-CTLA4 combined with 50 or 100 μCi 90 Y-NM600 treatment significantly reduced tumor growth (ANOVA test p < 0.05). 90 The Y-NM600 combined with anti-CTLA-4 treatment group showed an intermediate growth delay response, showing a dose-response trend.

[0388] like Figure 55 As shown, with 50μCi 90 Mice treated with Y-NM600 in combination with anti-CTLA4 exhibited significantly higher overall survival than mice treated with TRT or PBS vehicle alone (p<0.05). The Log Rank for the combination treatment was p=0.06 compared to anti-CTLA4 alone.

[0389] like Figure 56 As shown, all three combination treatments significantly improved survival. Notably, at therapeutic doses of 50 and 100 μCi 90At the Y-NM600 dose, there were 6 / 12 (50%) complete responders in the TRT+CTLA4 combination treatment group, compared to 0 / 24 complete responders in the non-combination control group (PBS, 50 μCi of TRT only, 100 μCi of TRT only, and anti-CTLA4 only).

[0390] These results illustrate the therapeutic potential of combining molecularly targeted radiotherapy with any agent that can induce immune checkpoint inhibition (ICI). The results show that the combination of molecularly targeted TRT and ICI has a synergistic effect relative to treatment with each agent alone. In addition to demonstrating significant tumor regression, this combination approach also has the potential to generate immune memory and ultimately provide an effective in situ carcinoma vaccine effect to prevent tumor recurrence.

[0391] Example 16:

[0392] Enhancement of systemic checkpoint inhibition efficacy in metastatic cancer models using molecularly targeted radiation therapy

[0393] In a follow-up study to Example 15, we provide extensively expanded supporting data demonstrating the effectiveness of the disclosed approach of combining systemic administration of immune checkpoint inhibitors with the systemic administration of the cytotoxic T-cell receptor used in the previous examples. 90 The combination of TRT with the Y-NM600 chelate demonstrated efficacy in mouse models of melanoma, neuroblastoma, and breast cancer, as well as in multiple tumor melanoma models with disseminated "cold" tumors.

[0394] Clinical studies have shown that some patients treated with immune checkpoint inhibitors (ICIs) experience durable and complete responses (CRs) to all sites of disease. However, ICIs are often ineffective in patients with immunologically "cold" tumors characterized by low levels of T cell infiltration and / or few mutation-generated neoantigens. In this example, we demonstrate the use of the disclosed combination approach to stimulate immune responses in such tumors and enhance responses in "hot" tumors. More specifically, we enhance the efficacy of systemic ICIs by combining it with systemic molecularly targeted radiation therapy (MTRT), which can deliver immunostimulatory, low-dose radiation to all sites of disease without causing the counterproductive systemic lymphoid depletion that can inhibit responses to anti-tumor immunotherapy.

[0395] method:

[0396] For tumor uptake studies of MTRT, flank tumors (n=3 for B78 melanoma and Panc02) were established in C57BL / 6 mice by injecting 1-2x106 cells in 100 μL of PBS under approved IACUC protocols. Both B78 and Panc02 tumors are weakly to moderately immunogenic, slow-growing, and radioresistant tumor lines, a profile that makes them very useful for MTRT studies, where slow growth provides time for MTRT to decay, and radioresistance combined with poor immunogenicity allows for testing the efficacy of MTRT+ICI combination therapy.

[0397] After the tumor is fully established, approximately 5 weeks after injection, a single IV 86 Animals were treated with Y-NM600 and serial PET / CT images were collected 1, 2, and 3 days after MTRT injection. PET uptake values ​​were compared with background activity areas including the heart and liver. Paired t-tests were performed to test for differences between background organs and tumor sites. 86 Significant differences in Y-NM600 uptake.

[0398] To prove 90 To investigate the ability of Y-NM600 and / or ICI to reduce the immunosuppressive Treg cell population within the flank tumor of B78 melanoma, we generated a flank tumor model of B78 melanoma (n=4 per group). Treatment groups included MTRT (50 μCi), anti-CTLA4 (200 μg, day 4, 7, 10), MTRT and CTLA4, and a PBS placebo control group. The effect of treatment on tumor immune cell populations was examined by collecting tumor tissue and freezing a portion for histology and preserving another portion for quantitative PCR on days 1, 7, and 14 after radiation or saline placebo delivery. The remaining tumor samples were prepared for mRNA and RT-PCR analysis. Quantitative RT-PCR was used to assess changes in immune sensitivity markers (such as Fas, MHC-1, and PD-L1) expressed in tumor cells.

[0399] For efficacy studies, a bilateral flank tumor model with two B78 melanoma tumors was generated in C57BL / 6 mice. Tumors grew to 80-120 mm. 3 Afterwards, they were randomly divided into the following treatment groups: anti-CTLA-4 was given alone at 200 μg IP on days 4, 7, and 10; 90 Y-NM600 IV (50 μCi) and anti-CTLA4, 12 Gy total body irradiation (EBRT) and anti-CTLA-4, 12 Gy EBRT + 50 μCi 90 Y-NM600, 12 Gy EBRT + 50 μCi 90Y-NM600 and anti-CTLA4. Tumor measurements were performed twice weekly for 30 days, and survival was followed up to 60 days with a euthanasia endpoint of 15 mm diameter to reduce tumor burden.

[0400] Mice that fully responded to therapy were treated with 2x10 6 B78 or 1x10 6 The opposite flank was re-challenged with Panc02 cells and then with Panc02 (B78 only) and B16 melanoma at day 120 to test tumor-specific immune memory responses.

[0401] result:

[0402] Our MTRT reagent was validated in both B78 and Panc02 tumor models. 90 Selective uptake of Y-NM600. For B78 melanoma, tumor uptake of 90Y-NM600 showed that after the initial injection, most of the agent was in the blood pool as expected, but by 48 hours after injection, most of the agent was retained in the tumor or elimination organs (liver, kidney). Gamma counts of tissue sections taken on day 48 confirmed that PET imaging uptake values ​​with higher radioactivity counts in tumor tissue increased over time, while radioactivity counts in the bone marrow space were lower, which decreased over time. Monte Carlo radioactivity measurements performed as a result of the collaboration showed that when our experimental dose was 50 μCi, approximately 2-3 Gy would be delivered over the life cycle of the MTRT agent. PET uptake and tissue biodistribution studies in Panc02 pancreatic cancer also showed that higher uptake in tumor tissue was observed compared to bone marrow tissue at 72 hours. 90 Increased uptake and retention of Y-NM600.

[0403] To investigate the effects of treatment on tumor immune cell populations, tumor tissue samples were collected at different time points after irradiation. 90 On day 14 after Y-NM600, we found that the combination of MTRT and anti-CTLA4 significantly increased the ratio of effector / suppressive immune T cells, as measured by the degree of CD4 / FoxP3 and CD8 / FoxP3 infiltration in tumor tissue. Quantitative PCR (qPCR) studies of gene expression also showed increased expression of inflammatory genes, including those that are part of the stimulator of interferon genes (STING) pathway. Compared with PBS controls, the levels of Mx1, IFNα, IFNβ, and PDL1, all downstream of STING activation, were upregulated.

[0404] Next, we established a single B78R flank tumor in mice once they reached approximately 80 mm 3We randomized them into treatment groups with 25, 50, and 100uCi MTRT doses given on day 1, with and without anti-CTLA4 on days 4, 7, and 7, respectively, as well as PBS and anti-CTLA4 alone as controls. We found that the combination of MTRT with anti-CTLA4 at dose levels of 50 and 100uCi showed significantly improved tumor growth delay compared to the other groups ( Figure 57 ) and survival ( Figure 58 At 25 uCi of MTRT, responses were moderate. Furthermore, the only mouse to fully respond to treatment was in the combined treatment group, with 66%, 33%, and 16% of animals in the 50 uCi, 100 uCi, and 25 uCi MTRT dose groups, respectively. All fully responding mice were challenged with contralateral B78 cells on day 60 after MTRT injection, and rejection was 100% compared to the naive control group, demonstrating that our treatment was able to generate an immune memory response.

[0405] The study was repeated and showed a similar trend, with survival rates in both studies showing that mice treated with the combination of MTRT (50, 100 uCi) and anti-CTLA4 had significantly improved overall survival compared to the other groups.

[0406] Next, we extended this study to mouse models of neuroblastoma (NXS2) and breast cancer (4T1). Figure 59 (NXS2) and Figure 60 As shown in (4T1), the CTLA4 MTRT combination was again the only group that significantly reduced tumor growth (actually, tumor volume).

[0407] Next, we expanded our study to demonstrate improved response rates in mice with multiple bulky tumors. We designed a study of MTRT in a dual-tumor mouse model with the goal of treating mice with multiple bulky tumors, which would correspond to patients with bulky metastatic disease at multiple sites. For this experiment, we examined whether MTRT could improve response rates over the current clinical paradigm of delivering EBRT to a single site combined with immune checkpoint blockade.

[0408] Two tumor models of Panc02 and B78 melanoma were established. First, in B78 melanoma, conventional immunosensitizing EBRT (12 Gy) to the primary site of disease (secondary sites were shielded) was combined with anti-CTLA4 and compared with radiotherapy alone, MTRT and anti-CTLA4, or a combination of EBRT to the primary site and MTRT and anti-CTLA4 to all sites. Tumor growth curves showed that the triple combination therapy improved primary ( Figure 61 ) and secondary ( Figure 62) tumors. Furthermore, triple therapy significantly improved survival compared to the dual-combination therapy group (p < 0.01). Triple therapy resulted in a 40% complete response rate in all responding animals with tumor-specific immune memory against B78 or related B16 melanoma (CR was 16% in the MTRT+anti-CTLA4 group and 0% in the other groups).

[0409] Finally, we mimicked advanced, multisite "cold" cancer (i.e., multisite tumors that do not elicit a robust immune system response and are therefore largely resistant to checkpoint inhibition) using a mouse model with two distant, bulky tumors and disseminated micrometastases.

[0410] To establish large primary tumors, mice were injected in one flank with 2 x 10 6 To form small secondary tumors, mice were injected with 5x10 B78 melanoma tumor cells in the contralateral flank twelve days later. 5 On day 17 (day 1), mice were injected intravenously with 2 x 10 5 B16 melanoma cells.

[0411] Mice were exposed to various monotherapy or combination therapies: PBS control injection; MTRT, 50 μCi IV on day 1; ICI, anti-CTLA4 / PD1 on days 4, 7, and 10; and in situ vaccine (IS), 12 Gy local RT on day 1 plus intratumoral injection of anti-GD2 mAb and IL2 on days 6-10. The monotherapy and combination therapies tested were PBS, MTRT, ICI, IS, MTRT+ICI, MTRT+IS, ICI+IS, and MTRT+IS+ICI. Mice were monitored for tumor growth and animal survival starting on day 60, and tumor-free mice were re-challenged with B78 on day 90.

[0412] At day 90, less than 20% of ICI mice were alive, while approximately half of the MTRT+IS and ICI+IS mice were alive (with a higher survival rate for MTRT+IS). Surprisingly, 100% of the MTRT+IS mice survived (all other groups had zero survival). Notably, 83% of these mice were found to be tumor-free, demonstrating a complete remission (CR) with immune cell memory (i.e., cured), while the remainder retained uncontrolled secondary tumors.

[0413] We have also demonstrated 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 carcinoma (MOC-2). In addition to tumor uptake and dosimetry, toxicity analyses were performed, and no radiation-induced bone marrow toxicity (measured as serum leukocytes or lymphocytes) was observed at our therapeutic radiation dose of 50 μCi (2-3 Gy tumor dose). We also irradiated mice with external beam radiation and varying doses of 90Y-NM600 and collected tissue for histology stained with IHC and for mRNA analysis using PCR. Data from these studies showed upregulation of the interferon signaling pathway and increased PDL1 expression with 50 μCi of 90Y-NM600. Additionally, we have found that tumor-infiltrating regulatory T cells are reduced in response to molecularly targeted radiation therapy.

[0414] In summary, our conclusions from this study suggest that low-dose NM600 MTRT, combined with checkpoint blockade, can enhance distant tumor responses. Notably, the NM600 MTRT radiotherapy delivery agent demonstrated the ability to improve responses in “cold” tumors that are typically unresponsive to immune checkpoint blockade alone. Furthermore, the relatively low MTRT dose of 50 μCi (2.5 Gy tumor dose) was sufficient to achieve immune stimulatory effects to enhance ICI efficacy without the need for systemic lymph node dissection. MTRT can be added to single-site EBRT and checkpoint blockade to achieve greater tumor responses and cure rates at local and distant tumor sites. Our results suggest that MTRT has great potential to improve the therapeutic efficacy of immunotherapy in patients.

[0415] Conclusion of the Example

[0416] These embodiments illustrate anticancer strategies based on the synergistic and widely applicable combination of targeted systemic delivery of radiotherapy with systemic delivery of immunostimulants such as immune checkpoint inhibitors. Since the disclosed metal chelated and radiohalogenated alkylphosphocholine analogs can target cancers of almost any histology, systemic administration of monoclonal antibodies or small molecules (immune checkpoint inhibitors) against immune checkpoints can be used for almost any type of cancer (tumor-reactive monoclonal antibodies have been approved or are in clinical trials for almost all histological types of cancer). Therefore, the clinical translation of the two different combination strategies has broad application to almost all high-risk cancers.

[0417] Other embodiments and applications of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention described herein. All references cited herein for any reason, including all journal articles and U.S. / foreign patents and patent applications, are specifically and entirely incorporated herein by reference. It should be understood that the present invention is not limited to the specific reagents, formulations, reaction conditions, etc. illustrated and described herein, but also includes modifications thereof within the scope of the following claims.

Claims

1. A method of treating cancer comprising one or more solid malignant tumors in a subject, the method comprising systemically administering to the subject: (a) an immunomodulatory dose of a targeted radiotherapy (TRT) agent that is differentially taken up by and retained in solid malignant tumor tissue; and (b) one or more immunostimulants; Cancer is thereby treated in the subject.

2. The method according to claim 1, wherein The one or more immunostimulatory agents are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules.

3. The method according to claim 2, wherein: The one or more checkpoint molecules that the checkpoint inhibitor can target 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 25) 2), GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T-cell costimulatory molecule), ICOSL (inducible T-cell costimulatory ligand), B7H3 (CD276; cluster of differentiation 276), B7H4 (VTCN1; V-set domain-containing inhibitor of T-cell activation 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 inhibitor of T-cell activation).

4. The method according to claim 2 or 3, wherein: The one or more immune checkpoint inhibitors include: one or more anti-immune checkpoint molecule antibodies or one or more small molecule immune checkpoint inhibitors that block one or more immune checkpoint molecules.

5. The method of claim 4, wherein: The one or more anti-immune checkpoint molecule antibodies are selected from: anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-KIR antibodies, anti-A2AR antibodies, anti-BTLA antibodies, anti-CD40 antibodies, anti-CD27 antibodies, anti-CD28 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-OX40L antibodies, GITR antibodies, GITLL antibodies, ICOS antibodies, ICOSL antibodies, B7H3 antibodies, B7H4 antibodies, IDO antibodies, TIM-3 antibodies, Gal-9 antibodies and VISTA antibodies; or wherein the one or more small molecule immune checkpoint inhibitors that block one or more immune checkpoint molecules include small molecule PD-L1 inhibitors.

6. The method according to any one of claims 1 to 5, wherein The TRT reagent is: (1) meta-iodobenzylguanidine (MIBG), where the iodine atom in MIBG is a radioactive iodine isotope; (2) radiolabeled tumor-targeting antibodies; (3) Radioactive radium isotopes; or (4) Phospholipid ether metal chelates or radioactive halogenated phospholipid ethers having the following formula: or a salt thereof, wherein: R1 comprises (a) a chelating agent that chelates with a metal atom, wherein the metal atom is an alpha, beta, or Auger emitting metal isotope with a half-life greater than 6 hours and less than 30 days; or (b) a radioactive halogen isotope; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is selected from: -H, -OH, -COOH, -COOX, -OCOX and -OX, wherein X is alkyl or aralkyl; R2 is selected from: -N + H3, -N + H2Z,-N + HZ2 and -N + Z3, wherein each Z is independently alkyl or aryl; and b is 1 or 2, provided that if R1 comprises a radioactive halogen isotope, then b is 1.

7. The method of claim 6, wherein (1) a metal isotope 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; (2) The radioactive halogen isotope is selected from: 123 I, 124 I, 125 I, 131 I, 211 At, 77 Br and 76 Br; or (3) The radioactive radium isotope is Ra-223.

8. The method according to claim 6 or 7, wherein: The chelating agent is selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) and its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives; 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-diacetic acid (NODAGA) and its derivatives; 1,4,7,10-tetraazacyclodecane, 1-pentanedioic acid-4,7,10-triacetic acid (DOTAG A) and its derivatives; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) and its derivatives; diethylenetriaminepentaacetic acid (DTPA), its diesters, and its derivatives; 2-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A"-DTPA) and its derivatives; deferoxamine (DFO) and its derivatives; 1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H2dedpa) and its derivatives; and DADA and its derivatives, wherein DADA comprises the structure:

9. The method of any one of claims 6 to 8, wherein: (a) m is 0; or (b) b is 1; or (c) n is 18; or (d) R2 is -N + Z3; or (e) Any combination of two or more of (a)–(d).

10. The method of claim 9, wherein: Each Z is independently -CH2CH3 or -CH3.

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