JP2021502368A - Use of Targeted Radiation Therapy (TRT) to Promote Antitumor Immune Responses to Immunotherapy - Google Patents

Abstract

Methods of the present disclosure for treating malignant solid tumors in a subject include immunomodulatory doses of radioactive phospholipid ether metal chelate, radioactive halogenated phospholipid ether, or the like that are differentially retained in malignant solid tumor tissue. It includes a step of administering a targeted radiotherapy (TRT) agent to a subject and a step of performing immunotherapy in the subject by systemically administering an immunostimulatory agent, for example, an immune checkpoint inhibitor. In a non-limiting example, the radioactive phospholipid metal chelate compound has formula (I), in which R1 comprises a chelating agent chelated to a metal atom, the metal atom being longer than 6 hours 30. It is an alpha, beta or Auger-emitting metal isotope with a half-life of less than a day, or 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. FIG. 57.

Description

Cross-references to related applications This application claims the benefit of US Patent Application Publication No. 15/809427 filed November 10, 2017, the full text of which is incorporated herein by reference.

Federally Funded Research or Development Description The invention was made with government support under OD024576 and CA197078 given by the National Institutes of Health. The United States Government reserves certain rights to the present invention.

The present disclosure generally relates to methods of treating cancer. In particular, the present disclosure describes cancers that include one or more malignant solid tumors in a subject as follows: (1) immunomodulatory doses of targeted radiotherapy (TRT) agents such as radiometal chelate compounds, radiohalogenated compounds, radiolabeled antibodies. Alternatively, a radioisotope that is differentially taken up and retained by solid tumor tissue is systemically administered to a subject, and (2) one or more immunostimulators, for example, one or more immune checkpoint inhibitors are targeted. It relates to a method of treating by systemic administration to.

Current cancer treatments generally involve systemic chemotherapy, which allows non-targeted small molecules or antibody-directed cytotoxic agents to preferentially invade or bind to cancer cells by a variety of mechanisms (antibodies). (In the case of directional drugs) to kill cancer cells. External beam radiotherapy (xRT), often combined with chemotherapy, kills cancer cells by inducing cleavage of the nuclear DNA duplex, resulting in cell cycle death. Unlike systemic chemotherapy, xRT depends on the ability to accurately determine the anatomical site of the tumor. Surgical resection of a tumor also depends on the ability to examine the tumor and its complete removal, as residual tumor cells rapidly reconstruct the tumor after surgery. Since surgery and xRT are usually limited to the topical treatment of malignant tumors, there are limits to the treatment of disseminated or metastatic disease. This is why chemotherapy is often used in conjunction with these modes of treatment. Systemic chemotherapy can reach many distant metastases, with the exception of brain metastases, but for too many patients, the response is generally short-term (months to years) and final. Causes tumor recurrence.

Immunological techniques are rapidly becoming widespread in the cancer treatment paradigm, as the body's innate immune system can also destroy cancer cells after they are recognized. However, some cancer cells, and to a greater extent, cancer stem cells, initially attempt to circumvent immune surveillance, evolve by remaining relatively immunologically invisible, and eventually realize the ability to survive. [Gaipi et al., Immunotherapy 6: 597-610, 2014].

One particular immunological technique that is being investigated is "in-situ vaccination," that is, enhancing tumor immunogenicity, producing tumor-infiltrating lymphocytes (TILs), and "not vaccinated." It is a strategy that seeks to elicit a systemic anti-tumor immune response against disseminated tumors. In situ vaccination, malignant solid tumors are injected (or with it) one or more agents that promote the release of tumor antigens, while simultaneously providing pro-inflammatory signals that reverse the microenvironment that tolerates the tumor's immunity. Treat) [Pierce et al., Human Vaccines & Immunotherapeutics 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].

Another completely different approach is systemic immunotherapy. In systemic immunotherapy, an immunostimulant, such as an immune checkpoint inhibitor, is administered to circulate throughout the body (eg, intravenously) rather than being injected locally into the tumor. Such agents can be used to treat tumors that have an anti-tumor immune response but are "exhausted" or ineffective. In the case of checkpoint inhibitors, tumor cells express "checkpoint ligands" or other checkpoint molecules that interact with the "checkpoint receptors" of existing anti-tumor immune cells, inactivating these cells. cause. By blocking this interaction, systemically administered checkpoint inhibitors turn on the exhausted existing immune response of cancer patients and promote a more effective attack on cancer cells by the patient's own immune system. ..

Although recent data from clinical trials and preclinical models indicate the potential of these techniques, there is a great need for systemic immunotherapy methods that show improved systemic efficacy in the art. ing.

Radiation hormesis is a decade-old hypothesis that low-dose ionized RTs can be beneficial by stimulating the activation of natural protective and repair mechanisms that are not activated without ionized RTs [Cameron and Molder, Med. Phys. 25: 1407, 1998]. Preliminary repair mechanisms are hypothesized to be sufficiently effective when stimulated to not only counteract the adverse effects of ionized RT, but also to control diseases not associated with RT exposure. Perhaps related, the Abscopal effect is a phenomenon reported in the 1950s in which xRT treatment of one tumor actually causes shrinkage of another tumor outside the RT treatment area. Although rare, this phenomenon is thought to depend on the activation of the immune system. Taken together, the hormesis and abscopal effects support the potential for immune system interactions and stimulatory effects by low-dose (immune-stimulating but non-cytotoxic) RT. This can then be combined with other immunological techniques such as systemic immunotherapy.

We found that in the presence of a single tumor, a combination of local xRT + insitu vaccination and / or systemic checkpoint inhibitor immunotherapy is potent in treating large established tumors in mice. It was previously announced to be synergistic [Morris et al., Cancer Res; 76 (13); 3929-41, 2016]. Surprisingly, however, we have found that the combination of in situ vaccination and xRT does not suppress tumor growth in the presence of a second non-irradiated tumor. Apparently, non-irradiated tumors exhibit a suppressive effect on the immunomodulatory effects of xRT and in situ vaccines on irradiated tumors (we call them "accompanying immune tolerance").

When xRT is administered to the entire area of the tumor, this associated immune tolerance can be overcome, enabling the effectiveness of in situ vaccination. However, xRT can deliver xRT to all sites of the tumor if multiple tumors are present, especially if the number of tumors is not small, or if the site of one or more tumors is not exactly known. If this is not possible, it cannot be effectively used in combination with the insitu vaccination method. In addition, administration of xRT to all tumor sites in patients with metastatic disease is likely to result in systemic immunosuppression, which is not the main purpose of systemic immunotherapy.

Therefore, there is a need for an improved method of delivering immunomodulatory doses of RT to all tumors in a subject in combination with systemic immunotherapy, regardless of the number and anatomical site of the tumor.

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 full text of which is incorporated herein by reference, Weichert et al., The main agent 18- (p-iodophenyl) octadecyl for the detection, localization and treatment of various malignant solid tumors. The use of an analog of phosphocholine (NM404; see FIG. 1) is disclosed. If the iodine moiety is an imaging-optimized radionuclide, eg iodine-124 ([ ¹²⁴ I] -NM404), this analog is positron emission tomography of solid tumors-computed tomography (PET / CT). Alternatively, it can be used in single photon emission computed tomography (SPECT) imaging. Alternatively, the iodine moiety is ideal for delivering therapeutic doses of RT to solid tumor cells that incorporate analogs, such as iodine-125 or iodine-131 ([ ¹²⁵ I] -NM404 or [ ¹³¹ I] -NM404). If it is an iodinated radioactive nuclei, this analog can be used to treat solid tumors.

Such analogs not only target a wide variety of solid tumor types in vivo, but also undergo long-term selective retention in tumor cells, thus providing high potential as RT agents. In addition, tumor uptake is limited to malignant cancers, not premalignant or benign lesions.

However, there are metal isotopes that have better imaging and / or RT properties than the radioactive iodine isotopes used in the previously disclosed alkylphosphocholine analogs. For example, as an imaging isotope, I-124 has insufficient positron emission (only about 24% of the radiation is positron) and is further confused with gamma radiation (600 KeV). This actually interferes with normal 511 KeV PET detection. Certain positron emitting metals have good imaging properties. As another example, as an RT isotope, I-131 produces other non-therapeutic releases at other energies, which adds unwanted radiation dosimetry to adjacent normal tissues, including bone marrow. The beta particle range of I-131 is also very long, causing off-target toxicity. Some metal radiotherapy isotopes provide a cleaner release profile and shorter pathway lengths, thus less latent toxicity.

We have improved to include chelated radioactive metal isotopes in place of radioactive iodine isotopes (see, eg, US Pat. No. 2018/0022768, the full text of which is incorporated herein by reference). Alkylphosphocholine analogs have been developed. Since this analog contains the same skeleton as the previously disclosed radioiodinated compounds, they are still selectively taken up and retained by tumor cells. However, chelated radioactive metal isotopes have improved release for imaging and / or radiotherapy applications. Such agents are less than cytotoxic but very effective in delivering immunomodulatory doses of ionized RT to all malignancies present in the subject, with or without the number and anatomical site of the tumor. Suitable for.

Therefore, in the first aspect, the present disclosure includes a method of treating a cancer, including one or more malignant solid tumors, in a subject. This method involves systemic administration of immunomodulatory doses of targeted radiotherapy (TRT) and (b) one or more immunostimulants that are (a) differentially uptake and retained by malignant solid tumor tissue in the subject. Includes the step of administration.

In some embodiments, one or more immunostimulants are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules.

Non-limiting examples of one or more immune checkpoint inhibitors include the following checkpoint molecules: A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-related protein 4). ), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), PD-1 (programmed cell death receptor 1), PD-L1 (programmed cell death ligand 1), CD40 (differentiation antigen) Group 40), CD27 (differentiation antigen group 27), CD28 (differentiation antigen group 28), CD137 (differentiation antigen group 137), OX40 (CD134; differentiation antigen group 134), OX40L (OX40 ligand; differentiation antigen group 252), GITR (Glucocorticoid-induced tumor necrosis factor receptor-related protein), GITRL (Glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (Inducible T cell costimulation), ICOSL (Inducible T cell costimulation) Ligands), B7H3 (CD276; differentiation antigen group 276), B7H4 (VTCN1; V-set domain-containing T cell activation inhibitor 1), IDO (indolamine 2,3-dioxygenase), TIM-3 (T cell immunity) Drugs capable of targeting one or more of globulin and mutin domains 3), Gal-9 (galectin-9), or VISTA (V domain Ig suppressor for T cell activation).

In some embodiments, one or more immune checkpoint inhibitors include one or more anti-immune checkpoint molecular antibodies. In some such embodiments, one or more anti-immune checkpoint molecular antibodies include at least one monoclonal antibody.

In some embodiments, one or more immune checkpoint inhibitors include one or more small molecules capable of suppressing or blocking one or more immune checkpoint molecules. Non-limiting examples of such small molecule checkpoint inhibitors include CA-170 and CA-327, both of which target PD-L1.

In some embodiments, one or more anti-immune checkpoint molecular 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. Antibodies, 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, Included are anti-IDO antibody, anti-TIM-3 antibody, anti-Gal-9 antibody, or anti-VISTA antibody.

In some embodiments, the TRT agent is metaiodobenzylguanidine (MIBG), in which the iodine atom in the MIBG is a radioactive iodine isotope.

In some embodiments, the TRT agent is a radiolabeled tumor targeting antibody.

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

In some embodiments, the TRT agent has the formula:

A radioactive phospholipid ether metal chelate or a salt thereof. R ₁ contains a chelating agent chelated to a metal atom, which is an alpha, beta or Auger emitting metal isotope with a half life of more than 6 hours and less than 30 days, where a is 0 Or 1, n is an integer of 12-30, m is 0 or 1, Y is -H, -OH, -COOH, -COOX, -OCOX, or -OX, and X is alkyl or Aryl, R ₂ is -N ⁺ H ₃ , -N ⁺ H ₂ Z, -N ⁺ HZ ₂ , or -N ⁺ Z ₃ , each Z is independently alkyl or alloalkyl, 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.

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-triaza. Cyclononan-1,4-diacetic acid (NODA) or one of its derivatives, 1,4,7-triazacyclononan-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-glutaric acid-4,7 -Diacetic acid (NODAGA) or one of its derivatives, 1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-Triacetic acid (DOTAGA) or one of its derivatives, 1 , 4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (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, one of deferoxamine (DFO) or derivatives thereof, 1,2 - one of [[6-carboxy-2-yl] methylamino] ethane _(H 2 _dedpa) or a derivative thereof, And selected from the group consisting of one of DADA or its derivatives, DADA has the following structure:

Have.

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

In some embodiments, m is 1 (acylphospholipid series). In some such embodiments, n is an integer between 12-20. In some embodiments, Y is -OCOX, -COOX or -OX.

In some embodiments, X is -CH ₂ CH ₃ or -CH ₃ .

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

In some embodiments, b is 1.

In some embodiments, n is 18.

In some embodiments, R ₂ is −N ⁺ Z ₃ . In some such embodiments, each Z is independently -CH ₂ CH ₃ or -CH ₃ . In some such embodiments, each Z is -CH ₃ .

In some embodiments, the chelating agent chelated to a metal atom is:

In some embodiments, the radioactive phospholipid ether metal chelate is one of the following compounds in which the selected compound is chelated to a metal atom:

In some embodiments, in the phospholipid ether metal chelate structure, a is 1, b is 1, m is 0, n is 18, and R ₂ is −N ⁺ (CH ₃ ) _3. Is. In some such embodiments, the phospholipid ether metal chelate is an NM600 chelated to a metal atom, such as (but not limited to) ⁹⁰ Y-NM600.

In some embodiments, the TRT agent has the formula:
A radioactive halide phospholipid ether having the above or a salt thereof. R ₁ contains a radioactive halogen isotope, a is 0 or 1, n is an integer of 12-30; m is 0 or 1, Y is -H, -OH, -COOH, -COOX. , -OX, and -OCOX, where X in the above equation is alkyl or arylalkyl, R ₂ is -N ⁺ H ₃ , -N ⁺ H ₂ Z, -N ⁺ HZ ₂ , and Selected from the group consisting of −N ⁺ Z ₃ , each Z in the above equation is independently alkyl or aryl.

In some embodiments, the radioactive halogen isotope is ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At, ⁷⁶ Br, or ⁷⁷ Br.

In some embodiments, a is 1 and m is 0.

In some embodiments, n is 18.

In some embodiments, R ₂ is −N ⁺ (CH ₃ ) ₃ . In some embodiments, a is 1, m is 0, and n is 18. In some such embodiments, the radioactive halogen isotope is the radioactive halogen isotope ¹²³ I, ¹²⁴ I, ¹²⁵ I, or ¹³¹ I (radiohalogenated 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).

In some embodiments, a TRT agent, an immunostimulant, or both are administered intravenously.

In some embodiments, the subject is a human.

Non-limiting examples of cancers shown as malignant solid tumors that can be treated using this method include melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, Subcutaneous cancer, cutaneous or head and neck squamous cell carcinoma, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing sarcoma, rhizome myoma, osteosarcoma, Wilms tumor , And childhood brain tumors.

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

In some embodiments, the anti-GD2 antibody is not administered to the subject.

In a second aspect, the disclosure includes the use of a TRT agent and an immunostimulant to treat a cancer, including one or more malignant solid tumors, in a subject, as further described above.

In a third aspect, the disclosure, as further described above, is a TRT agent and / or one or more immunity for the manufacture of a drug for treating a cancer containing one or more malignant solid tumors in a subject. Includes the use of activators.

Other objects, features and advantages of the present invention will become apparent after reviewing the specification, claims and drawings.

The patent or application documents include at least one colored drawing. A copy of the patent or publication of the application, including this one or more color drawings, can be obtained by requesting the Patent Office and paying the required fee.

It is a figure which shows the chemical structure of the main agent 18- (p-iodophenyl) octadecylphosphocholine (NM404). It is a figure which shows the graph which shows that xRT + IT-IC induces in situ tumor vaccination. More specifically, FIG. 2A shows a tumor growth curve showing a synergistic effect between xRT and IT-hu14.18-IL2. 71% (22/31) of mice treated with xRT + IT-IC are disease-free. It is a figure which shows another graph which shows that xRT + IT-IC induces in situ tumor vaccination. More specifically, FIG. 2B shows the Kaplan-Meier survival curve showing a synergistic effect between xRT and IT-hu14.18-IL2. 71% (22/31) of mice treated with xRT + IT-IC are disease-free. It is a figure which shows another graph which shows that xRT + IT-IC induces in situ tumor vaccination. More specifically, FIG. 2C shows that 90% of treated mice reject subsequent engraftment by B78 melanoma. It is a figure which shows the graph which shows the accompanying immune tolerance. The response of the primary tumor is shown. Untreated distant tumors suppress the response to xRT + IT-IC in a two-tumor B78 melanoma model. This suppression can be overcome by irradiating a second tumor (overcome be radiating). FIG. 5 shows a graph showing that the associated immune tolerance is due to Tregs. The response of the primary tumor is shown. Untreated distant tumors suppress the response to xRT + IT-IC in a two-tumor B78 melanoma model, which suppresses Treg depletion (using transgenic DEREG mice expressing diphtheria toxin receptors on Tregs, diphtheria It can be overcome by (depleting Tregs by administering toxins). It is a figure which shows the image which shows the selective uptake of ¹²⁴ I-NM404 by B78 melanoma. Mice with a B78 tumor of about 200 mm ³ were intravenously administered IV ¹²⁴ I-NM404 and subjected to continuous PET / CT scans. This image at 71 hours shows selective uptake by the tumor and some residual background uptake by the heart and liver. FIG. 5 shows a graph showing that in situ vaccination can be induced in the presence of residual levels of molecularly targeted radiation therapy (TRT). Combination treatment with xRT + IT-IC is equally effective with or without 3 μCi ¹³¹ I-NM404. This is the residual radiation of TRT that would be present when we deliver xRT (day 0) followed by IT-IC (days 6-10), as described in Example 4. It is almost the same as Noh. It is a figure which shows the transition of the MRI image with time which shows the growth of the tumor (T) up to 24 hours after the injection of Gd-NM600 of the mouse having a tumor. FIG. 5 shows tumor-specific suppression of primary tumor response to a combination of local RT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice with (GD2 +) primary flank tumors expressing allogeneic disialogangliosides +/- contralateral abdominal secondary tumors, as shown, on day 1 only for primary tumors. Treated by xRT and on days 6-10 by intratumoral (IT) injection of 50 mcg of anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2). The average volume of the primary tumor is shown in FIG. 8A. More specifically, FIG. 8A shows that in mice with primary B78 melanoma tumors, the presence of untreated secondary B78 tumors antagonized the primary tumor response to RT + IT-IC. We describe this effect as "accompanying immune tolerance", an antagonism of untreated distant tumors to the local response of treated tumors to xRT + IT-IC. Another figure showing tumor-specific suppression of primary tumor response to a combination of local RT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice with (GD2 +) primary flank tumors expressing allogeneic disialogangliosides +/- contralateral abdominal secondary tumors, as shown, on day 1 only for primary tumors. Treated by xRT and on days 6-10 by intratumoral (IT) injection of 50 mcg of anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2). More specifically, FIG. 8B shows the Kaplan-Meier survival curve for mice and repeat experiments. Almost all mice were euthanized due to the progression of the primary tumor. Another figure showing tumor-specific suppression of primary tumor response to a combination of local RT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice with (GD2 +) primary flank tumors expressing allogeneic disialogangliosides +/- contralateral abdominal secondary tumors, as shown, on day 1 only for primary tumors. Treated by xRT and on days 6-10 by intratumoral (IT) injection of 50 mcg of anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2). More specifically, FIG. 8C shows the presence of untreated Panc02 secondary tumor in mice with primary Panc02-GD2 + pancreatic tumor, with or without secondary Panc02-GD2-tumor on the contralateral abdomen. It shows that the response of the primary Panc02-GD2 + tumor to RT + IT-IC was suppressed. Another figure showing tumor-specific suppression of primary tumor response to a combination of local RT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice with (GD2 +) primary flank tumors expressing allogeneic disialogangliosides +/- contralateral abdominal secondary tumors, as shown, on day 1 only for primary tumors. Treated by xRT and on days 6-10 by intratumoral (IT) injection of 50 mcg of anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2). More specifically, FIG. 8D shows that in mice with a primary B78 melanoma tumor, the secondary B78 tumor suppressed the primary tumor response to RT + IT-IC, whereas the secondary Panc02-GD2 + pancreatic tumor exerted this effect. Indicates that it did not work. Another figure showing tumor-specific suppression of primary tumor response to a combination of local RT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice with (GD2 +) primary flank tumors expressing allogeneic disialogangliosides +/- contralateral abdominal secondary tumors, as shown, only in the primary tumor on day 1 Treated by xRT and on days 6-10 by intratumoral (IT) injection of 50 mcg of anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2). More specifically, FIG. 8E shows that in mice bearing the primary Panc02-GD2 + tumor, the secondary Panc02-GD2-tumor suppressed the primary tumor response to the combination of xRT and IT-hu14.18-IL2. It shows that the B78 secondary tumor was not suppressed. n = number of mice per group. NS = not significant, ^*** p <0.001. FIG. 5 shows immunohistochemical images (left and center) and graphs (right) showing that associated immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, FIG. 9A shows mice having one or two tumors (FIG. 9A, leftmost panels A1 and A2) or two tumors (FIG. 9A, central panels A3 and A4) 6 days after xRT. The immunohistochemistry of FoxP3, a Treg marker of the evaluated tumor (typical 400x image is shown) is shown. Mice did not receive xRT or received xRT only for primary tumors. Primary tumors are shown in panels A1 to A3 of FIG. 9A, and secondary tumors are shown in panels A4 of FIG. 9A. Small arrows point to some FoxP3 + cells (brown core = FoxP3 +, blue = hematoxylin counterstain). The graph on the right shows the blind test quantification of FoxP3 + cell count per 200x field of view, corresponding to the conditions shown in panels A1, A2, A3 and A4 of FIG. 9A, respectively. FIG. 5 shows immunohistochemical images (left and center) and graphs (right) showing that associated immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, FIG. 9A shows mice having one or two tumors (FIG. 9A, leftmost panels A1 and A2) or two tumors (FIG. 9A, central panels A3 and A4) 6 days after xRT. The immunohistochemistry of FoxP3, a Treg marker of the evaluated tumor (typical 400x image is shown) is shown. Mice did not receive xRT or received xRT only for primary tumors. Primary tumors are shown in panels A1 to A3 of FIG. 9A, and secondary tumors are shown in panels A4 of FIG. 9A. Small arrows point to some FoxP3 + cells (brown core = FoxP3 +, blue = hematoxylin counterstain). The graph on the right shows the blind test quantification of FoxP3 + cell count per 200x field of view, corresponding to the conditions shown in panels A1, A2, A3 and A4 of FIG. 9A, respectively. It is another graph showing that the associated immune tolerance is avoided by the specific depletion of regulatory T cells (Tregs). More specifically, FIG. 9B shows that DEREG mice express diphtheria toxin receptors under the control of the Treg-specific FoxP3 promoter, allowing specific depletion of Treg during IP injection of diphtheria toxin. DEREG mice with primary and secondary B78 melanoma tumors were treated with xRT + IT-IC into the primary tumor and IP injection of either diphtheria toxin or PBS (first repeat experiment shown). Concomitant immune tolerance is eliminated after Treg depletion in these mice, improving the response of primary tumors (Fig. 9B). n = number of mice per group. ^** p <0.01, ^*** p <0.001. It is another graph showing that the associated immune tolerance is avoided by the specific depletion of regulatory T cells (Tregs). More specifically, FIG. 9C shows that DEREG mice express diphtheria toxin receptors under the control of the Treg-specific FoxP3 promoter, allowing specific depletion of Treg during IP injection of diphtheria toxin. DEREG mice with primary and secondary B78 melanoma tumors were treated with xRT + IT-IC into the primary tumor and IP injection of either diphtheria toxin or PBS (first repeat experiment shown). Concomitant immune tolerance is eliminated after Treg depletion in these mice, improving the response of secondary tumors (Fig. 9C). n = number of mice per group. ^** p <0.01, ^*** p <0.001. FIG. 5 shows a graph showing that associated immune tolerance is overcome by delivering xRT to both tumor sites. In mice with primary and secondary B78 tumors, the secondary tumor suppresses the primary tumor response to primary tumor treatment with xRT + IT-IC. This was overcome by delivering 12 Gy xRT to both primary and secondary tumors and IT-IC to the primary tumor, resulting in a repeat-to-primary tumor response (first repeat). Is shown) has been improved (Fig. 10A). n = number of mice per group. ^** p <0.01, ^*** p <0.001. FIG. 5 shows another graph showing that concomitant immune tolerance is overcome by delivering xRT to both tumor sites. In mice with primary and secondary B78 tumors, the secondary tumor suppresses the primary tumor response to primary tumor treatment with xRT + IT-IC. This was overcome by delivering 12 Gy xRT to both primary and secondary tumors, and IT-IC to primary tumors, resulting in increased total animal survival from repeated experiments. Improved (Fig. 10B). n = number of mice per group. ^** p <0.01, ^*** p <0.001. Graphs showing that low doses of xRT alone do not induce in situ vaccination but actually overcome the associated immune tolerance when delivered to a distant tumor site with 12 Gy + IT-IC treatment at the in situ vaccine site. It is a figure which shows. More specifically, FIG. 11A shows that in mice with only primary B78 tumors, 12Gy + IT-IC induced in situ vaccination (as already shown) and complete tumor regression in most mice (book). Experiments have shown that it results in 4/6) and a memorable 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. Another indication that low doses of xRT do not induce in situ vaccination alone, but actually overcome the associated immune tolerance when delivered to a distant tumor site with 12 Gy + IT-IC treatment at the in situ vaccine site. It is a figure which shows two graphs. More specifically, FIG. 11B shows that in mice with primary and secondary B78 melanoma tumors, low doses xRT (2 Gy or 5 Gy) delivered to the secondary tumors were associated with immune tolerance in the primary tumors. It shows that its ability to overcome is comparable to 12 Gy. Another indication that low doses of xRT do not induce in situ vaccination alone, but actually overcome the associated immune tolerance when delivered to a distant tumor site with 12 Gy + IT-IC treatment at the in situ vaccine site. It is a figure which shows two graphs. More specifically, FIG. 11C shows that overcoming the associated immune tolerance by delivering a low dose xRT to a secondary tumor in these same animals rescues the systemic response to IT-IC immunotherapy. Indicates that is clear. In this regard, when xRT is delivered to all tumor sites, IT-IC injection of the primary tumor causes a systemic antitumor effect, resulting in a secondary tumor response to 2 Gy or 5 Gy of the primary tumor. Greater than the response to 12 Gy xRT without IT-IC injection. In the figure showing PET images showing that low dose TRT and ¹³¹ I-NM404 effectively deplete tumor infiltrating FoxP3 + Treg without systemic leukopenia or without depleting tumor infiltrating CD8 + effector T cells. is there. In most clinical scenarios, delivering external beam radiation to all tumor sites without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression, is not feasible, even at low doses. is there. Here, can the inventors specifically administer TRT systemically to specifically deplete tumor infiltration-suppressing immune cells (Tregs) without causing systemic immune cell depletion and leukopenia? I tested it. More specifically, FIG. 12A shows that dosimetric studies in this B78 melanoma tumor model using positron emission ¹²⁴ I-NM404 confirm tumor selective uptake of NM404. C57BL / 6 mice with B78 tumors were treated with 60 μCi ¹³¹ I-NM404. This radioactivity is approximately the same as the amount of ¹³¹ I-NM404 required to deliver approximately 2 Gy of TRT to the B78 tumor. Peripheral blood and tumor samples were collected in untreated control mice (named C) and then at 8-day intervals (T1 = d8, T2 = d16, T3 = d24, T4 = d32). FIG. 6 shows a bar graph showing that low dose TRT and ¹³¹ I-NM404 effectively deplete tumor infiltrating FoxP3 + Treg without systemic leukopenia or without depleting tumor infiltrating CD8 + effector T cells. .. In most clinical scenarios, delivering external beam radiation to all tumor sites without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression, is not feasible, even at low doses. is there. Here, can the inventors specifically administer TRT systemically to specifically deplete tumor infiltration-suppressing immune cells (Tregs) without causing systemic immune cell depletion and leukopenia? I tested it. More specifically, FIG. 12B shows that this dose of TRT did not cause significant systemic leukopenia. Another bar graph showing that low dose TRT and ¹³¹ I-NM404 effectively deplete tumor infiltrating FoxP3 + Treg without systemic leukopenia or without depleting tumor infiltrating CD8 + effector T cells. It is a figure. In most clinical scenarios, delivering external beam radiation to all tumor sites without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression, is not feasible, even at low doses. is there. Here, can the inventors specifically administer TRT systemically to specifically deplete tumor infiltration-suppressing immune cells (Tregs) without causing systemic immune cell depletion and leukopenia? I tested it. More specifically, FIG. 12C shows that this dose of TRT had no significant effect on the level of tumor infiltrating CD8 + effector T cells (ANOVA p = 0.25). Another bar graph showing that low dose TRT and ¹³¹ I-NM404 effectively deplete tumor infiltrating FoxP3 + Treg without systemic leukopenia or without depleting tumor infiltrating CD8 + effector T cells. It is a figure. In most clinical scenarios, delivering external beam radiation to all tumor sites without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression, is not feasible, even at low doses. is there. Here, can the inventors specifically administer TRT systemically to specifically deplete tumor infiltration-suppressing immune cells (Tregs) without causing systemic immune cell depletion and leukopenia? I tested it. More specifically, FIG. 12D shows that the tumor infiltrating FoxP3 + Treg was significantly depleted by this dose of TRT (ANOVA p = 0.03; ^* p <0.05). FIG. 5 shows a graph showing that low-dose TRT and ¹³¹ I-NM404 effectively overcome associated immune tolerance and rescue the systemic antitumor effect of in situ vaccination. Low dose ¹³¹ I-NM404 TRT is, mouse taking into account the ability to deplete tumor infiltrating Treg without the leukopenia, we effectively immune tolerance to low dose ¹³¹ I-NM404 is accompanied Tested to see if it could be overcome. C57BL / 6 mice with two B78 tumors were treated with 60 mcCi ¹³¹ I-NM404 on day 1 as shown (NM404). After one half-life (8 days), animals were given 12 Gy of xRT to the primary tumor (in situ vaccine site) or no xRT. Control mice that did not receive ¹³¹ I-NM404 were treated as shown (0, 2, or 12 Gy) for secondary tumors. On days 13-17, mice were administered to the primary tumor (in situ vaccine site) as indicated by daily IT injections of IC. More specifically, FIG. 13A demonstrates a primary tumor response, effectively overcoming the immune tolerance associated with administration of low dose TRT and relieving the systemic antitumor effect of in situ vaccination. To do. FIG. 5 shows another graph showing that low-dose TRT and ¹³¹ I-NM404 effectively overcome associated immune tolerance and rescue the systemic antitumor effect of in situ vaccination. Low dose ¹³¹ I-NM404 TRT is, mouse taking into account the ability to deplete tumor infiltrating Treg without the leukopenia, we effectively immune tolerance to low dose ¹³¹ I-NM404 is accompanied Tested to see if it could be overcome. C57BL / 6 mice with two B78 tumors were treated with 60 mcCi ¹³¹ I-NM404 on day 1 as shown (NM404). After one half-life (8 days), animals were given 12 Gy of xRT to the primary tumor (in situ vaccine site) or no xRT. Control mice that did not receive ¹³¹ I-NM404 were treated as shown (0, 2, or 12 Gy) for secondary tumors. On days 13-17, mice were administered to the primary tumor (in situ vaccine site) as indicated by daily IT injections of IC. More specifically, FIG. 13B demonstrates a secondary tumor response, effectively overcoming the immune tolerance associated with administration of low dose TRT and relieving the systemic antitumor effect of in situ vaccination. To do. It is a figure which shows the chemical structure of the alkylphosphocholine metal chelate ( ⁶⁴ Cu-NM600) which becomes an example. Other metals may be used in place of ⁶⁴ Cu. It is a figure which shows the PET / CT image from the scan performed 48 hours after the injection with ⁸⁶ Y-NM600 of two 1-tumor B78 mice. FIG. 5 shows PET / CT images from a scan performed 48 hours after injection in ⁸⁶ Y-NM600 of two 2-tumor B78 mice. FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection of U87MG mice with ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection of 4T1 mice with ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans of HCT-116 mice performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection in ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans of A549 mice performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection in ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) of PC-3 mice injected with ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection of HT-29 mice with ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) of MiaPaca mice after injection with ⁶⁴ Cu-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection of 4T1 mice with ⁸⁶ Y-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel) and 48 hours (right panel) after injection of 4T1 mice with ⁸⁹ Zr-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 4 hours (left panel) and 1 day (right panel) after injection of HT-29 mice with ⁵² Mn-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown on the far right). FIG. 5 shows PET / CT images from scans performed 4 hours (left panel) and 1 day (right panel) after injection of PC-3 mice with ⁵² Mn-NM600. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown to the right of each image). HT-29 mice were injected 2 days (left panel), 3 days (second panel from left), 5 days (second panel from right) and 7 days (right panel) after injection with ⁵² Mn-NM600. It is a figure which shows the PET / CT image from a broken scan. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown to the right of the image). Lined 2 days (left panel), 3 days (second panel from left), 5 days (second panel from right) and 7 days (right panel) after injection of PC-3 mice with ⁵² Mn-NM600. It is a figure which shows the PET / CT image from a broken scan. These images show tissue radioactivity calculated as injection volume / g tissue percentage (% ID / g, scale shown to the right of the image). ^It is a graph which shows the PET quantitative range of the target data (chelate uptake as a function of time) about 4T1 tumor tissue of 4T1 mouse injected with ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600 and ⁸⁹ Zr-NM-600. .. FIG. 5 is a graph showing the PET quantification range of target data (chelate uptake as a function of time) for heart tissue of 4T1 mice injected with ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600 and ⁸⁹ Zr-NM-600. FIG. 5 is a graph showing the PET quantification range of target data (chelate uptake as a function of time) for liver tissue of 4T1 mice injected with ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600 and ⁸⁹ Zr-NM-600. It is a figure which shows the PET quantification range of the target data (chelate uptake as a function of time) about the whole body of 4T1 mice injected with ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600 and ⁸⁹ Zr-NM-600. Exvivo of healthy and tumor tissue of 4T1 mice 48 hours ( ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600, ⁸⁹ Zr-NM-600 and ¹⁷⁷ Lu-NM600) and 96 hours ( ¹⁷⁷ Lu-NM600) after injection of metal chelate It is a figure which shows the bar graph which shows the distribution in the chelate body in. It is a figure which shows the chemical structure of the alkylphosphocholine metal chelate ( ¹⁷⁷ Lu-NM600) which becomes an example. Other metals may be used in place of ¹⁷⁷ Lu. It is a figure which shows the audioradiograph image (audioradiographic image) of three B78 mice taken 48 hours after the injection of ⁹⁰ Y-NM600. Xenograft B78 tumors appear as large black dots in the lower right corner of each mouse image. It is a figure which shows the audio-radiography image of three B78 mice taken 96 hours after injection of ⁹⁰ Y-NM600. Xenograft B78 tumors appear as large black dots in the lower right corner of each mouse image. It is a figure which shows the audioradiography image of the B78 mouse taken on the 5th day after injection by ¹⁷⁷ Lu-NM600. Xenograft B78 tumors appear as two black dots in the lower part of the mouse. It is a figure which shows the audioradiography image of the B78 mouse taken on the 13th day after the injection by ¹⁷⁷ Lu-NM600. Xenograft B78 tumors appear as two black dots in the lower part of the mouse. It is a figure which shows the audio-radiography image of the MiaPaca mouse taken 10 days after the injection by ¹⁷⁷ Lu-NM600. The location of xenograft MiaPaca tumors is indicated by arrows and dashed circles. It is a figure which shows the audioradiography image of three 4T1 mice taken 48 hours after the injection of ¹⁷⁷ Lu-NM600. The location of the xenograft 4T1 tumor is indicated by an arrow and a dashed circle. It is a figure which shows the audioradiography image of three 4T1 mice taken 96 hours after the injection of ¹⁷⁷ Lu-NM600. The location of the xenograft 4T1 tumor is indicated by a dashed circle. It is a figure which shows the audio-radiography image of three 4T1 mice taken 4 hours after the injection of ⁹⁰ Y-NM600. The location of the xenograft 4T1 tumor is indicated by an arrow and a dashed circle. It is a figure which shows the audio-radiography image of three 4T1 mice taken 48 hours after the injection of ⁹⁰ Y-NM600. Xenograft 4T1 tumors appear as large black dots in the lower right corner of each mouse image. It is a figure which shows the audio-radiography image of three 4T1 mice taken 96 hours after the injection of ⁹⁰ Y-NM600. Xenograft 4T1 tumors appear as large black dots in the lower right corner of each mouse image. FIG. 5 shows a graph illustrating the effect of radiotherapy on ⁹⁰ Y-NM600 at two different doses (150 μCi and 300 μCi) in a B78 xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. FIG. 5 shows a graph illustrating the effect of a single 500 μCi dose of ¹⁷⁷ Lu-NM600 radiotherapy in a B78 xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. FIG. 5 shows a graph illustrating the effect of a single 400 μCi dose of ¹⁷⁷ Lu-NM600 radiotherapy in a MiaPaca xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. FIG. 5 shows a graph illustrating the effect of a single 500 μCi dose of ¹⁷⁷ Lu-NM600 radiotherapy in a 4T1 xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. ^* P <0.05; ^** P <0.01; ^*** P <0.001. FIG. 5 shows a graph illustrating the effect of radiotherapy on two consecutive doses (500 μCi and 250 μCi) of ¹⁷⁷ Lu-NM600 in a 4T1 xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. FIG. 5 shows a graph illustrating the effect of radiotherapy on two different doses (500 μCi and 250 μCi) of ¹⁷⁷ Lu-NM600 in a 4T1 xenograft mouse model compared to controls (excipients only). The data are presented as a measured tumor volume (mm ³ ) as a function of time expressed in days after injection. It is a figure which shows the influence of the tumor amount on the comparison of the therapeutic effect of ⁹⁰ Y-NM600 and ¹³¹ I-NM404 in the conventional TRT. FIG. 6 shows a bar graph comparing the average albumin binding energies of three different metal chelate analogs of NM404, along with amine analogs. For comparison, the I-NM404 binding energy is shown as a dotted line. Tumor volume in B78 melanoma flank tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or targeted radiotherapy (TRT) agent Y90-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi) It is a figure which shows the graph which shows mm ³ ) as a function of time (day). Control mice were dosed with vehicle (PBS) without anti-CTLA4 or TRT agents. After 18 days, combined treatment with 50 or 100 μCi Y90-NM600 and anti-CTLA4 significantly reduced tumor growth (p <0.05 at ANOVA) compared to PBS, Y90-NM600 alone, or anti-CTLA4 alone. I let you. The 25 μCi Y90-NM600 combination treatment group with anti-CTLA-4 showed a moderate growth retardation response that tended to dose response. Overall animal survival of mice treated with the combination of TRT (50 μCi Y90-NM600) and checkpoint block (anti-CTLA4) compared to mice treated with TRT only, checkpoint block only (anti-CTLA4), or PBS vehicle. It is a figure which shows the graph which shows. FIG. 5 shows a graph showing overall animal survival of mice treated with three different combinations of checkpoint blockade (anti-CTLA4) and TRT (25 μCi, 50 μCi, and 100 μCi Y90-NM600). Tumors of B78 melanoma flank tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or molecular-targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi) It is a figure which shows the graph which shows the volume (mm ³ ) as a function of time (day). Control mice received a vehicle (PBS) without anti-CTLA4 or MTRT agents. Combination treatment with 50 or 100 μCi ⁹⁰ Y-NM600 and anti-CTLA4 significantly reduced tumor growth (p <0.05 at ANOVA) compared to PBS, ⁹⁰ Y-NM600 alone, or anti-CTLA4 alone. It was. Total of B78 melanoma flank tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or molecular-targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi) It is a figure which shows the graph which shows the animal survival rate. Mice treated with the combination of MTRT (50 μCi ⁹⁰ Y-NM600 or 100 μCi ⁹⁰ Y-NM600) and checkpoint blockade (anti-CTLA4) showed significantly increased survival rates compared to the other groups. Tumor volume (mm ³ ) of NXS2 neuroblastoma tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi molecular targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600 time (day) It is a figure which shows the graph which shows as a function of. Control mice were dosed with vehicle (PBS) without anti-CTLA4 or TRT agents. Combination treatment with ⁹⁰ Y-NM600 MRTT and anti-CTLA4 significantly reduced tumor growth compared to PBS, ⁹⁰ Y-NM600 alone, or anti-CTLA4 alone. Tumor volume (mm ³ ) of 4T1 breast cancer tumor mice treated with anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi molecular targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600 as a function of time (days) It is a figure which shows the graph which shows. Control mice were dosed with vehicle (PBS) without anti-CTLA4 or TRT agents. Combination treatment with ⁹⁰ Y-NM600 MRTT and anti-CTLA4 significantly reduced tumor growth compared to PBS, ⁹⁰ Y-NM600 alone, or anti-CTLA4 alone. In the figure which shows the tumor volume (mm ³ ) of the irradiated primary B78 tumor as a function of time (day) of the B78 melanoma flank tumor mouse having both the primary tumor and the secondary (distant) tumor. is there. Mice were subjected to primary tumor-only EBRT (12 Gy, secondary tumor shielded), anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi molecular-targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600. Treated in various combinations. The combination treatment of 12 Gy EBRT, ⁹⁰ Y-NM600 MRT, and anti-CTLA4 significantly reduced primary tumor growth compared to the other groups. FIG. 5 shows a graph showing the tumor volume (mm ³ ) of a shielded secondary (distant) B78 tumor in B78 melanoma flank tumor mice with both primary and secondary tumors as a function of time (days). .. Mice were subjected to primary tumor-only EBRT (12 Gy, secondary tumor shielded), anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi molecular-targeted radiotherapy (MTRT) agent ⁹⁰ Y-NM600. Treated in various combinations. Combined treatment with EBRT, ⁹⁰ Y-NM600 MRTT, and anti-CTLA4 for primary tumors significantly reduced secondary tumor growth compared to the other groups.

I. Overview It is understood that the specific methodologies, protocols, materials, and reagents described are subject to change and this disclosure is not limited thereto. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is limited only by non-provisional applications filed later. ..

As used herein and in the appended claims, the singular forms "a", "an", and "the" include references to the plural form unless explicitly indicated in the context. .. Similarly, the terms "a" (or "an"), "one or more" and "at least one" may be used synonymously herein. The term "comprising" and its variants have no limiting meaning as long as these terms appear herein and in the claims. Therefore, the terms "comprising", "inclusion", and "having" can be used synonymously.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any method or material similar or equivalent to that described herein can be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications and patents specifically referred to herein describe and disclose the chemicals, instruments, statistical analyzes and methodologies reported in publications that may be used in connection with the present invention. Incorporated by reference for any purpose, including. All references cited herein are to be construed as indicating the skill of one of ordinary skill in the art.

The terminology described herein is for illustration purposes only and should not be construed as limiting the invention as a whole. Unless otherwise noted, "a," "an," "the," and "at least one" are used synonymously to mean one or more.

The present disclosure comprises the compounds described herein (including intermediates) in any of their pharmaceutically acceptable forms, in which isomers (eg, diastereomers and enantiomers), each other. Includes variants, salts, solvates, polymorphs, prodrugs and the like. In particular, when the compound is optically active, the invention specifically comprises each of the enantiomers of the compound, as well as a racemic mixture of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated (although occasionally "salt" is explicitly stated). ..

As used herein, "pharmaceutically acceptable" means that the compound or composition or carrier achieves the treatment described herein without unduely harmful side effects in view of the need for treatment. Means that it is suitable for administration to a subject.

The term "effective amount" as used herein refers to the amount or dose of a compound that elicits a biological or medical response to a subject, tissue or cell sought by a researcher, veterinarian, physician or other clinician. ..

As used herein, "pharmaceutically acceptable carriers" include all kinds of dry powders, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption retarders and the like. A pharmaceutically acceptable carrier is a useful substance for administering a compound in the methods of the invention. The material is preferably non-toxic, may be a solid, liquid or gaseous material, is otherwise inert and pharmaceutically acceptable and compatible with the compounds of the invention. Examples of such carriers include, but are not limited to, various lactose, mannitol, oils such as corn oil, buffers such as PBS, physiological saline, polyethylene glycol, glycerin, polypropylene glycol, dimethylsulfoxide, etc. Examples include amides such as dimethylacetamide, proteins such as albumin, and surfactants such as Tween80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.

The term "administering" or "administration" as used herein provides a compound or pharmaceutical composition of the invention to a subject suffering from or at risk of a disease or condition to be treated or prevented. It means to do.

In pharmacology, the route of administration is the route by which a drug is taken up by the body. Routes of administration are usually classified according to the site to which the substance is applied. Common examples include oral and intravenous administration. Pathways can also be classified based on where the target of action is. The action is local, enteral (systemic action, but delivered through the gastrointestinal tract), or parenterally via the lungs by inhalation (systemic action, but delivered by a route other than the gastrointestinal tract). It may be there.

In enteral administration, the desired effect is systemic (nonlocal) and the substance is administered via the gastrointestinal tract. With parenteral administration, the desired effect is systemic and the substance is administered by routes other than the gastrointestinal tract.

Enteral administration may be administration that involves any part of the gastrointestinal tract and has a systemic effect. Examples include administration of many drugs by mouth (oral), tablets, capsules, or drops, administration of many drugs and enteral nutrition by gastric feeding tube, duodenal feeding tube, or gastrostomy. , As well as administration of various drugs in suppositories into the rectum.

Examples of parenteral administration are intravenous (intravenous) administration, eg, many drugs, intraarterial (intravenous) administration of central arterial feeding, eg, vasodilators in the treatment of vasospasm. And thrombolytic agents for the treatment of embolism, intraosseous injection (intramedullary), intramuscular, intracerebral (intracranial), intraventricular (intraventricular), Intramuscular administration (injection into the spinal canal) and subcutaneous (under the skin) administration can be mentioned. Of these, intraosseous infusion is essentially indirect intravenous access because the bone marrow is drained directly into the venous system. Intraosseous infusions are occasionally used for drugs and fluids in emergency medicine and pediatrics when intravenous access is difficult.

The following abbreviations are used in this disclosure: ADCC, antibody-dependent cell-mediated cytotoxicity; anti-CTL4, targeting cytokinetic T-lymphocyte-related antigen 4 (CTLA4) found in cytotoxic T-lymphocytes (CTL). Antibodies; B16, C57Bl / 6 mice and syngeneic melanoma; B78, variants of B16 expressing GD2 by transfection with GD2 synthase; D, Sun; Hu14.18-IL2, disclosed in Examples. Primary immunocytokines used in the study (reacts to GD2); ICs, immunocytokines (fusion proteins of tumor-reactive mAbs bound to IL2); ICIs, immune checkpoint inhibitors; IL2, interleukin-2; IT , Intratumor; IV, intravenous; mAb, monoclonal antibody; MAHA, mouse antihuman antibody; NM404, used to refer to the phospholipid ether shown in FIG. 1, selectively taken up and performed by most tumors. Used for TRTs in the studies disclosed in the examples; NM600, used to refer to the phospholipid ethers shown in FIG. 14, can be chelated with any metal, which is also selective by most tumors. Used in TRTs in studies disclosed in the Examples; NXS2, neuroblastomas of the same lineage as AJ mice; Panc02-GD2, C57Bl / 6 mice expressing GD2 for transfection with GD2 synthase. Pancreatic cancer of the same family; PLE, phospholipid ether; RT, radiotherapy; TRT, targeted radiotherapy; W, week; 9464D-GD2, syngeneic with C57Bl / 6 mice expressing GD2 for transfection with GD2 synthase Neuroblastoma.

II. The present invention relates to a method of treating any cancer present as one or more malignant solid tumors. The disclosed method combines the two treatment steps, resulting in a much improved effect on malignant solid tumors by an unexpected synergistic effect. Specifically, when an immunomodulatory dose of a radiophospholipid metal chelate compound or a radiohalide phospholipid compound, which is differentially taken up and retained by malignant solid tumor tissue, is administered to a patient, an immunostimulatory agent. Systemic administration (eg, by intravenous injection) of a composition comprising one or more agents capable of stimulating specific immune cells, with or without additional xRT for at least one of the malignant solid tumors treated in By doing so, further immunomodulation is performed.

Immunomodulatory doses of radiophospholipid metal chelate or radiohalogenated compounds are likely to reduce Treg levels (and other immunosuppressive factors) and xRT is used for one tumor and one or more additional It prevents the deterioration of the immune system (accompanying immune tolerance) that occurs when the tumor is not irradiated, but understanding of the mechanism is not necessary for the practice of the present invention, and the present invention is not limited to a specific mechanism of action.

A. Systemic immunotherapy: An example immunocheckpoint inhibitor as an immunostimulant A method of immunoactivation by direct administration of an immunomodulator to a tumor (eg, in situ shown in some of the examples below). In contrast to intratumoral immunization by vaccination), systemic immunotherapy is performed by systemic administration of an immunostimulant. The immunostimulant circulates throughout the subject and stimulates the body's natural immune response.

Immune checkpoint inhibitors are an unrestricted example of such an immunostimulant. Activated T cells are co-suppressive receptors for multiple immunosuppressive receptors such as lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and cytotoxic T lymphocyte-related protein 4 (CTLA4). Express the body. These and other immune checkpoint molecules have been shown to regulate T cell responses to tumor antigens in the tumor microenvironment via unique non-overlapping pathways.

More specifically, cancer growth is partially mediated by cancer-induced immunosuppression. Tumors can activate immunosuppressive checkpoint pathways to reduce the general immune response to tumors. Therefore, blocking of the major immune checkpoint pathway can induce anti-tumor immunity promoted by the patient's own immune system.

CTLA4 was the first immune checkpoint molecule to be clinically targeted by administration of a (anti-CLA4) mAb that targets CTLA4. So far, the most promising immune checkpoint inhibitor strategy for the treatment of cancer involves administration of mAbs that target CTLA-4 and / or PD-1 / PD-L1. Other immune checkpoint inhibitor strategies are currently under development and the concomitant methods of the present disclosure are not limited to targeting specific immune checkpoint pathways.

A series of reviews covering checkpoint inhibitors and cancer immunotherapy was recently published in Volume 276 of Immunological Reviews. An introductory overview, Sharpe, A. et al. H. , "Introduction to checkpoint inhibitors and cancer immunotherapy", Immunol Rev. 276 (March 4, 2017): These reviews, including 5-8, are incorporated herein by reference in their entirety.

B. Immunomodulatory doses of radioactive phospholipid metal chelate compounds The radioactive phospholipid metal chelate compounds used are RTs released by metal isotopes chelated to metal chelate compounds, which substantially impose other histological types on the released RTs. A wide range of solid tumor cell types should be selectively targeted so that they can be directed to malignant solid tumor tissues without exposure. The radioactive metal isotope included in the radioactive phospholipid metal chelate compound may be any radioactive metal isotope known to release ionized RT in a manner that results in immunoactivation of the cells that take up 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, Examples include Ac-225, Pb-212, or Th-227.

The immunomodulatory RT dose of the radioactive phospholipid metal chelate compound (as opposed to the infusion volume) is much lower than the dose traditionally used for RT for malignant solid tumors. Specifically, the dose does not eliminate the desired immune cells responsible for the immunostimulatory effect, while at the immune cells within the tumor microenvironment (perhaps by reducing immunosuppressive Treg levels and other immunosuppressive cells or molecules). It should be sufficient to stimulate the response.

The appropriate immunomodulatory dose can be calculated from the image data obtained after administration of the "promoting detection" dose of the radioactive metal chelate compound. The detection-promoting dose can be quite different from the immunomodulatory dose, and the radiometal isotopes incorporated into the radiometal chelate compound can be different (but the rest of the compound structure should be the same). .. The radioactive metal isotope used in the detection steps and dosimetry calculations may be any radioactive metal isotope known to emit RT in a form immediately detectable 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, Examples include Ga-67, In-111, or Tc-99m.

C. Metal chelate of PLE analog The disclosed structure utilizes an alkylphosphocholine (APC) carrier backbone. Once synthesized, the agent should have formulation properties that preserve tumor selectivity as previously demonstrated with the associated radiohalogenated compounds while at the same time making them suitable for injection. The disclosed structures include chelating moieties that chelate with radioactive metal isotopes to produce the final imaging or therapeutic agent.

D. Examples of Methods for Synthesizing M-PLE Analogs The proposed synthesis of Compound 1 is shown below. The first steps of synthesis are similar to those described in Org Synth, 2008, 85, 10-14. Synthesis starts with cyclen and converts it to DO3A tris-Bn ester. This intermediate is then coupled with NM404 in the presence of base and Pd catalysts. Finally, the benzyl protecting group is removed by catalytic hydrogenation.

The synthesis of compound 2 is shown below. Synthesis begins with the DO3A tris-Bn ester, which is alkylated with 3- (bromo-propa-1-ynyl) -trimethylsilane. After alkylation, the trimethylsilyl group is removed and the intermediate acetylene is coupled to NM404 by a sonogashira reaction. The benzyl group is removed and the triple bond is simultaneously hydrogenated in the final step of synthesis.

Compounds 5 and 6 can be synthesized from the same precursors, DTPA dianhydride and 18-p- (3-hydroxyethyl-phenyl) -octadecylphosphocholine, as shown in the scheme below.

The NOTA-NM404 conjugate can be synthesized in a similar manner. One example is the following NOTA-NM404 conjugate 7.

E. Dosage Form and Method of Administration Any route of administration will be suitable for synergistic targeting RT. In one embodiment, the disclosed alkylphosphocholine analogs may be administered to the subject via intravenous injection. In another embodiment, the disclosed alkylphosphocholine analogs are presented via any other suitable systemic delivery, such as parenteral, nasal, sublingual, rectal, or transdermal administration. May be administered to.

In another embodiment, the disclosed alkylphosphocholine analogs may be administered to the subject via the nasal system or mouth, eg, by inhalation.

In another embodiment, the disclosed alkylphosphocholine analogs may be administered to the subject via intraperitoneal or IP injection.

In certain embodiments, the disclosed alkylphosphocholine analogs may be provided as pharmaceutically acceptable salts. However, other salts may be useful in the preparation of alkylphosphocholine analogs or pharmaceutically acceptable salts thereof. Suitable pharmaceutically acceptable salts include, but are not limited to, solutions of alkylphosphocholine analogs, hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, etc. Examples include acid addition salts that can be formed by mixing with a solution of a pharmaceutically acceptable acid such as benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

If the disclosed alkylphosphocholine analogs have at least one asymmetric center, they may be present as enantiomers accordingly. If the disclosed alkylphosphocholine analogs have more than one asymmetric center, they may be present as diastereoisomers accordingly. It is understood that all such isomers and mixtures thereof in any proportion are included within the scope of the present disclosure.

The disclosure also includes methods of using pharmaceutical compositions comprising one or more disclosed alkylphosphocholine analogs with a pharmaceutically acceptable carrier. Preferably, these compositions are tablets, rounds, capsules, powders, granules, sterile parenteral solutions or suspensions, quantitative aerosols or liquid sprays, drops, ampoules, automatic injection devices or suppositories. It is in the form of a unit dosage form for parenteral administration, intranasal administration, sublingual or rectal administration, or administration by inhalation or spraying, such as a drug.

To prepare solid compositions such as tablets, the main active ingredient is a pharmaceutically acceptable carrier, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, stearic acid. A solid that is mixed with magnesium, dicalcium phosphate or rubber, and other pharmaceutical diluents such as water to contain a homogeneous mixture for the compounds of the invention, or pharmaceutically acceptable salts thereof. Preliminary preparation composition of. When these prescription compositions are referred to as homogeneous, it is easy to make the composition into equally effective unit dosage forms such as tablets, pills and capsules because the active ingredient is evenly dispersed throughout the composition. It means that it can be subdivided. This solid prescription composition is then subdivided into the above types of unit dosage forms containing 0.1 to about 500 mg of the active ingredient of the invention. A typical unit dosage form contains 1-100 mg, eg, 1, 2, 5, 10, 25, 50 or 100 mg of active ingredient. The tablets or pills of the new composition may be coated or otherwise formulated to provide a dose that gives the benefit of long-term action. For example, tablets or pills can contain an inner and outer dosage component, the latter in the form of a jacket over the former. The two components can be separated by an enteric layer, which helps resist decomposition in the stomach and helps the inner component enter or delay release into the duodenum without damage. A variety of materials can be used for such enteric layers or coatings, such materials include a large number of polymeric acids, as well as polymeric acids and such materials as cellac, cetyl alcohol and cellulose acetate. Contains a mixture of.

Liquid forms in which alkylphosphocholine analogs may be incorporated for oral or injectable administration include aqueous solutions, appropriately flavored syrups, aqueous or oily suspensions, and cottonseed oil, sesame oil, coconut oil or peanut oil. Flavored emulsions containing edible oils such as, as well as elixirs and similar pharmaceutical excipients. Suitable dispersants or anti-precipitation agents for aqueous suspensions include synthetic and natural rubbers such as tragacant rubber, arabic rubber, alginate, dextran, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone or gelatin.

The disclosed alkylphosphocholine analogs are particularly useful when formulated in pharmaceutically injectable dosage forms, including combinations with injectable carrier systems. As used herein, the injectable and injectable dosage forms (ie, parenteral dosage forms) are lipid bilayers with phospholipids that enclose a liposome injection or active drug substance, but not limited to. Multilayer vesicles can be mentioned. Injections include sterile preparations intended for parenteral use.

There are five different classes of injections as defined by USP: emulsions, lipids, powders, solutions and suspensions. Emulsion injections include emulsions containing sterile pyrogen-free preparations intended for parenteral administration. Lipid complexes and powders for solution injection are sterile preparations intended for reconstitution to form solutions for parenteral use. Suspension injection powders are sterile preparations intended for reconstitution to form suspensions for parenteral use. The lyophilized powder for liposomal suspension injection is a sterile freeze-dried preparation intended for reconstitution for parenteral use in which the formulation is formed upon reconstitution, which can be in a lipid bilayer or It is formulated to allow the incorporation of liposomes, such as lipid bilayer vesicles, that have phospholipids used to encapsulate the active drug substance in the aqueous space. A lyophilized powder for solution injection involves the process of removing water from the frozen product at very low pressure, and subsequent addition of the liquid produces a solution that is in all respects compatible with the requirements of the injection. A dosage form for solutions prepared by lyophilization (“freeze-drying”). A lyophilized powder for suspension injection is a liquid preparation intended for parenteral use that contains a solid suspended in a suitable liquid medium, and it freezes the agent intended for suspension. It meets all the requirements of sterile suspensions prepared by drying. Solution injections include liquid preparations containing one or more APIs dissolved in a mixture of suitable or miscible solvents suitable for injection.

Solution concentrate injections include sterile preparations for parenteral use that, when the appropriate solvent is added, produce a solution that meets the requirements of the injection in all respects. Suspension injection is a liquid preparation (suitable for injection) containing solid particles dispersed throughout the liquid phase, in which the particles are insoluble and the oil phase is dispersed throughout the aqueous phase or vice versa. including. Suspended liposome injections form liposomes (lipid bilayer vesicles usually containing phospholipids used to encapsulate the active drug substance either in the lipid bilayer or in the aqueous space). As such, it is a liquid preparation (suitable for injection) in which the oil phase is dispersed throughout the aqueous phase. Suspension sonication injections are liquid preparations (suitable for injection) containing solid particles dispersed throughout the liquid phase, in which the particles are insoluble. Moreover, the product may be sonicated as the gas is blown into the suspension, resulting in the formation of microspheres of solid particles.

Parenteral carrier systems include one or more pharmaceutically suitable excipients such as solvents and co-solvents, solubilizers, wetting agents, suspensions, thickeners, emulsifiers, chelating agents, buffers, pH adjusters. Includes agents, antioxidants, reducing agents, antibacterial preservatives, fillers, protective agents, isotonic agents, special additives and the like.

The following examples are provided for explanatory purposes only and are by no means intended to limit the scope of the invention. Indeed, in addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the above description and examples below and fall within the appended claims. ..

III. Examples Introduction to Examples These examples show the potential to combine two very different cutting-edge disciplines in cancer treatment research that take advantage of unexpected and very strong synergies. These disciplines are 1) systemic TRT and 2) locally administered antibody-mediated cancer immunotherapy or systemic cancer immunotherapy. The data presented herein suggest that a strong synergistic effect is created by combining these methods. Taken together, these two strategies are strong immunity in which destroyed cancer cells generate tumor-specific T-cell immunity that can eradicate persistent residual metastatic disease against any type of solid tumor at any site. It can be used to destroy visible macroscopic tumors in a way that allows it to function as an activator.

Our ongoing preclinical studies have shown that the combination of tumor-specific mAbs and IL2 (to activate innate immune cells) enhances antibody-dependent cell-mediated cytotoxicity (ADCC). [1, 2]. This is a process that has already been converted into clinical benefits for children with neuroblastoma [3]. Recent preclinical data show stronger antitumor efficiency when the mAb-IL2 fusion protein is injected intratumorally (IT) [4, 5]. Notably, large tumors that do not respond to these mAb / IL2 injections and continue to grow when treated with topical xRT alone can be completely eradicated when xRT is combined with mAb / IL2 treatment. Most mice have healed and developed T-cell memory that rejects re-attacks by similar tumor cells [6]; the combined xRT + mAb / IL2 acts as a potent "in situ" anticancer vaccine. It shows that it is.

An important limitation is that if these animals receive xRT + mAb / IL2 treatment for the primary (first) tumor and another macroscopic tumor is present, the second tumor will continue to grow and be surprising. In particular, it suppresses the immune response and prevents the tumor from shrinking after the first treatment. This "accompanying immune tolerance" is due in part to the inhibitory regulatory T cells (Tregs) of the second tumor. Delivery of RT alone to both tumors has minimal antitumor effects, but depletes these Tregs. Thus, when the first tumor is treated with xRT + mAb / IL2, adding RT to the second tumor avoids this immune tolerance and allows eradication of both tumors [7]. These findings indicate a limitation of in situ tumor vaccination in metastatic situations, but also suggest a strong ability of RT to overcome this limitation.

xRT cannot generally be delivered to all metastatic sites without banned normal tissue toxicity and immunosuppression. However, failure to deliver xRT to all sites of gross disease can leave the inhibitory immune system intact, which provides an immunological response to our local xRT + mAb / IL2 immunotherapy. It can be suppressed. Therefore, what is needed is a means of delivering RT to all tumor sites in cancer patients in a targeted manner.

We have developed a TRT excipient that can target systemically administered RT to both primary and metastatic cancers. One such TRT reagent, ¹³¹ I-NM404, is an intravenously (IV) administered phospholipid ether (PLE) analog that is a nearly universal tumor target in over 60 in vivo cancer and cancer stem cell models. The chemical characteristics were shown. This reagent is currently being clinically evaluated in multiple imaging and clinical trials [8, 9]. ¹³¹ Systemic injection of I-NM404 disrupts intratumoral immunosuppressive pathways that can be concentrated in all tumors, regardless of anatomical site, and prevent the generation of an effective tumor eradication immune response. Provides sufficient RT internally. A unique feature of this approach is the near-universal tumor targeting capacity of NM404, as well as the ability to deliver sublethal immunomodulatory doses of RT to all tumor sites, which is generally not feasible with xRT. .. What is new about this is that our TRT agents can immunomodulate all tumors, regardless of anatomical site, and overcome the associated tolerance, thereby local xRT and subsequent. A long-term inventor tumor vaccination effect is provided after injection of tumor-specific mAb + IL2. As more and more tumor-specific mAbs are being approved for clinical use, this combination strategy provides an extension of the approach to any tumor type that can be targeted by tumor-reactive mAbs. To do. Moreover, this approach can be easily generalized to all in situ tumor vaccination strategies.

In recent years, the inventors have found that iodine in ¹³¹ I-NM404 can be replaced with a chelating agent capable of having a wide variety of metal imaging (MRI and PET) and TRT radiotherapy moieties. In these examples, the combined treatment of ¹³¹ I-NM404 (and associated metal chelate analogs) with xRT + immunotherapy allows it to elicit a strong radioimmune-promoting response to cancerous solid tumors (timours). We describe a method of assessing the ability to initiate a systemic immunomodulatory response required for this. Similar techniques can be used in combination with TRT delivered to PLE analogs with other immunotherapies used for cancerous solid tumors. For example, the inventors have found that the combination method may use an immunomodulation step that is significantly different from local in-situ tumor vaccination, i.e. systemic administration of an immunostimulant agent such as an immune checkpoint inhibitor. Is shown below.

In short, we disclose here a therapeutic and research process that combines two different methods, which are seemingly separated from the discipline of cancer treatment, into one integrated treatment. The data presented in these examples show that these two methods can be synergistically combined to effectively eliminate malignant solid tumors and prevent tumor recurrence.

In Example 1, the inventors present background data obtained from the B78 GD2 + model as support for the method.

In Example 2, we determine the dose of xRT required for optimal in situ vaccine effect on primary tumors and the minimum dose of xRT required for distant tumors to prevent concomitant immune tolerance. Provide guidance.

In Example 3, we determine a dose of ¹³¹ I-NM404 that is close to the required dose of xRT for metastasis, as determined in Example 2, followed by a dose of ¹³¹ I-NM404. Provides guidance for assessing the effects of iodine on in vivo immune function. Such guidance may be similarly applied when using the disclosed radioactive phospholipid metal chelate compounds as TRT agents.

In Example 4, we used the data obtained from Examples 2 and 3 to destroy locally treated tumors in mice with two or more tumors and of all distant tumors. Provides guidance for designing / testing / developing a dosing regimen of ¹³¹ I-NM404 + topical xRT + IT-mAb / IL2 to induce T cell-mediated eradication. A significant issue of TRT and xRT dose and time has been optimized for antitumor efficiency. Again, such guidance can be applied similarly when the disclosed radioactive phospholipid metal chelate compounds are used as TRT agents.

In Example 5, the inventors provide an exemplary synthesis that also finds application in the synthesis of analog compounds chelating radioactive metal isotopes.

In Example 6, we found that analogs with chelating agents and chelated metals used in place of the iodine moiety of NM404 (Gd-NM600) were incorporated into solid tumor tissue (and within solid tumors). Can be imaged in) and thus provides a proof of concept for the use of disclosed metal chelates as TRT agents.

In Examples 7, 8, 9 and 10, the inventors provide information and specific data obtained from empirical studies conducted according to the guidance of Examples 1-4.

In Examples 11 and 12, we found that additional analogs in which the chelating agent and chelated metal were replaced with the iodine moiety of NM404 were incorporated into various solid tumors in an in vivo model and various solids. It can be imaged in tumors and can be used therapeutically for TRT in various solid tumors, thus providing further proof of concept for the use of the disclosed metal chelates as TRT agents in the disclosed methods. Show that.

In Example 13, we will consider how dosimetry can be used in combination with known radiosensitivity to allow one of ordinary skill in the art to optimize treatment doses for any solid tumor.

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

Examples 15 and 16 show that TRT in combination with systemic immunotherapy is also more effective in treating solid tumors than in situ vaccination. The systemically administered immunostimulant may be an immune checkpoint blocker or inhibitor (in this case, anti-CTLA4).

Example 1: Background-supporting data The Sondel lab found that tumor-specific mAb + IL2 activates innate immune cells to mediate ADCC in mice [2] and is clinically beneficial for children with neuroblastoma. [3] It was shown that. In mice, IV administration of hu14.18-IL2 was more potent than IV administration of anti-GD2 mAb + IL2 [2, 10]. This technique can provide dramatic antitumor effects on tiny, recently established GD2 + tumors or tiny microscopic level metastases, in patients in remission but at high risk of recurrence. Potentially describes the clinical use of [3]. Stronger antitumor efficiency for measurable macroscopic tumors [ie, GD2 + tumors of about 50 mm ³ ] can be achieved when the IC is intratumorally injected (IT-IC) rather than IV [4, 5]. ].

We are currently looking at ways to benefit in the context of much larger macroscopic tumors. Mice with a medium-sized (200 mm ³ ) B78 melanoma tumor established 5 weeks ago did not respond to IV-IC, although IT-IC slowed its growth. , The tumor continues to grow. These same 200 mm ³ tumors also grow after 12 Gy xRT. In contrast, the combination of IT-IC and xRT eliminated 73% of animal tumors and appeared to cure the disease (FIGS. 2A and 2B). These mice then show T cell-mediated rejection of reloading by the same tumor (Fig. 2C). Therefore, IT-IC + xRT exerts a synergistic effect and induces the tumor to become an "in situ tumor vaccine" [6].

To simulate clinical metastasis, we inoculate B78 into the flank of mice on day 1 and the other flank on week 2. At 5 weeks, the first tumor is 200 mm ³ and the second tumor is 50 mm ³ . We predicted that xRT + IT-IC would destroy the first tumor and the resulting T cell response would then destroy the second tumor. However, the addition of IT-IC to xRT had virtually no effect on either 50 mm ³ tumors or 200 mm ³ tumors (Fig. 3). This has shown an important limitation to the therapies provided by the inventors; that is, if another tumor is present when these mice receive xRT + IT-IC in the first tumor, a second tumor Is to cause a systemic tumor-specific concomitant immune tolerance effect and prevent the shrinkage of both tumors. Importantly, we first developed an immune response in which local xRT (12 Gy) to the first and second tumors simultaneously suppressed this tolerant effect and eradicated both tumors in most mice. It was found that it induces IT-IC for tumors (Fig. 4) [7]. Recent data using transgenic mice (FIG. 4) [7] that allow Treg depletion mAbs (not shown) or selective Treg depletion show that this immune tolerance is partially mediated by regulatory T cells (Tregs). Indicates that it will be done. RTs to the first and second tumors potentially deplete these Tregs and potentially explain how irradiation of both tumors avoids tolerant effects [7].

Local xRT for both first and second tumors avoids tolerance, but clinical metastatic disease is often present at several sites. All gross metastatic diseases must undergo RT to prevent immune tolerance and allow xRT + IT-IC to effectively eradicate all tumor sites. However, delivering 12 Gy xRT to all diseased sites can be close to "systemic RT" with major dose-dependent (potentially lethal) toxicity and severe systemic immunosuppression.

Previously, the Weichert laboratory pioneered the development of TRT to deliver RT to all systemic tumor sites while at the same time minimizing "off-target" RT to normal tissue, especially bone marrow and immune tissue. did.

Based on the finding that tumor cells contain excess phospholipid ethers (PLE) [11], we aim to identify analogs that selectively target tumors with over 30 radioactive iodines. A PLE analog was synthesized [12]. One of these, NM404, showed almost universal tumor uptake in all but three of the more than 70 in vivo models investigated, regardless of anatomical sites including brain metastases and cancer stem cells. Instead, when they invaded tumor cells, they were selectively retained for long periods of time [8]. These diagnostic and therapeutic PLE analogs are unique in that they avoid premalignant and inflammatory lesions. Surface lipid raft, which is overexpressed in cancer cells compared to normal cells, serves as a gateway for PLE containing NM404 to enter cancer and cancer stem cells [8]. Radioiodinated NM404s (I-124 and I-131) have now been evaluated in five Phase 1 and Phase 2 PET imaging studies and three Phase 1 TRT radiotherapy trials, respectively, in more than a dozen humans. It exhibits similar tumor uptake and retention properties in cancer types [8]. Excellent tumor uptake in the cancer model associated with these examples (B78 GD2 + mouse melanoma) was confirmed by ¹²⁴ I-NM404 PET imaging (FIG. 5).

Example 2: Determining xRT Dosage Our data suggest these four hypotheses: (1) The dose of xRT used to treat one tumor is modest direct in vivo tumor death. And increase susceptibility to immune-mediated death (due to both ADCC and T cells); (2) The strong T cell response elicited by the addition of IT-IC rather than IT mAbs in the presence of IL2. It is suggested that mAbs that bind to irradiated tumor cells promote enhanced antigen presentation and induction of adaptive immunity; (3) immunosuppressive cells present in the second tumor in the presence of the second tumor. Due to the tolerability primarily caused by systemic effects of [eg Treg and possibly myeloid suppressor cells (MDSCs)], xRT + IT-IC on the first tumor does not cause virtually any antitumor effect; This tolerance can be avoided by depletion of Treg (FIG. 4) or irradiation of the second tumor (FIG. 3); (4) the RT required in the second tumor to avoid tolerance. The dose may be much lower than the xRT dose required for the first tumor to become an "in situ vaccine" [14].

Optimization of xRT dose at the site of the primary (“in situ”) tumor site

Our in vivo study of xRT + IT-IC has focused on a single dose of 12 Gy for the first tumor. This was linked to our in vivo data demonstrating that the in situ vaccine effect of xRT + IT-IC requires mice with functional Fas-L, in vitro RT of Fas in B78 tumor cells. Based on our data showing that it induces dose-dependent functional upregulation (near the peak of> 12 Gy) (6). We conducted an in vivo pilot study before selecting a dose of 12 Gy. It showed that high doses (16 Gy) or increased split flank RT were toxic (dermatitis, ulcers, and hindlimb edema) and showed no improvement in tumor response. We have selected a 12 Gy single fraction xRT for in vivo studies to safely and effectively induce in situ vaccine effects as we proceed to clinical interpretation. It would be beneficial to better understand the mechanism of the local xRT effect and its dose conditions.

Our mouse data (FIGS. 2A, 2B and 2C) show that 12 Gy xRT + IT-IC can induce a potent vaccine effect, even if 12 Gy xRT alone does not cause tumor shrinkage. Show; it only slows progressive growth. We believed that there would be as strong an in situ vaccine effect as when lower doses of RT were used. To test this, we inventor a series of xRT doses (4-16 Gy) as a single dose in mice with a B78 tumor of about 200 mm ³ followed by our standard IT-IC. The dosing regimen (50 mcg / day on days 6-10) is evaluated. We determine which xRT dose results in optimal tumor eradication and T cell memory when used in combination with IT-IC. Where doses less than 12 Gy are less toxic and show comparable efficacy, such low doses are better targets for our xRT dose to the "in situ vaccine" site of Examples 3 and 4. Will be. Dosing may be optimized for a particular target or subject using similar techniques.

Optimization of xRT dose in distant tumors to prevent tolerance from blocking "in situ vaccination".

Treatment of both the first and second tumors with 12 Gy (FIG. 3) allows IT-IC to the first tumor to induce a strong response to eradicate both tumors. .. An object of the present inventors is to provide xRT + IT-IC to one tumor, and at the same time, it is possible to achieve this same in situ vaccine effect by using the minimum required RT dose at the metastatic site to avoid tolerance. Is to do. We recognize that xRT itself can be bone marrow / immunosuppressive, especially if it is widespread. Therefore, we are tracking TRT in Examples 3 and 4. The TRT makes some systemic delivery of RT, even if it is targeted. To minimize systemic immunosuppression by TRT, we need to effectively suppress tumor-induced immune tolerance without causing systemic RT-induced global immunosuppression. He wants to administer as low a dose of TRT as possible. Therefore, to deliver to distant tumors, higher xRT doses to the first tumor, when used in combination with IT-IC to the first tumor, to allow it to function as an in situ vaccine. It is best to choose the minimum dose of xRT required.

As an example optimization experiment, mice with a first B78 tumor of 200 mm ^{3 and} a second B78 tumor of about 50 mm ³ had 12 Gy on day 0 (about 5 weeks after transplantation of the first B78 tumor). XRT is administered to the first tumor. Following this, on days 6-10, the standard IT-IC administration schedule of the present inventors is performed. Different groups of mice receive different doses of xRT on the second tumor. B. shows that 3 Gy systemic xRT can prevent immunosuppressive effects in myeloma models. Based on data from Johnson's lab (15), we evaluate doses of 0, 1, 5 and 8 Gy (in addition to the 12 Gy doses currently known to be effective). We will determine whether doses substantially less than 12 Gy for the second tumor are as effective as total doses of 12 Gy in eliminating immune tolerance.

Once we have selected a marginal dose of xRT that loses its beneficial effect, we perform the following analysis to better optimize the marginal dose. For example, if 5 Gy is as effective as 12 Gy, but 1 Gy is not much different from 0 Gy, we have two tumor models in which 12 Gy + IT-IC is administered to the first tumor. 3 and 4 Gy are compared to determine the marginal minimum effective RT dose required to eliminate tolerance and gain efficacy.

This to the second tumor is then performed in a repetitive study, where the dose to the first tumor is not the 12 Gy dose but the minimum working dose in one tumor model (tested in Example 2 above). See if the minimum working dose still enables the in situ vaccine. In short, the study of Example 2 optimizes the minimum dose of xRT for the first and second tumors at 12 Gy for both without losing the efficacy we have demonstrated.

Begin study of xRT dose required for first and second tumors in mice with tumors other than B78.

We will initiate the analysis of RT + IT-IC with additional models of GD2 + tumors so that our mouse studies can show more clinical generalizability. We have published an IT-IC with hu14.18-IL2 IC in AJ mice with GD2 + NXS2 neuroblastoma [5]. We also developed IT-IC with this same IC in C57BL / 6 mice with GD2 + 9464D-GD2 neuroblastoma and Panc02-GD2 pancreatic cancer that expresses GD2 by inserting the GD2 synthase gene. I'm evaluating. For Example 2, for each model, we determine the minimum effective xRT dose required for primary and secondary tumors to retain in situ vaccine efficacy.

Example 3: Determination of dose of ¹³¹ I-NM404 and measurement of immune function dose by TRT in C57BL / 6 mice and evaluation of effect on immunosuppression from TRT
¹³¹ I-NM404 showed selective uptake in vitro in> 95% of tumor strains (human and mouse), low uptake by non-malignant cells, and similar tumor specificity in vivo. This includes in vivo selective uptake by B78 tumors (Fig. 5). In a preliminary dosimetry study, we administered ¹²⁴ I-NM404 to C57BL / 6 mice and characterized the time course of TRT exposure by continuous PET / CT imaging (as in FIG. 5). Monte Carlo dosimetry calculations based on this study [16-18] showed that approximately 60 μCi of ¹³¹ I-NM404 was required to deliver approximately 3 Gy to established B78 tumors over a 4-week decay period. It was. After these 4 weeks, the remaining TRT dose to the B78 tumor will be less than 0.25 Gy. We replicate the data obtained in the two-tumor model using xRT (Fig. 3), but it is possible to effectively eliminate tumor-induced tolerance at all sites of distant disease. To use the minimum possible dose of the targeted ¹³¹ I-NM404 TRT. However, unlike xRT, which occurs after delivering all doses in minutes, TRT has both a biological and physical half-life of the target isotope (8 days t1 / 2 for ¹³¹ I). Correspondingly, the dose is accumulated over time. We are seeking an initial TRT effect at a distant tumor site to eradicate immune tolerance. However, we want to minimize the immunosuppressive effect of TRT when IT-IC is administered to induce ADCC and in situ vaccine antitumor effects. This is essential for complete tumor destruction at all sites.

Using dosimetry calculations obtained from our preliminary data, we found that a dose of 3 μCi, ¹³¹ I-NM404, should deliver an amount equivalent to about 0.2 Gy to the tumor site. Estimated. The doses we have assumed should not be immunosuppressive and should not prevent lymphocyte-mediated tumor destruction. As mentioned above, this is the dose estimated by us to not yet be delivered 28 days after the first ¹³¹ I-NM404 dose of 60 μCi. In this way, we evaluated a group of mice with a single 200 mm ³ B78 tumor. On day 0, all mice received 12 Gy xRT into their tumors, and on days 6-10, all mice received 50 mcg / day IT-IC. One group also received 3 μCi of ¹³¹ I-NM404 on day 0 (approximately 0.2 Gy). ^6, 131 I-NM404 group administered ^is indicative that showed tumor eradication comparable to the group not administered the 131 I-NM404, is "residual" TRT of the low dose of the tumor, Demonstrate that it does not prevent immune-mediated disruption by the RT + IT-IC in situ vaccine. Therefore, we used an initial dose of 60 μCi of ¹³¹ I-NM404 TRT on day 22 to effectively block the immunotolerant effect of distant tumors and further on day 0 for the first tumor. It is assumed that xRT of the eye and IT-IC on days 6-10 (28 days after TRT) function as an inventor vaccine and then induce an adaptive response to eradicate all tumors.

The experiments outlined in this example optimize the dose correlation tested in FIG. In our one-tumor B78 model, we test the dose range of ¹³¹ I-NM404 TRT to prevent the desired in situ vaccine effect (and thereby delay the eradication of the first tumor? Or select the best TRT dose that results in undesired systemic immunosuppression sufficient to prevent). This is important for Example 4. This is because it can be confirmed that the residual radioactivity of TRT is attenuated to be smaller than this value when IT-IC to the first tumor is started in the mouse having a distant disease. We also evaluated the rate of TRT response after various TRT doses and administered "TRT doses that interfere with tolerance" to animals with multiple tumors before RT + IT-IC of the first tumor. Select the optimal time period to wait so that treatment can still induce in situ vaccine efficacy and eradicate primary and distant tumors.

A related study also looks at which dose of TRT given as a monotherapy is most beneficial for slowing, shrinking, or eradicating a single B78 tumor. The dose of TRT that is most beneficial for eliminating tumor-induced immune tolerance will be substantially less than the TRT dose required to actually induce complete tumor lysis (from TRT alone).

Finally, once the effects of various optimized doses of TRT are determined in a single tumor model, we will evaluate the sera of these subjects for the immune response of the IC to the human IgG component to reduce the amount of TRT. Evaluate the immunosuppressive effect. We have shown that immunoqualified mice produce levels of mouse anti-human antibody (MAHA) that are readily quantified after treatment with these humanized ICs (19). We use this as a means of determining the dose at which TRT is expected to cause a detectable dose-dependent decrease in the strength of the mouse's immune response, and these mice receive from this TRT. The overall immunosuppressive effect of systemic administration of RT is measured. The low TRT doses we need to block tumor-induced immune tolerance will cause minimal systemic immunosuppression.

Example 4: Development of optimal dosing regimen for ¹³¹ I-NM404 + local xRT + IT-mAb / IL2 in mice with 2 or more tumors 2 Examination of the efficacy of TRT + RT + IT-IC in a tumor B78 model.

The dose and timing information obtained from the studies outlined in Examples 2 and 3 provides the information needed to optimize the TRT dosing and timing required for efficacy in our two-tumor model. Bring. C57BL / 6 mice are simultaneously inoculated with B78 on the left (L) and right (R) flanks. Each tumor should be about 50 mm ³ after 2 weeks and about 200 mm ³ after 5 weeks. If we believe that a 60 μCi TRT needs to be delivered to a second tumor to approach a 3 Gy RT in the dosimetric calculation of Example 3 (to prevent immune tolerance), we These external irradiation xRT studies predict that this dose should have a minimal slowing effect on tumor growth. We would plan to treat different groups of mice with 30, 60 or 90 μCi at 2 weeks (if the tumor is about 50 mm ³ ). After 3 weeks, the tumor should be about 200 mm ³ ; at that time we administer xRT (the dose determined as outlined in Example 2) followed by 6 days (of TRT). Approximately 28 days later), the tumor in the L flank is administered with IT-IC injection 5 times daily to induce the in situ vaccine effect. Predicting the absence of in situ vaccine in control mice due to tolerance from distant tumors, TRT is not administered and only xRT and IT-IC are administered to the L flank. In another group, local xRT is administered to both tumors and IT-IC is administered to the L flank, predicting the eradication of both tumors due to the in situ vaccine effect. Another group receives TRT + IT-IC, but not local xRT, in anticipation of incomplete vaccine efficacy.

Follow-up experiments further evaluate the timing variations between various doses of TRT and TRT and local xRT + IT-IC to the primary tumor (L flank). The readings are (A) eradication of primary tumors; (B) eradication of secondary tumors; and (C) systemic immunosuppression by ELISA analysis of MAHA responses. Our aim is to add a topical xRT + IT-IC dosing regimen that can eradicate both tumors in most subjects while minimizing systemic immunosuppression (as measured by the MAHA response). , Identifying optimal TRT dose and timing by specific subject and disease model.

Optimization of TRT + xRT + IT-IC in mice with 3 or more B78 tumors.

This section of Example 4 has a relevant clinical context, ie, injectable tumors that can be used as in situ vaccine sites, but each has multiple distant metastases that can cause tumor-induced immune tolerance. Most similar to patients with. These studies will reproduce the conditions found to be most effective in the first part of Example 4 (above). An important difference is that each of these subjects has four separate tumors on the flanks of L and R and on the lateral shoulders of L and R. TRT is administered at the dose and timing found to be most effective in the study outlined in the first section of Example 4, with xRT + IT-IC subsequently administered only to L flank lesions. Since TRT effectively eliminates tumor-induced immune tolerance caused by three sites not receiving xRT, the objective here is to enable the most effective in situ vaccine of TRT dose and timing. Choosing a problem. The measure of efficacy is the removal of all four tumors in most subjects. Test TRT dose and timing modifications to develop the most effective optimized dosing regimen. Such dosing regimens are found to be used in the clinic for patients with multiple distant metastases. All of the multiple distant metastases cannot be irradiated by an external beam, but can be irradiated by TRT when used in combination with a local xRT + IT-IC to the "in situ vaccine" site.

Example 5: Synthesis of Metal Chelated NM600 In this example, we present a synthetic scheme used to synthesize one example phospholipid chelate, Gd-NM600. Analogs incorporating various radioisotopes can be synthesized in a similar manner, and the radioisotope in question is used in place of Gd.

Scheme for synthesizing Gd-NM600 (disclosed radioactive metal isotopes can be used in place of Gd):

Example 6: Proof of Concept by In vivo Imaging In this example, we use Gd-NM600 as an MRI contrast agent to show successful in vivo MRI imaging of tumors. The data demonstrate that skeletal phospholipids and chelators are taken up and retained by solid tumors, and that such chelates incorporating various radioactive metals disclosed herein exhibit similar properties. To demonstrate.

Proof of concept for tumor uptake of Gd-NM404 agents For in vivo imaging, thymus-deficient nude mice with xenografts of flank A549 tumor (non-small cell lung cancer) were scanned. Gd-NM600 (2.7 mg) was delivered by tail intravenous injection. Mice were anesthetized and scans were performed prior to contrast administration and 1, 4, 24, 48, and 72 hours after contrast delivery. Imaging was performed with a 4.7T Varian preclinical MRI scanner equipped with a quadrature volume coil. The following pulse sequence parameters: repeat time (TR) = 206 ms, echo interval = 9 ms, echo train length = 2, effective echo time (TE) = 9 ms, average number 10 and field of view 40 x 40 mm ² , 192 × 192 matrices, 10 sections each 1 mm thick were used to obtain T1-weighted images at all imaging time points.

As can be seen in FIG. 7, MRI imaging of the tumor was significantly enhanced by 24 hours after injection.

These results demonstrate that differential uptake and retention of alkylphosphocholine analogs is maintained with respect to the metal chelated analogs disclosed herein. Therefore, the disclosed metal chelates can be readily applied for clinical treatment and imaging applications.

Example 7: Experiment to determine the dose of xRT required for optimal insitu vaccine effect on primary tumors and the minimum dose of xRT required for distant tumors to prevent concomitant immunological tolerance Follow-up studies on Examples 1-4 Dose titration experiments were performed to evaluate various xRT doses for mice with 1 or 2 tumors. The first objective is to test the dose of xRT required to promote synergistic effects and "in situ vaccine" containing tumor-reactive mAbs bound to IT-IC, IL2 in mice with one tumor. Was that. In initial experiments, 12 Gy RT alone did not eradicate or regress the established B78 melanoma tumor (complete regression was 0%), while 12 Gy + IT-IC in mice with one tumor. We have confirmed our previous findings that it results in complete regression of most B78 tumors (66%). On the other hand, 2 Gy + IT-IC, as compared to IT-IC alone slow the progression of tumors (mean tumor size at day 32, respectively, 472Mm ³ and 1214mm ³⁾ is any mice did not become disease-free (perfectly Regression 0%).

In our "two-tumor model", treatment of one "primary" tumor with xRT + IT-IC is effective in treating either treated primary tumor or untreated "secondary" tumor. We have previously shown that there is no such thing. In fact, in this two-tumor model, we have observed that the presence of a second tumor eliminates the efficacy of IT-IC injections after xRT. We have named this phenomenon "accompanying immune tolerance" (CIT), which arises, at least in part, from regulatory T cells (Tregs) of distant (non-irradiated) secondary tumors that circulate throughout the body. , XRT treatment / IT-IC-injected primary tumors demonstrated to reassemble. These Tregs returning to the primary tumor appear to interfere with the desired "in situ" effect.

We have now confirmed our previous findings that CIT can be overcome by delivering 12 Gy of xRT to both primary and secondary tumors. Importantly, Tregs are considered to be fairly sensitive to RT and to overcome CIT and rescue the response to insitu vaccination in primary tumors (primary tumors treated with 12 Gy + IT-IC). We hypothesized that lower doses of RT could be delivered to secondary tumors. Therefore, we tested this and found that a xRT dose of 2 Gy or 5 Gy to a secondary tumor was comparable to 12 Gy in its ability to blunt CIT and rescue the response to primary tumor treatment with 12 Gy + IT-IC. Observed. These important experiments were repeated twice, and the dose of xRT that had to be administered to the distant tumor to prevent CIT (as assumed) was the primary injection of IT-IC for the purpose of producing an in situ vaccine effect. It suggests that the dose is much lower than the dose required at the tumor site.

This supports our comprehensive hypothesis in the disclosure, and in animals with multiple tumors, we use the Targeted Radiation Therapy Agent (TRT) NM600 to treat all sites of the disease. Since relatively low doses of RT can be delivered, it is suggested that CIT can be overcome when used in combination with local xRT and IT-IC injection at one tumor site (inventor vaccine site).

Example 8: As determined above, a ¹³¹ I-NM404 dose close to the required dose of xRT for metastasis is determined and then the effect of the ¹³¹ I-NM404 dose on in vivo immune function is evaluated. Experiments Based on the above preliminary data of Examples 1 to 4, studies have been conducted to transfer these concepts to in vivo tests using TRT. Dosimetry studies were performed in mice with one or two B78 tumors (the tumor model we are using to demonstrate our best in situ vaccine approach and CIT hurdles). .. This was done to estimate the amount of ¹³¹ I-NM404 needed to approach xRT of about 2 Gy.

Two separate approaches have then been pursued to determine if an equivalent dose of about 2 Gy of ¹³¹ I-NM404 has the desired effect on intratumoral lymphoid cells, especially Tregs. .. First, we administered this dose of ¹³¹ I-NM404 to mice with radiosensitive lymphoma tumors showing NM404 uptake comparable to B78 tumors. Following this, we have shown that strong lymphoid tumor shrinkage / dose-dependent suppression under conditions that do not cause substantial shrinkage / slowdown of B78 tumors or overt depletion of circulating lymphoid cells. (Measured by peripheral total lymphocyte count). These data are consistent with the fact that lymphoid cells are much more sensitive to low doses of RT than typical solid tumor cells, and selective uptake of TRT in tumors is systemic lymph. It suggests that it may allow intratumoral lymphocytic cell depletion without hypocytopenia. These studies also suggest that such lymphoid tumors can serve as an in vivo biological "dosemeter" for identifying and monitoring the effects of TRT on intratumoral lymphoid cells.

The second approach involved treating mice with B78 tumors with these same doses of ¹³¹ I-NM404. These animals were then sacrificed at half-life (8 days) intervals and delayed sufficiently for radioactive decay before staining tumors for the presence of effector T cells and Tregs by immunohistochemistry. Interestingly, animals treated with ¹³¹ I-NM404 in this initial experiment did not show systemic lymphopenia at any time (depending on peripheral total blood cell count) and were intratumoral with a two-half-life following TRT administration. It showed a decrease in FoxP3 + Treg. At this half-life, we also observed a decrease in intratumoral effector CD8 + T cells. However, importantly, at subsequent 3 and 4 half-life, we found that both intratumoral CD8 + effector T cells were compared to untreated baseline and second half-life levels. An increase was observed, but a further decrease in intratumoral Treg levels was observed. Again, our hypothesis that it would be feasible to use TRTs to overcome Treg-mediated CITs in order to rescue in situ vaccine effects in animals with multiple tumors. To support.

Finally, to characterize the immunological effects of TRT on immune cells within tumors, we treat mice bearing B78 with ¹³¹ I-NM404, pretreatment and subsequent half-life (8 days). Tumor tissue was collected at intervals of. These tissues were then analyzed by RT-PCR for gene expression in the panel of immune signatures. The results show that TRT treatment alone causes the expression of immune-sensitive tumor cell markers and significant changes in genes normally expressed only by immune cells, with the gene manifesting a decrease in expression followed by overexpression of rebound. Shows a change over time.

Example 9: Using the data obtained from Examples 5 and 6, a dosing regimen of ¹³¹ I-NM404 + local xRT + IT-mAb / IL2 in mice with two or more tumors was developed and T cells from all distant tumors. Experiments to Induce Mediated Eradication This example illustrates treating animals with tumors at at least two sites. Our strategy is to use topical IT-IC at xRT and in situ vaccine sites in combination with systemic TRT to suppress CIT to enhance anti-tumor immune activity at all tumor sites. including. Significant TRT and xRT dose and timing issues are optimized for antitumor efficiency.

Using the data summarized in Examples 7 and 8, studies were performed on mice with two separate B78 tumors. Mice received an estimated required systemic ¹³¹ I-NM404 dose followed by local immunotherapy to the xRT and in situ vaccine sites. With proper control, this dose of ¹³¹ I-NM404 appeared to weaken the CIT as required in mice with two tumors. Moreover, in mice with one tumor, this TRT dose did not appear to interfere with the local in situ vaccine effect (as assumed and desired). Further studies and some modifications of the experimental variables are underway to maximize the desired effect of blocking CIT without suppressing the in situ vaccine effect. Further details regarding these experiments are disclosed in Example 10 below.

Example 10: Data obtained from mice with tumors of 2 or more Tumor-specific suppression of the primary tumor response to a combination of local xRT + IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models.

C57BL / 6 mice with syngeneic GD2 + primary flank tumor +/- contralateral abdominal secondary tumor, as shown, to primary tumor only, by xRT on day "1", and days 6-10. The eyes were treated with an IT injection of 50 mcg of anti-GD2 IC, hu14.18-IL2.

In mice with primary B78 melanoma tumors, the presence of untreated secondary B78 tumors antagonized the primary tumor response to xRT + IT-IC (FIG. 8A). We describe this effect as "accompanying immune tolerance", an antagonism of untreated distant tumors to the local response of treated tumors to xRT + IT-IC. Kaplan-Meier survival curve. Obtained for these mice and repeat experiments (Fig. 8B). Almost all mice were euthanized due to the progression of the primary tumor.

In mice with primary Panc02-GD2 + pancreatic tumors, the presence of untreated Panc02 secondary tumors, with or without secondary Panc02-GD2-tumors on the contralateral abdomen, is associated with primary Panc02-GD2 + tumors for xRT + IT-IC. Response was suppressed (Fig. 8C). In mice with primary B78 melanoma tumors, secondary B78 tumors suppressed the primary tumor response to xRT + IT-IC, whereas secondary Panc02-GD2 + pancreatic tumors did not exert this effect (Fig. 8D). In mice with primary Panc02-GD2 + tumors, secondary Panc02-GD2-tumor suppressed the primary tumor response to the combination of xRT and IT-hu14.18-IL2, but not B78 secondary tumor (). FIG. 8E).

Concomitant immune tolerance is avoided by specific depletion of regulatory T cells (Tregs).

Immunohistochemical images of the tumor examined 6 days after xRT in mice with 1 or 2 tumors for the Treg marker FoxP3 were obtained (FIG. 9A). Mice did not receive xRT or received xRT only for primary tumors. DEREG mice express diphtheria toxin receptors under the control of the Treg-specific FoxP3 promoter, allowing specific Treg depletion at the same time as IP injection of diphtheria toxin (FIGS. 9B and 9C). DEREG mice with primary and secondary B78 melanoma tumors were treated with xRT + IT-IC into the primary tumor and IP injection of either diphtheria toxin or PBS. Concomitant immune tolerance is eliminated after Treg depletion in these mice, resulting in improved primary (FIG. 9B) and secondary (FIG. 9C) tumor responses.

Concomitant immune tolerance is overcome by delivering xRT to both tumor sites.

In mice with primary and secondary B78 tumors, the secondary tumor suppresses the primary tumor response to treatment of the primary tumor with xRT + IT-IC. This was overcome by delivering 12 Gy xRT to both primary and secondary tumors and IT-IC to the primary tumor, resulting in improved primary tumor response from repeated experiments (FIG. 10A). ), The aggregate animal survival was improved (FIG. 10B).

Low-dose xRT alone does not induce in situ vaccination, but does actually overcome the associated immune tolerance when delivered to a distant tumor site with 12 Gy + IT-IC treatment of the in situ vaccine site.

In mice with only primary B78 tumors, 12Gy + IT-IC induces in situ vaccination (as previously shown), resulting in complete tumor regression (FIG. 11A) and memory immune response in most mice (FIG. 11A). 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.

In mice with primary and secondary B78 melanoma tumors, the low dose xRT (2 Gy or 5 Gy) delivered to the secondary tumor is comparable to 12 Gy in its ability to overcome the immune tolerance associated with the primary tumor (2 Gy or 5 Gy). FIG. 11B). It is clear that overcoming the associated immune tolerance by delivering low dose xRT to secondary tumors in these same animals rescues the systemic response to IT-IC immunotherapy (Fig. 11C). In this regard, when RT is delivered to all tumor sites, IT-IC injection of the primary tumor causes a systemic antitumor effect, resulting in a secondary tumor response to 2 Gy or 5 Gy of the primary tumor. Greater than the response to 12 Gy RT without IT-IC injection.

Low-dose TRT and ¹³¹ I-NM404 effectively deplete tumor-infiltrating FoxP3 + Treg without systemic leukopenia or without depleting tumor-infiltrating CD8 + effector T cells.

In most clinical scenarios, delivering external beam radiation to all tumor sites without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression, is not feasible, even at low doses. is there. Here, can the inventors specifically administer TRT systemically to specifically deplete tumor infiltration-suppressing immune cells (Tregs) without causing systemic immune cell depletion and leukopenia? I tested it. A dosimetric study in this B78 melanoma tumor model was performed with positron emission ¹²⁴ I-NM404 to confirm tumor selective uptake of NM404 (FIG. 12A). C57BL / 6 mice with B78 tumors were treated with 60 μCi ¹³¹ I-NM404. This radioactivity is approximately the same as the amount of ¹³¹ I-NM404 required to deliver approximately 2 Gy of TRT to the B78 tumor. Peripheral blood and tumor samples were collected in untreated control mice (C) and then at 8-day intervals (T1 = d8, T2 = d16, T3 = d24, T4 = d32). This dose of TRT did not result in significant systemic leukopenia (Fig. 12B) and had no significant effect on the level of tumor-infiltrating CD8 effector T cells (Fig. 12C). However, the tumor infiltrating FoxP3 + Treg was significantly depleted by this dose of TRT (Fig. 12D).

Low-dose TRT and ¹³¹ I-NM404 effectively overcome associated immune tolerance and rescue the systemic antitumor effect of in situ vaccination.

Low dose ¹³¹ I-NM404 TRT is, mouse taking into account the ability to deplete tumor infiltrating Treg without the leukopenia, we effectively immune tolerance to low dose ¹³¹ I-NM404 is accompanied Tested to see if it could be overcome. C57BL / 6 mice with two B78 tumors were treated with 60 μCi ¹³¹ I-NM404 on day 1 as shown (NM404). After one half-life (8 days), animals were given 12 Gy of xRT to the primary tumor (in situ vaccine site) or no xRT. Control mice that did not receive ¹³¹ I-NM404 were treated as shown (0, 2, or 12 Gy) for secondary tumors. On days 13-17, mice were administered to the primary tumor (in situ vaccine site) as indicated by daily IT injections of IC. Primary tumor (Fig. 13A) and secondary tumor (Fig. 13B) responses effectively overcome the immune tolerance associated with administration of low-dose TRT and rescue the systemic antitumor effect of in situ vaccination. Demonstrate.

References cited in Examples 1-4 and 7-10:
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Example 11: In vivo uptake of multiple NM600 metal chelates in xenografted mice with eight different solid tumor types demonstrated by PET imaging In this example, we present PET / CT of such tumors. As shown by imaging, it shows different uptake of NM600 chelated with four different metals in various solid tumors in vivo. These data provide further support for the use of metal chelated alkylphosphocholine analogs as TRT agents to eliminate tumor-induced immune tolerance, as disclosed herein. Structure of NM600 ^is shown in Figure 14 as a chelated exemplary species in ^{64 Cu (64 Cu-NM600)} ; however, any metal can also be readily chelated to NM600.

Specifically, eight different solid tumor cell lines (B78 (melanoma), U87MG (glioblastoma), 4T1 (breast cancer), HCT-116 (colorectal cancer), A549 (lung cancer), PC-3 ( One of Prostate Cancer), HT-29 (Colorectal Adenocarcinoma), or MiaPaca (Pancreatic Cancer) was heterologously transplanted into mice. For each xenografted mouse, a cell suspension containing tumor cells was inoculated into the subcutaneous tissue of one or both flanks of the mouse. Once the xenograft tumors reached their maximum size, each mouse was injected by lateral tail vein injection with NM600 radiolabeled with ⁶⁴ Cu, ⁸⁹ Zr, ⁸⁶ Y, or ⁵² Mn between 150 and 300 μCi. After the uptake period, PET imaging was performed on Inveon micro PET / CT. Immediately prior to each scan, mice were anesthetized with isoflurane (2%) and placed on the scanner in the prone position. Static PET scans of 4000-80 million matching events over time were taken at 3, 12, 24, and 48 hours after injection of the radiotracer and images were reconstructed using the OSEM3D / MAP reconstruction algorithm.

FIG. 15 shows images obtained 48 hours after injection of 1 tumor B78 mice injected with ⁸⁶ Y-NM600; FIG. 16 shows images obtained 48 hours after injection of 2 tumor B78 mice injected with ⁸⁶ Y-NM600. Images obtained; FIG. 17 shows images obtained 3, 24 and 48 hours after injection of U87MG mice injected with ⁶⁴ Cu-NM600; FIG. 18 shows images of 4T1 mice injected with ⁶⁴ Cu-NM600. Images obtained at 3, 24 and 48 hours post-injection; FIG. 19 shows images obtained at 3, 24 and 48 hours post-injection of HCT-116 mice injected with ⁶⁴ Cu-NM600; FIG. 20 Shows images obtained 3, 24 and 48 hours after injection of A549 mice injected with ⁶⁴ Cu-NM600; FIG. 21 shows 3, 24 and 48 hours after injection of PC-3 mice injected with ⁶⁴ Cu-NM600. shows the images obtained at 48 hours; Figure 22 ^shows an image obtained on the 64 Cu-NM600 injected HT-29 mice after injection 3,24 and 48 hours; Figure 23 is ^a 64 Cu-NM600 Images obtained at 3, 24 and 48 hours after injection of MiaPaca mice injected with ⁸⁶ Y-NM600; FIG. 24 shows images obtained at 3, 24 and 48 hours after injection of 4T1 mice injected with ⁸⁶ Y-NM600. Shown; FIG. 25 shows images obtained 3, 24 and 48 hours after injection of 4T1 mice injected with ⁸⁹ Zr-NM600.

^For HT-29 and PC3 mice injected with ⁵² Mn-NM600, PET images were taken 4 hours and 1 day after injection (FIG. 26 for HT-29; FIG. 27 for PC3), and 2, 3, 5 and after injection. Obtained on day 7 (FIG. 28 for HT-29; FIG. 29 for PC-3).

As seen in FIGS. 15-29, the scanned mice produced PET / CT three-dimensional volume rendering showing a concentrated absorbed dose distribution in xenografted tumors. These results confirm the differential uptake of metal chelated NM600 into xenografted solid tumor tissues, and the availability of NM600 analogs incorporating radioactive metal isotopes in the disclosed treatment methods. The sex is demonstrated.

Quantitative region of interest analysis of the images was performed by manually contouring the tumor and other organs of interest. Quantitative data were expressed as a percentage of injection volume per gram of tissue (% ID / g). Illustrative data show that 4T1 tumor tissue increased its uptake over time and effectively retained all three NM600 chelates tested ( ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600 and ⁸⁹ Zr-NM600, (See FIG. 30), healthy heart (FIG. 31), liver (FIG. 32) and systemic tissue (FIG. 33) all show a significant decrease in uptake / retention over time.

Biological distribution analysis with Exvivo was performed after the last PET scan over time. Mice were anesthetized, tissue was harvested, wet weight was measured and counted with an automatic gamma counter (Wizard 2480, Perkin Elmer). Illustrative biodistribution data show significant uptake and retention in tumor tissue (4T1) for different NM-600 chelates ( ⁸⁶ Y-NM600, ⁶⁴ Cu-NM600, ⁸⁹ Zr-NM600 and ¹⁷⁷ Lu-NM600, see FIG. 34). ).

Taken together, these results demonstrate that the metal chelates of the present disclosure can be readily used in the TRT step of the therapeutic methods of the present disclosure.

Example 12: Demonstration of antitumor activity and tumor autoradiography with two different NM600 metal chelate for multiple solid tumor types of heterologous transplanted mice In this example, three different solid tumor models were used and alkylphosphocholine metals We show that chelate analogs can be effectively used to promote conventional TRT. These results further demonstrate the potential for using metal chelates in the TRT steps of the therapeutic methods disclosed herein.

Subcutaneous abdominal xenografts of B78, MiaPaca and 4T1 were introduced into mice as described above. Mice were then administered therapeutic doses (250-500 μCi) of ⁹⁰ Y-NM600, ¹⁷⁷ Lu-NM600, or control solutions by lateral tail vein injection.

Plane 2D phosphor images of the biodistribution of the drug were imaged using a Cyclone Phosphorimager (Perkin Elmer). Mice were anesthetized and placed in direct supine contact with a fluorophore plate, where the mice were left in place for 15-30 minutes; then the plates were read with a phosphoimager. Various images were recorded between 4 and 96 hours after injection of radiation dose. The resulting autoradiographic images demonstrate rapid and selective uptake and long-term retention of chelates in all of the solid tumor tissue types tested (see Figures 40, 41, 42, 43, 44 and 45).

Tumor response was evaluated by comparing tumor growth in treated and control mice. Tumor volume was determined by measuring the length and width of the tumor with a caliper and calculating the volume using the formula for calculating the volume of the ellipsoid. Mouse weight was also recorded. The humanitarian endpoint was defined as: tumor volume> 2500 m ³ or significant weight loss of less than 13 g.

As seen in FIGS. 46, 47, 48, 49, 50 and 51, these results show that the two tested NM600 chelate had a statistically significant in vivo therapeutic effect when compared to controls. As a result, the average tumor volume was reduced for double doses of ¹⁷⁷ Lu-NM600 in 4T1 xenografts (see Figure 50), and MiaPaca, 4T1 or B78 xenografts were administered with a single dose of ¹⁷⁷ Lu-NM600. Reduce the growth of pieces (see Figures 47, 48, and 49) or B78 or 4T1 xenografts (see Figures 46 and 51) administered with a single dose of ⁹⁰ Y-NM600 to near zero or reduce their growth rate. Demonstrate slowing down.

These results further demonstrate the effectiveness of delivering TRTs using the disclosed alkylphosphocholine metal chelates to efficiently treat various types of solid tumors.

Example 13: Coupling Dosimetry and Radiation Sensitivity Index to Predict TRT Responses in a Wide Range of Solid Tumor Types In this example, we set the TRT steps of the methods disclosed in various solid tumor types. Consider the factors that determine the appropriate chelate dose.

Estimating Tumor Absorbed Dose Whether the amount of ¹⁷⁷ Lu / ⁹⁰ Y-NM600 administered is immunostimulatory or cytotoxic depends on the tumor absorbed dose. Tumor dosimetry was estimated utilizing the diagnostic and therapeutic properties of the NM600, each of which could use ⁶⁴ Cu / ⁸⁶ Y-NM600 as a substitute for imaging the therapeutic metal ¹⁷⁷ Lu / ⁹⁰ Y-NM600. Finally, PET / CT of ⁶⁴ Cu / ⁸⁶ Y-NM600 was used to quantitatively measure the biodistribution in vivo to determine the dose-limiting organs and the potential tumor efficacy of ¹⁷⁷ Lu / ⁹⁰ Y-NM600 TRT. Estimated radiation dosimetry that could help identify.

The general concept is as follows: (1) Quantify the concentration of ⁶⁴ Cu / ⁸⁶ Y-NM600 in the tumor over time using long-term PET / CT imaging, (2) ⁶⁴ Cu / ^86. Attenuation correction of the concentration of Y-NM600 will explain the difference in attenuation between ⁶⁴ Cu / ⁸⁶ Y-NM600 and ¹⁷⁷ Lu / ⁹⁰ Y-NM600, (3) ¹⁷⁷ Lu / ⁹⁰ Y-NM600 in tumors. Concentrations are time-integrated to calculate cumulative radioactivity or total decay. (4) Accumulation of radionuclide decay is modeled and quantified within the tumor.

Steps (1) to (3) can be performed with a medical image processing software package, but step (4) requires high performance dosimetry software. OLINDA / EXM (Stabin, Sparks and Crown 2005) is a 510 (k) approved dosimetry estimation software that uses a format developed by the Medical Internal Exposure Dose (MIRD) Commission of the American Society of Nuclear Medicine. (Bolch et al., 2009). The MIRD approach estimates the average absorbed dose received by a tissue or organ due to radiation emitted from within the organ itself or from another source organ. The simplest form of the MIRD formula,

Gives the absorbed dose, D [mGy], from the radionuclide radioactivity s in the source region to the target region t. The radionuclide radioactivity of s is the cumulative radioactivity

Expressed as, this is the total number of radionuclide decays expressed in MBq-s. S factor, S (t ← s) [ mGy / MBq-s] were normalized by the mass _{m t} of the target region t, is deposited in the target region t, the collapse of one radionuclide in the source region s The percentage of energy released by. Factor S is a tabular value calculated using Monte Carlo in a set of standard phantoms and organs. In general, we present the dose per unit of injected radioactivity,

I am interested in [mGy / MBq]. This equation relates to residence time, τ _h , [MBq−s / MBq _inj ].

The above formula is the ratio of cumulative radioactivity and injectable radioactivity, A _inj [MBq].

It is expressed as.

When calculating tumor dosimetry, OLINDA / EXM models the tumor as an isolated unit density sphere whose volume has been estimated from the tumor region of interest (ROI) generated as part of step (1). To become. The concentration of NM600 (% ID / g) in the tumor was determined at each time point and corrected for attenuation. Cumulative radioactivity was then calculated by integrating the concentrations over time using a trapezoidal compartmentalized integral.

The results of radiation dosimetry for many cell lines are shown in Table 1. This information can be used to estimate absorbed doses for radiotherapy studies aimed at either eradicating tumors or stimulating the immune system.

Radiation sensitivity index for predicting dose response

Inherent radiosensitivity is an important factor underlying the radiation therapy response, and knowing it in advance about the cancer type predicts how it will respond to radiation from the TRT. Can help. However, since there is no method for its routine evaluation in tumors, radiosensitivity is measured as viability (between 0 and 1) after irradiation with 2 Gy (SF2) by the clonogenicity test. The relative radiosensitivity of the cancer cell phenotype ranges from very low radiosensitivity (pancreas, colorectal polyps, gliomas and breasts) to high radiosensitivity (lymphoma). Cancers can be classified or ranked by their radiosensitivity index (Table 2).

We have used APC metal chelate to perform good tumor uptake and growth in highly radiosensitive tumors such as lymphoma and highly radioresistant tumors such as glioma, breast, pancreas or colonic rectum. If these agents can target tumors in vivo, where suppression can be shown, then these agents have an SF ₂ value (0.3-0.82) between lymphoma and glioma. It can be meant to be effective against any tumor that has. In that case, the amount of radiation required to eradicate glioma tumor cells is also expected to be higher than the amount required to treat more radiosensitive lymphoma cells.

We currently have in vivo imaging to confirm tumor selectivity and therapeutic response (tumor growth inhibition) data for all tumor cell lines listed in Table 2. In some cases, it may be necessary to administer multiple APC chelates to induce sufficient cancer cell death. By using quantitative imaging in conjunction with radiation dose measurement calculations, we either kill cancer cells (high dose) or stimulate the immune system (low dose), as disclosed herein. The tumor absorbed dose required for (dose) can be estimated.

Combining dosimetric estimates from various cancer cell lines (Table 1) with their respective radiosensitivity indices (Table 2) supports the establishment of a dose response landscape for the NM600. By knowing the tumor targeting properties and efficacy of NM600 within a series of cell lines, it is possible to estimate the absorbed tumor dose and potential efficacy of cell lines with similar radiosensitivity indices. In addition, the therapeutic dose can be increased or decreased linearly according to Table 1 depending on the desired outcome of tumor eradication or immune stimulation (as disclosed herein).

¹ Tagian, Hearing et al., "In vivo radiation sensitivity of glioblastoma multiforme." International Journal of Radiation Oncology ^* Biology ^* Physics ^* Physics ^* Physics ^* Physics.
² Ramsay, J. et al. , R. Ward, and N.M. M. Bleehen. "Radiosensitivity testing of human mariginant gliomas." International Journal of Radiation Oncology ^* Biology ^* Physics 24.4 (1992): 675-680.
³ Fertil, B.I. , And E. P. 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.
⁴ Wollin, Michael et al., "Radio sensitivity of human prostate cancer cell lines." Radiotherapy and Oncology 15.3 (1989).
⁵ Kodym, Elisabeth et al., "The small-molecule CDK inhibitor, SNS-032, enchance cellular radiosensitivity in quiescent and hypoxic non-c.
⁶ Unkel, Stephen, Claus Belka, and Kirsten Lauber. "On the analytical of clonogenic surveillance data: Static alternates to the linear-quadratic model." Radiation Oncology 11.1 (2016): 11.
⁷ EP Malaise, Patrick J. et al. Deschavanne, and Bernard Fertil. "Intrinsic radiosensitivity of human cells." Advances in radiobiology 15 (2016): 37-70.
⁸ Series, E.I. Et al., "Relationship beween p53 status and radiosensitivity in human tumor cell lines." British journal of cancer 73.5 (1996): 581-588.

References cited in Example 13:
Bolch, W. et al. E. , K. F. Eckerman, G.M. Sgouros, and S. R. Thomas. 2009. "MIRD Pamphlet No. 21: A Generalized Schema for Radiopharmaceutical Dosimetry --- Standardization of Nomenclature." Journal of Nuclear-M. doi: 10.2967 / jnumed. 108.056036.

Stabin, MG, RB Sparks, and E Crown. 2005. "OLINDA / EXM: The Second-Generation Personal Computer Software for International Dose Assessment in Nuclear Medicine." J Nucl Med 46 (6): 1023-27.

Example 14: Advantages and Differences When Using Alkylphosphocholine Metal Chelate Instead of Radio-iodinated Compounds, eg, those exemplified in Examples 1-4 and 7-10 In this example, we are radioactive. Consider the advantages of using APC metal chelates instead of iodinated compounds (compounds exemplified in Examples 1-4 and 7-10). We also consider factors that should be considered by those of skill in the art in optimizing the dose of the metal chelate that will be used in the TRT step of the disclosed method.

Chelate allows the use of a wide variety of stable or radioactive metal ions for imaging and treatment. They can be combined with any wide variety of alpha, beta, auger, gamma and positron emitters, but iodine is one positron (I-124), one beta (I-131), one gamma ( Limited to I-123) and one Auger (I-125) isotope.

Metal isotopes are more diagnostically and therapeutically effective than I-131 and I-124.

Lu-177 is preferred for SPECT imaging and dosimetry because of its low high energy gamma. However, its beta energy is slightly lower than I-131, making it ideal for the treatment of small tumors.

I-131 and Lu-177 are comparable in "horsepower" of therapeutic effect, but the contribution of Lu-177 to the overall dose from gamma release is significantly less. In the case of Y-90, the contribution of radiation dose from gamma emission is negligible.

Compared to I-131, Y-90 is more effective than I-131 in killing cancer cells by conventional TRT, as seen in FIG. 52 and further discussed below.

The Medical Internal Exposure Dose (MIRD) Committee is developing standard methods, models, assumptions and mathematical schemas for assessing internal radiation doses from administered radiopharmaceuticals. This MIRD approach simplifies the problem of assessing radiation levels for many different radionuclides and is implemented in the widely used 510 (k) approved software, OLINDA / EXM1. Along with its many standard anthropomorphic phantoms, OLINDA / EXM has a Spheres Model that can be used to estimate tumor doses. The Spheres Model assumes that the radiopharmaceuticals are evenly distributed within unit density spheres of various tumor volumes (0.01-6,000 g).

Using this standard model, we compared Y-90 and I-131 with respect to the radioactivity normalized by the administered radioactivity. The results of this comparison are shown in FIG. 52 for tumor volumes between 1-100 g. The ratio of Y-90 to I-131 reaches 4 with 4 g of tumor, stays between 4.0-4.2 up to 100 g of tumor, and on an mCi / mCi basis, Y-90 is up to 10 g in size. It should be noted that it strongly suggests that tumors are 3.6 to 4.1 times more cytotoxic than I-131 and about 4.1 times more effective in tumors larger than 10 g.

Different pharmacokinetic properties Unlike iodinated analogs, APC chelates are too large to fit in known albumin-binding pockets of plasma, thus exhibiting different in vivo pharmacokinetics and biodistribution profiles (see Figure 53). Low binding energies increase the proportion of free molecules in plasma, which allows for faster tumor uptake. Some APC chelates are removed via the renal system, while iodinated analogs are removed by the hepatobiliary system. APC chelates also accumulate in tumors and are removed from the blood faster than iodinated analogs. Faster blood purification is directly associated with bone marrow loss and off-target toxicity of therapeutic radiopharmaceuticals.

These differences in PK and biodistribution profile result in various dose-restricted organ toxicity and ultimate utility. Transferring from hematological toxicity to dose-limiting toxicity to the kidney or liver will increase the usefulness of radioactive metal chelates for TRT.

Moreover, the pharmacokinetic profile of APC chelates can be easily manipulated by slight changes in the structure of the chelate (eg, chelate charge). The range of chelating agents to choose from is enormous. The faster the clearance from normal tissue, the better the image contrast and treatment window, resulting in a higher maximum permissible dose.

APC chelates have physicochemical properties that differ from iodinated analogs. APC chelates are much more water soluble and therefore do not require a surfactant to make them suitable for intravenous injection. APC chelates are based on ionic bonds to metal chelates, whereas iodinated compounds form covalent bonds with their carrier molecules. Deiodination in vivo is very common with alkyl iodides, but chelates tend to be very stable in vivo.

When deiodination occurs, free iodide accumulates rapidly in the thyroid gland and has a very long elimination half-life thereafter, whereas free radioactive metals are usually much more rapidly excreted from the body or detoxified. Will be done.

Both metals and chelates contribute to the tumor targeting properties of APCs, as the in vivo distribution of APC chelates can vary considerably depending on the metal ion. Not all chelates target tumors. Tumor targeting depends on the cumulative properties of the APC carrier, the type of chelate (linear chelate undergoes rapid renal removal, but macrocyclic chelate undergoes hepatic bile excretion), and metal ions. Even the slightest change in chelate structure results in significant changes in in vivo properties. Simple changes in isotopes can change tumor targeting by more than 50%.

Radioactive APC-metal chelates are easily radiolabeled in near quantitative (> 98%) yields under convenient conditions, whereas iodinated analogs have much lower radioiodination yields (generally I-). About 50% for 131, 60% for I-124). In addition, high specific activity can be achieved with chelates. Synthesis can be performed using radiolabeling kits in any nuclear pharmacy without the need for elaborate ventilation equipment or training. Radioiodination must be done in a draft equipped with wastewater monitoring equipment due to the volatility of radioactive iodine during the labeling reaction.

Contrast does not always make a good remedy, and vice versa.

Good tumor uptake with contrast agents cannot be assumed to mean that treatment is obvious. In addition to having good tumor uptake, the therapeutic agent must have long-term tumor retention compared to normal tissue and must be rapidly removed from the blood to reduce bone marrow exposure and associated toxicity. It doesn't become. Iodinated analogs remain in the blood for extended periods of time, resulting in dose-limited myeloid toxicity. In contrast, our APC chelates are most likely to exhibit much faster blood clearance kinetics due to the low albumin binding in plasma, as mentioned above.

Finally, due to the shorter pathway length and physical properties of metal beta and alpha radiators compared to iodine-131, there is no concern of exposure to healthcare professionals or families after injection. Patients receiving I-131 treatment often have to be housed in a lead-shielded room for some time (up to a week) before being released from the hospital before being discharged. Patients injected with radioactive alpha and beta-releasing APC chelates will not need to continue hospitalization.

Example 15: TRT delivered by Y90-NM600 in combination with administration of an anti-CLA4 immune checkpoint inhibitor synergistically inhibits cancer in an in vivo melanoma model. In this example, we disclose. Demonstrate the effectiveness of the combined method. Here, 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 ⁹⁰ Y-NM600 chelate used in the previous example. Will be.

Subcutaneous abdominal xenografts of B78 melanoma were transplanted into male C57BL / 6 mice as described above. Mice were then randomized and treated with various doses (25 μCi, 50 μCi, or 100 μCi) of ⁹⁰ Y-NM600 (day 1) with and without anti-CTLA4 antibody (immune checkpoint inhibitor). In some cases (200 μg on days 4, 7, and 11) (n = 6 for each experimental group). Both agents were administered via lateral tail vein injection (ie, intravenously). Control groups with PBS treatment only and anti-CTLA4 only were also included. Tumors were measured twice weekly with calipers and animal survival was monitored for 60 days.

As shown in FIG. 54, the three combination therapies (three different doses of anti-CTLA4 + ⁹⁰ Y-NM600) are single therapies (three different doses of anti-CTLA4 or ⁹⁰ Y-NM600 only) or PBS. Compared with the control, it showed a substantial suppression of tumor growth. After 18 days, combination treatment with 50 or 100 μCi ⁹⁰ Y-NM600 and anti-CTLA4 significantly increased tumor growth (p <0.05 at ANOVA) compared to PBS, ⁹⁰ Y-NM600 alone, or anti-CTLA4 alone. ) Reduced. The 25 μCi ⁹⁰ Y-NM600 combination treatment group with anti-CTLA-4 showed a moderate growth retardation response that tended to dose response.

As seen in FIG. 55, mice treated with 50 μCi ⁹⁰ Y-NM600 in combination with anti-CTLA4 showed significantly higher overall survival than mice treated with TRT alone or with PBS vehicles (p <0). .05). The log rank of the combination treatment (treatment) was p = 0.06 compared to anti-CTLA4 alone.

As seen in FIG. 56, all three combination treatments significantly improved survival. Significantly, ⁹⁰ of the therapeutic doses of 50 and 100 μCi compared to the full responder of the non-combination control arm (PBS, 50 μCi TRT only, 100 μCi TRT only, and anti-CTLA4 only) 0/24. With the Y-NM600 and TRT + CTLA4 combined arm, the complete responder was 6/12 (50%).

These results indicate the therapeutic potential of a molecular-targeted radiotherapy agent in combination with any agent that causes immune checkpoint inhibition (ICI). The results show that the combination of molecularly targeted TRT and ICI provides a synergistic effect compared to treatment with each drug alone. In addition to exhibiting significant tumor retraction, this combination may ultimately provide a potent in situ cancer vaccine effect that produces immune memory and prevents tumor recurrence.

Example 16: Utilization of Molecular Targeted Radiation Therapy to Increase the Effectiveness of Systemic Checkpoint Inhibition in Metastatic Cancer Models In a follow-up study of Example 15, the ⁹⁰ Y-NM600 chelate used in the previous example was used. We provide significantly expanded supporting data demonstrating the effectiveness of the methods of the present disclosure combining systemic administration of immune checkpoint inhibitors and TRT performed by systemic administration. Efficacy has been demonstrated in mouse melanoma, neuroblastoma, and breast cancer models, as well as multiple tumor melanoma models with disseminated "cold" tumors.

Clinical trials have shown that a subset of patients treated with immune checkpoint inhibitors (ICIs) experience a durable complete remission (CR) at all disease sites. However, ICI is generally ineffective for patients with immunologically "cold" tumors characterized by neoantigens produced by low levels of T cell infiltration and / or a small number of mutations. In this example, we demonstrate using the combined methods of the present disclosure to stimulate the immune response of such tumors and enhance the response in "hot" tumors. More specifically, we present low-dose immunostimulatory radiation to all diseases without resulting in systemic lymphocyte depletion, which is counterproductive in the generation of antitumor immunotherapeutic responses. The effectiveness of systemic ICI was enhanced by combining systemic molecular targeted radiotherapy (MTRT), which can be delivered to the site, with systemic ICI.

Method:
For tumor uptake studies MTRT, the (n = 3 for each of the B78 melanoma and Panc02) flank tumors by injecting with IACUC protocol approved 1-2 × 10 ⁶ cells 100μLPBS in C57BL / 6 mice Established. Both B78 and Panc02 tumors are low to moderately immunogenic, slow-growing, and radiation-resistant tumor strains, so this profile can be used in MTRT studies. The slow growth slows down MTRT, and radiation resistance and low immunogenicity make it possible to test the co-improvement of efficacy with combined MTRT + ICI.

After the tumor was well established, about 5 weeks after injection, animals were treated with a dose of IV ⁸⁶ Y-NM 600 and continuous PET / CT images were collected 1, 2, and 3 days after MRT injection. PET uptake values were compared to areas of background radioactivity, including the heart and liver. A paired t-test was performed to test for significant differences in ⁸⁶ Y-NM600 uptake between background organs and tumor sites.

To demonstrate the ability of ⁹⁰ Y-NM600 and / or ICI to reduce immunosuppressive Treg cell populations within B78 melanoma flank tumors, we present a model of B78 melanoma flank tumors (n for each group). = 4) was generated. MTRT (50 μCi), anti-CTLA4 (4th, 7th, 10th, 200 μg), MTRT and CTLA4, and PBS placebo controls were our treatment group. The effect of treatment on tumor immune cell populations was shown on days 1, 7, and 14 days after irradiation or saline placebo delivery, tumor tissue was harvested, partially frozen for histology, and for quantitative PCR. Investigate by preserving another part. The remaining tumor samples were prepared for mRNA and RT-PCR analysis. Quantitative RT-PCR was used to assess changes in tumor cell expression of immunosensitivity markers (eg, Fas, MHC-I, and PD-L1).

Two bilateral abdominal tumor models of B78 melanoma were generated in C57BL / 6 mice for efficacy studies. Once the tumor had grown to 80-120 mm ³ , the tumor was randomized to the next treatment group: only 200 μg IP anti-CTLA-4 on days 4, 7, and 10, ⁹⁰ Y-NM600 IV (50 μCi) on day 1. ), And anti-CTLA4, 12 Gy total body irradiation (EBRT) and anti-CTLA4, 12 Gy EBRT + 50 μCi ⁹⁰ Y-NM600, 12 Gy EBRT + 50 μCi ⁹⁰ Y-NM600 and anti-CTLA4. Tumor measurements were taken twice weekly for 30 days, survival was followed up to 60 days, and the euthanasia endpoint was a tumor load 15 mm in diameter.

Mice in complete remission to treatment were reloaded with 2x10 ⁶ B78 or 1x10 ⁶ Panc02 cells 90 days after MTRT on the contralateral flank, and then again on day 120 Panc02 (for B78 only) and B16 melanoma. Was reloaded to test tumor-specific immune memory responses.

result:
Selective uptake of our MTRT agent, ⁹⁰ Y-NM600, was confirmed in both B78 and Panc02 tumor models. In B78 melanoma, due to tumor uptake of ⁹⁰ Y-NM600, most of the drug was in the blood pool as expected after the first injection, but within 48 hours after injection, most of the drug was in the tumor or excretory organs ( It was demonstrated that it was retained in the liver and kidneys). The gamma count of the tissue section taken on day 48 confirms the uptake value of PET imaging. Radioactivity counts are high in tumor tissue and increase over time, and low in the medullary cavity and decrease over time. Monte Carlo dosimetry, performed as a collaborative attempt, shows that when 50 μCi of our experimental dose is delivered, about 2-3 Gy is delivered over the life of the MRT agent. Studies of PET uptake and tissue biodistribution in Panc02 pancreatic cancer also demonstrated increased uptake and retention of ⁹⁰ Y-NM600 in tumor tissue compared to 72 hours of bone marrow tissue.

Tumor tissue samples were collected at various time points after irradiation to study the therapeutic effect on tumor immune cell populations. Effector T cells / immunosuppressive T cells determined by CD4 / FoxP3 and CD8 / FoxP3 infiltrates of tumor tissue by a combination of MTRT and anti-CTLA4 on day 14 after MTRT treatment (50 μCi ⁹⁰ Y-NM600) We have found that the proportion of cells is significantly increased. Quantitative PCR (qPCR) studies of gene expression have also shown increased expression of inflammatory genes, including genes that are part of the stimulatory factor of the interferon gene pathway (STING). Levels of Mx1, IFNα, IFNβ, and PDL1, all downstream of STING activation, were upregulated compared to PBS controls.

Next, we established a single B78 R flank tumor in mice, and when they reached about 80 mm ³ , randomized them to MTRT doses of 25, 50, and 100 uCi on day 1. The treatment was divided into groups that received anti-CTLA4 on days 4, 7, and 10 days, groups that did not receive anti-CTLA4, and groups that received only PBS and anti-CTLA4 as controls. The combination of MTRT with dose levels of 50 and 100 uCi and anti-CTLA4 demonstrated significant improvement in tumor growth delay (FIG. 57) and survival (FIG. 58) compared to other groups. The inventors have found. At the 25uCi MTRT, there was an intermediate response. In addition, mice that showed complete remission to treatment were only in the combination treatment group, with 66%, 33, and 16% of the animals in the 50uCi, 100, and 25uCi MTRT dose groups. When B78 cells were loaded into the contralateral abdomen of all mice that were in complete remission 60 days after MTRT injection, there was a 100% rejection rate compared to naive controls, and the treated immune memory response of the present inventors was It demonstrates that it was able to be generated.

Since then, this study has been reproduced and showed similar trends, with survival rates in both studies showing overall survival rates in mice treated in combination with MTRT (50, 100 uCi) and anti-CTLA4 by log rank tests. It was shown that there was a significant improvement compared to the other groups.

The inventors then extended this study to similar mouse models of neuroblastoma (NXS2) and breast cancer (4T1). As seen in FIGS. 59 (NXS2) and 60 (4T1), the combination of CTLA4 and MTRT was also the only group showing a significant reduction in tumor growth (in fact, reduction in tumor volume).

Next, we extended this study to demonstrate improved response rates in mice with multiple giant tumors. We designed a study of MTRT treatment in a two-tumor mouse model. The goal is to treat mice with multiple giant tumors, which corresponds to patients with giant metastatic disease at multiple sites. In this experiment, we investigated whether MTRT could improve response rates over the current clinical paradigm of delivering EBRT to a single site in combination with immune checkpoint blockade.

Two tumor models of Panc02 and B78 melanoma were established. Initially for B78 melanoma, conventional immunosensitized EBRT (12 Gy) was administered to the primary site of the disease (shielding the secondary site) in combination with anti-CTLA4, radiation only, to MTRT and anti-CTLA4, or to the primary site. EBRT in combination with MTRT and anti-CTLA4 in all sites. The tumor growth curve shows that triple-drug treatment results in improved tumor regression in both primary and secondary tumors (FIG. 62) compared to other groups. Furthermore, the survival rate was significantly improved by the triple-drug treatment (p <0.01) as compared with the two-drug combination treatment group. The triple treatment resulted in a complete remission rate of 40% (16% complete remission for MTRT + anti-CTLA4, 0% in the other groups) and all responding animals were tumor specific for B78 or associated B16 melanoma. Had an immune memory.

Finally, using a mouse model with two macroscopic distant tumors and disseminated microscopic metastases, a checkpoint because it does not provoke an advanced multisite "cold" cancer (ie, a strong immune system response). A multi-site tumor that greatly resists inhibition) was simulated.

In order to form a large primary tumor, 2 × 10 ⁶ B78 melanoma tumor cells were injected into one flank of the mouse. Twelve days later, 5 × 10 ⁵ B78 melanoma tumor cells were injected into the opposite flank of the mice to form small secondary tumors. The coming after 17 days (day 1), in order to create a disseminated metastatic, 2 × 10 ⁵ B16 melanoma cells were injected intravenously into mice.

Mice were exposed to a variety of single or combination treatments: PBS control injection; MTRT on day 1, 50 μCi IV; ICI on days 4, 7, and 10, anti-CTLA4 / PD1; In Situ vaccine on day 1 ( IS), 12 Gy local RT + intratumoral injection of anti-GD2 mAb and IL2 on days 6-10. The single and combined treatments tested were PBS, MRT, ICI, IS, MRT + ICI, MRT + IS, ICI + IS, and MRT + IS + ICI. Tumor growth and animal survival of mice were monitored from day 60, and tumor-free mice were reloaded with B78 on day 90.

On day 90, less than 20% of ICI mice were alive, but about half of MRT + IS and ICI + IS mice were alive (MTRT + IS had slightly higher survival rates). Surprisingly, MTRT + IS mice were 100% alive (survival rates in all other groups were zero). In particular, 83% of these mice were found to be tumor-free and showed complete remission (CR) (ie, cure) with immune cell memory, while the rest maintained uncontrolled secondary tumors. It was.

In addition, the present inventors include neuroblastoma (NXS2, 9464D), rhabdomyosarcoma (M3-9-M), high-grade glioma, Lewis lung cancer, and head and neck cancer (MOC-2). We also confirmed uptake and dose delivery in various other cancers. In addition to tumor uptake and dosimetry, toxicity analysis was performed and radiation-induced myeloid toxicity (measured with serum leukocytes or lymphocytes) was observed at our therapeutic radiation dose of 50 μCi (tumor dose 2-3 Gy). Was not done. In addition, we irradiated mice with both an external beam and 90Y-NM600 at various doses and collected IHC and tissue-stained histology for mRNA analysis by PCR. The data obtained from these studies show up-regulation of the interferon signaling pathway and increased PDL1 expression with 50 μCi 90Y-NM600. Furthermore, we have found that tumor infiltration regulatory T cells are reduced by molecular-targeted radiotherapy.

In short, our findings from this study suggest that low-dose NM600 MRT can enhance tumor abscopal response when combined with checkpoint blockade. In particular, the NM600 MTRT radiotherapy delivery agent exhibits the ability to improve the response of "cold" tumors, which normally do not respond solely to immune checkpoint blockade. Moreover, a relatively low MTRT dose of 50 μCi (a tumor dose of 2.5 Gy) is sufficient to achieve an immunostimulatory effect that enhances the effectiveness of ICI without depletion of systemic lymphocytes. MTRT can be added to single-site EBRT and checkpoint blockade to achieve better tumor response and cure rates at both local and distant tumor sites. Our results show that MTRT has great potential to improve the therapeutic effect of immunotherapeutic treatments in patients.

Conclusion of Examples These examples demonstrate anti-cancer strategies based on a synergistic and widely applicable combination of targeted systemic delivery of radiation therapy and systemic delivery of immunostimulants such as immune checkpoint inhibitors. There is. Systemic administration of mAbs or small molecules (immune checkpoint inhibitors) that target immune checkpoints, as the disclosed metal chelated and radiohalogenated alkylphosphocholine analogs can target cancers of virtually any tissue. Has been found to be useful for virtually any cancer type (tumor-reactive mAbs have been approved or used in clinical trials for almost all cancer histological types). Therefore, the clinical transition of two different combination strategies has widespread use for virtually all high-risk cancers.

Other embodiments and uses of the invention will be apparent to those skilled in the art given the specification and practices of the invention disclosed herein. All references cited herein for any reason, including citations from all journals and US / foreign patents and patent applications, are specifically and fully incorporated herein by reference. It is understood that the present invention is not limited to the specific reagents, formulations, reaction conditions, etc. exemplified and described herein, and includes such modifications that fall within the claims below. To.

Claims (66)

A method of treating a cancer containing one or more malignant solid tumors in a subject.
Immunomodulatory doses of targeted radiotherapy (TRT) agents that are differentially uptake and retained by malignant solid tumor tissues, and (b) one or more immunostimulants,
Including systemic administration
A method of treating cancer in a subject thereby. The method of claim 1, wherein the one or more immunostimulants are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules. One or more checkpoint molecules that can be targeted by checkpoint inhibitors are A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-related protein 4), KIR ( Killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), PD-1 (programmed cell death receptor 1), PD-L1 (programmed cell death ligand 1), CD40 (differentiation antigen group 40), CD27 (differentiation antigen group 27), CD28 (differentiation antigen group 28), CD137 (differentiation antigen group 137), OX40 (CD134; differentiation antigen group 134), OX40L (OX40 ligand; differentiation antigen group 252), GITR (glucocorticoid induction) Tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T cell costimulatory), ICOSL (inducible T cell costimulatory ligand), B7H3 (CD276; Differentiation antigen group 276), B7H4 (VTCN1; V-set domain-containing T cell activation inhibitor 1), IDO (indolamine 2,3-dioxygenase), TIM-3 (T cell immunoglobulin domain and mutin) The method according to claim 2, which is selected from the group consisting of domains 3), Gal-9 (galectin-9), and VISTA (V domain Ig suppressor for T cell activation). Claim that one or more immune checkpoint inhibitors include one or more anti-immune checkpoint molecular antibodies, or one or more small molecule immune checkpoint inhibitors that act to block one or more immune checkpoint molecules. 2 or the method according to claim 3. One or more anti-immune checkpoint molecular antibodies are 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. Antibodies, anti-CD28 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-OX40L antibodies, GITR antibodies, GITRL antibodies, ICOS antibodies, ICOSL antibodies, B7H3 antibodies, B7H4 antibodies, IDO antibodies, TIM-3 antibodies, Gal-9 antibodies, and Claimed that one or more small immune checkpoint inhibitors selected from the group consisting of VISTA antibodies or acting to block one or more immune checkpoint molecules include small PD-L1 inhibitors. Item 4. The method according to item 4. TRT agent
(1) Metaiodobenzylguanidine (MIBG), in which the iodine atom in MIBG is a radioactive iodine isotope,
(2) Radiolabeled tumor-targeting antibody,
(3) Radioactive radium isotope; or (4) Equation:
[In the formula,
R ₁ is (a) a chelating agent chelated to a metal atom, an alpha, beta or Auger-releasing metal isotope in which the metal atom has a half-life of more than 6 hours and less than 30 days; or (b). ) Contains radioactive halogen isotopes
a is 0 or 1 and
n is an integer of 12 to 30
m is 0 or 1 and
Y is selected from the group consisting of -H, -OH, -COOH, -COOX, -OCOX, and -OX, where X is alkyl or arylalkyl.
R ₂ is selected from the group consisting of -N ⁺ H ₃ , -N ⁺ H ₂ Z, -N ⁺ HZ ₂ , and -N ⁺ Z ₃ , where each Z is independently alkyl or aryl and b. Is 1 or 2, where b is 1 if R ₁ contains a radioactive halogen isotope]
The method according to any one of claims 1 to 5, which is a phospholipid ether metal chelate or a radiohalated phospholipid ether or a salt thereof. (1) The metal isotopes are Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac- Select from the group consisting of 225, Pb-212, and Th-227,
(2) The radioactive halogen isotope is selected from the group consisting of ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At, ⁷⁷ Br, and ⁷⁶ Br, or (3) the radioactive radium isotope is Ra-. 223, the method of claim 6. The chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and its derivative, 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-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives, 1,4,7,10-tetra Azacyclodecane, 1-glutaric acid-4,7,10-triacetic acid (DOTOGA) 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, diethylenetriamine pentaacetic acid (DTPA), its diesters, and Derivatives thereof, 2-cyclohexyldiethylenetriamine pentaacetic acid (CHX-A "-DTPA) and its derivatives, deferroxamine (DFO) and its derivatives, 1,2-[[6-carboxypyridine-2-yl] methylamino] ethane (H). ₂ dedpa) and its derivatives, and DADA and its derivatives are selected from the group consisting of DADA with the following structure:
The method according to claim 6 or 7. (A) m is 0, or (b) b is 1, or (c) n is 18, or (d) R ₂ is -N ⁺ Z ₃ , or () e) Any combination of two or more of (a)-(d),
The method according to any one of claims 6 to 8. The method of claim 9, wherein each Z is independently -CH ₂ CH ₃ or -CH ₃ . 10. The method of claim 10, wherein each Z is −CH ₃ . A chelating agent chelated to a metal atom
The method according to any one of claims 6 to 11, which is selected from the group consisting of. Radioactive phospholipid ether metal chelate,
Has an expression selected from the group consisting of
The selected compound is chelated to a metal atom,
The method according to any one of claims 6 to 11. The invention according to any one of claims 6 to 12, wherein a is 1, b is 1, m is 0, n is 18, and R ₂ is −N ⁺ (CH ₃ ) _3. the method of. The method of claim 14, wherein the radioactive phospholipid ether metal chelate is NM600, which is a chelated metal atom, or the radioactive halide phospholipid ether is NM404. 15. The method of claim 15, wherein the radioactive phospholipid ether metal chelate is ⁹⁰ Y-NM600 or ¹⁷⁷ Lu-NM600. The radioactive halide phospholipid ethers are [ ¹²³ I] -NM404, [ ¹²⁴ I] -NM404, [ ¹²⁵ I] -NM404, [ ¹³¹ I] -NM404, [ ²¹¹ At] -NM404, [ ⁷⁷ Br] -NM404, Alternatively, the method of claim 15, wherein [ ⁷⁶ Br] -NM404. The method according to any one of claims 1 to 17, wherein the TRT agent, the immune checkpoint inhibitor, or both are administered intravenously. The method according to any one of claims 1 to 18, wherein the subject is a human. The cancers to be treated are melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, flat epithelial cancer of the skin or head and neck, intestinal cancer, retinal bud. Claims 1-19, selected from the group consisting of cell tumors, cervical cancers, gliomas, breast cancers, pancreatic cancers, soft tissue sarcomas, Ewing's sarcomas, rhizome myomas, osteosarcomas, Wilms tumors, and childhood brain tumors The method according to any one of the above. The method of any one of claims 1-20, wherein the cancer is treated without administering to the subject an antibody against a tumor antigen that is not a checkpoint molecule. The method according to any one of claims 1 to 21, wherein the cancer is treated without administering the anti-GD2 antibody to the subject. The use of (a) immunomodulatory doses of targeted radiotherapy (TRT) agents and (b) one or more immunostimulators to treat cancers, including one or more malignant solid tumors, in a subject. , Can be differentially taken up and retained by malignant solid tumor tissue, and both the TRT agent and one or more immunostimulatory agents are systemically administered to the subject.
Use, thereby treating cancer in the subject. 23. The use of claim 23, wherein the one or more immunostimulants are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules. One or more checkpoint molecules that can be targeted by checkpoint inhibitors are A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-related protein 4), KIR ( Killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), PD-1 (programmed cell death receptor 1), PD-L1 (programmed cell death ligand 1), CD40 (differentiation antigen group 40), CD27 (differentiation antigen group 27), CD28 (differentiation antigen group 28), CD137 (differentiation antigen group 137), OX40 (CD134; differentiation antigen group 134), OX40L (OX40 ligand; differentiation antigen group 252), GITR (glucocorticoid induction) Tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T cell costimulatory), ICOSL (inducible T cell costimulatory ligand), B7H3 (CD276; Differentiation antigen group 276), B7H4 (VTCN1; V-set domain-containing T cell activation inhibitor 1), IDO (indolamine 2,3-dioxygenase), TIM-3 (T cell immunoglobulin domain and mutin) The use according to claim 24, selected from the group consisting of domains 3), Gal-9 (Galectin-9), and VISTA (V domain Ig suppressor for T cell activation). 24. Claim 24, wherein one or more immune checkpoint inhibitors comprises one or more anti-immune checkpoint molecular antibodies or one or more small molecule immune checkpoint inhibitors that act to block one or more immune checkpoint molecules. Or the use according to claim 25. One or more anti-immune checkpoint molecular antibodies are 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. Antibodies, anti-CD28 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-OX40L antibodies, GITR antibodies, GITRL antibodies, ICOS antibodies, ICOSL antibodies, B7H3 antibodies, B7H4 antibodies, IDO antibodies, TIM-3 antibodies, Gal-9 antibodies, and Claimed that one or more small immune checkpoint inhibitors selected from the group consisting of VISTA antibodies or acting to block one or more immune checkpoint molecules include small PD-L1 inhibitors. Item 26. TRT agent
(1) Metaiodobenzylguanidine (MIBG), in which the iodine atom in MIBG is a radioactive iodine isotope,
(2) Radiolabeled tumor-targeting antibody,
(3) Radioactive radium isotope, or (4) Equation:
[In the formula,
R ₁ is (a) a chelating agent chelated to a metal atom, whether the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of more than 6 hours and less than 30 days. Or (b) contains a radioactive halogen isotope,
a is 0 or 1 and
n is an integer of 12 to 30
m is 0 or 1 and
Y is selected from the group consisting of -H, -OH, -COOH, -COOX, -OCOX, and -OX, where X is alkyl or arylalkyl.
R ₂ is selected from the group consisting of -N ⁺ H ₃ , -N ⁺ H ₂ Z, -N ⁺ HZ ₂ , and -N ⁺ Z ₃ , where each Z is independently alkyl or aryl and b. Is 1 or 2,
However, if R ₁ contains a radioactive halogen isotope, b is 1.]
The use according to any one of claims 23 to 27, which is a phospholipid ether metal chelate or a radiohalated phospholipid ether or a salt thereof. (1) The metal isotopes are Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac- Select from the group consisting of 225, Pb-212, and Th-227,
(2) The radioactive halogen isotope is selected from the group consisting of ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At, ⁷⁷ Br, and ⁷⁶ Br, or (3) the radioactive radium isotope is Ra-. 223, the use according to claim 28. The chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and its derivative, 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-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives, 1,4,7,10-tetra Azacyclodecane, 1-glutaric acid-4,7,10-triacetic acid (DOTAGA) 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, diethylenetriamine pentaacetic acid (DTPA), its diesters, and Derivatives thereof, 2-cyclohexyldiethylenetriamine pentaacetic acid (CHX-A "-DTPA) and its derivatives, deferroxamine (DFO) and its derivatives, 1,2-[[6-carboxypyridine-2-yl] methylamino] ethane (H). ₂ dedpa) and its derivatives, and DADA and its derivatives are selected from the group consisting of DADA with the following structure:
28 or 29 of the use according to claim 29. (A) m is 0, or (b) b is 1, or (c) n is 18, or (d) R ₂ is -N ⁺ Z ₃ , or () e) Any combination of two or more of (a)-(d),
Use according to any one of claims 28 to 30. 31. The use of claim 31, wherein each Z is independently -CH ₂ CH ₃ or -CH ₃ . 32. The use according to claim 32, wherein each Z is −CH ₃ . A chelating agent chelated to a metal atom
The use according to any one of claims 28-33, selected from the group consisting of. Radioactive phospholipid ether metal chelate,
Has an expression selected from the group consisting of
The selected compound is chelated to a metal atom,
Use according to any one of claims 28 to 34. 28 to 35, wherein a is 1, b is 1, m is 0, n is 18, and R ₂ is −N ⁺ (CH ₃ ) _3. Use of. 36. The use according to claim 36, wherein the radioactive phospholipid ether metal chelate is NM600, which is a chelated metal atom, or the radioactive halide phospholipid ether is NM404. 37. The use according to claim 37, wherein the radioactive phospholipid ether metal chelate is ⁹⁰ Y-NM600 or ¹⁷⁷ Lu-NM600. The radioactive halide phospholipid ethers are [ ¹²³ I] -NM404, [ ¹²⁴ I] -NM404, [ ¹²⁵ I] -NM404, [ ¹³¹ I] -NM404, [ ²¹¹ At] -NM404, [ ⁷⁷ Br] -NM404, Or [ ⁷⁶ Br] -NM404, the use according to claim 37. The use according to any one of claims 23 to 39, wherein the TRT agent, the immune checkpoint inhibitor, or both are administered intravenously. The use according to any one of claims 23-40, wherein the subject is a human. The cancers to be treated are melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, flat epithelial cancer of the skin or head and neck, intestinal cancer, retinal bud. 23-41, claim 23-41, selected from the group consisting of cell tumors, cervical cancers, gliomas, breast cancers, pancreatic cancers, soft tissue sarcomas, Ewing's sarcomas, rhizome myomas, osteosarcomas, Wilms tumors, and pediatric brain tumors. Use as described in any one of the items. The use according to any one of claims 23-42, wherein the cancer is treated without administering to the subject an antibody against a tumor antigen that is not a checkpoint molecule. The use according to any one of claims 23-43, wherein the cancer is treated without administering the anti-GD2 antibody to the subject. The use of (a) immunomodulatory doses of targeted radiotherapy (TRT) or (b) one or more immunostimulants in the manufacture of drugs to treat cancers, including one or more malignant solid tumors in a subject. ,
Use, in which the TRT agent can be differentially uptake and retained by malignant solid tumor tissue and the drug is systemically administered to the subject. The use according to claim 45, wherein the one or more immunostimulants are immune checkpoint inhibitors capable of targeting one or more checkpoint molecules. One or more checkpoint molecules that can be targeted by checkpoint inhibitors are A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-related protein 4), KIR ( Killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), PD-1 (programmed cell death receptor 1), PD-L1 (programmed cell death ligand 1), CD40 (differentiation antigen group 40), CD27 (differentiation antigen group 27), CD28 (differentiation antigen group 28), CD137 (differentiation antigen group 137), OX40 (CD134; differentiation antigen group 134), OX40L (OX40 ligand; differentiation antigen group 252), GITR (glucocorticoid induction) Tumor necrosis factor receptor-related protein), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand), ICOS (inducible T cell costimulatory), ICOSL (inducible T cell costimulatory ligand), B7H3 (CD276; Differentiation antigen group 276), B7H4 (VTCN1; V-set domain-containing T cell activation inhibitor 1), IDO (indolamine 2,3-dioxygenase), TIM-3 (T cell immunoglobulin domain and mutin) 46. The method of claim 46, selected from the group consisting of domains 3), Gal-9 (galectin-9), and VISTA (V domain Ig suppressor for T cell activation). 46. Claim 46, wherein the one or more immune checkpoint inhibitors comprises one or more anti-immune checkpoint molecular antibodies or one or more small molecule immune checkpoint inhibitors that act to block one or more immune checkpoint molecules. Or the use according to claim 47. One or more anti-immune checkpoint molecular antibodies are 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. Antibodies, anti-CD28 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-OX40L antibodies, GITR antibodies, GITRL antibodies, ICOS antibodies, ICOSL antibodies, B7H3 antibodies, B7H4 antibodies, IDO antibodies, TIM-3 antibodies, Gal-9 antibodies, and Claimed that one or more small immune checkpoint inhibitors selected from the group consisting of VISTA antibodies or acting to block one or more immune checkpoint molecules include small PD-L1 inhibitors. Item 48. TRT agent
(1) Metaiodobenzylguanidine (MIBG), in which the iodine atom in MIBG is a radioactive iodine isotope,
(2) Radiolabeled tumor-targeting antibody,
(3) Radioactive radium isotope, or (4) Equation:
[In the formula,
R ₁ is (a) a chelating agent chelated to a metal atom, whether the metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of more than 6 hours and less than 30 days. Or (b) contains a radioactive halogen isotope,
a is 0 or 1 and
n is an integer of 12 to 30
m is 0 or 1 and
Y is selected from the group consisting of -H, -OH, -COOH, -COOX, -OCOX, and -OX, where X is alkyl or arylalkyl.
R ₂ is selected from the group consisting of -N ⁺ H ₃ , -N ⁺ H ₂ Z, -N ⁺ HZ ₂ , and -N ⁺ Z ₃ , where each Z is independently alkyl or aryl and b. Is 1 or 2,
However, if R ₁ contains a radioactive halogen isotope, b is 1.]
The use according to any one of claims 45 to 49, which is a phospholipid ether metal chelate or a radiohalated phospholipid ether or a salt thereof. (1) The metal isotopes are Sc-47, Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac- Select from the group consisting of 225, Pb-212, and Th-227,
(2) The radioactive halogen isotope is selected from the group consisting of ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ²¹¹ At, ⁷⁷ Br, and ⁷⁶ Br, or (3) the radioactive radium isotope is Ra-. The use according to claim 50, which is 223. The chelating agent is 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-glutaric acid-4,7-diacetic acid (NODAGA) and its derivatives; 1,4,7,10-tetra Azacyclodecane, 1-glutaric acid-4,7,10-triacetic acid (DOTAGA) 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; diethylenetriamine pentaacetic acid (DTPA), its diesters, and its derivatives. Derivatives thereof; 2-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A "-DTPA) and its derivatives; deferroxamine (DFO) and its derivatives; 1,2-[[6-carboxypyridine-2-yl] methylamino] ethane (H) ₂ dedpa) and its derivatives; and selected from the group consisting of DADA and its derivatives, DADA has the following structure:
50 or the use according to claim 51, including. (A) m is 0; or (b) b is 1; or (c) n is 18; or (d) R ₂ is -N ⁺ Z ₃ ; or ( e) Any combination of two or more of (a)-(d),
Use according to any one of claims 50 to 52. The use according to claim 53, wherein each Z is independently -CH ₂ CH ₃ or -CH ₃ . Each Z is -CH _3, use of claim 54. A chelating agent chelated to a metal atom
The use according to any one of claims 50-55, selected from the group consisting of. Radioactive phospholipid ether metal chelate,
Has an expression selected from the group consisting of
The use according to any one of claims 50-56, wherein the selected compound is chelated to a metal atom. 5. One of claims 50-57, wherein a is 1, b is 1, m is 0, n is 18, and R ₂ is −N ⁺ (CH ₃ ) _3. Use of. The use according to claim 58, wherein the radioactive phospholipid ether metal chelate is NM600, which is a chelated metal atom, or the radioactive halide phospholipid ether is NM404. The use according to claim 59, wherein the radioactive phospholipid ether metal chelate is ⁹⁰ Y-NM600 or ¹⁷⁷ Lu-NM600. The radioactive halide phospholipid ethers are [ ¹²³ I] -NM404, [ ¹²⁴ I] -NM404, [ ¹²⁵ I] -NM404, [ ¹³¹ I] -NM404, [ ²¹¹ At] -NM404, [ ⁷⁷ Br] -NM404, Or the use of claim 59, which is [ ⁷⁶ Br] -NM404. The use according to any one of claims 45 to 61, wherein the TRT agent; an immune checkpoint inhibitor; or both; is administered intravenously. The use according to any one of claims 45 to 62, wherein the subject is a human. The cancers to be treated are melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, flat epithelial cancer of the skin or head and neck, intestinal cancer, retinal bud. 45-63 claims selected from the group consisting of cell tumors, cervical cancers, gliomas, breast cancers, pancreatic cancers, soft tissue sarcomas, Ewing's sarcomas, rhizome myomas, osteosarcomas, Wilms tumors, and pediatric brain tumors. Use as described in any one of the items. The use according to any one of claims 45-64, wherein the cancer is treated without administering to the subject an antibody against a tumor antigen that is not a checkpoint molecule. The use according to any one of claims 45-65, wherein the cancer is treated without administering the anti-GD2 antibody to the subject.