Use of targeted radiotherapy (TRT) to promote the antitumor immune response to immunotherapy

Abstract

To provide a method of treating a malignant solid tumor in a subject.SOLUTION: A method comprises the steps of administering to a subject an immunomodulatory dose of a radioactive phospholipid ether metal chelate, a radiohalogenated phospholipid ether, or other targeted radiotherapy (TRT) agent that is differentially retained within a malignant solid tumor tissue, and performing immunotherapy in the subject by systemically administering to the subject an immunostimulatory agent, such as an immune checkpoint inhibitor. In a non-limiting example, the radioactive phospholipid metal chelate compound has a structure expressed by a following formula. Here, R1 comprises a chelating agent that is chelated to a metal atom. The metal atom is an alpha, beta or Auger emitting metal isotope with a half-life of greater than 6 hours and less than 30 days. Alternatively, R1 comprises a radioactive halogen isotope.SELECTED DRAWING: Figure 57

Description

[Technical Field]

[0001] Cross-reference of related applications This application was filed November 10, 2017, and its entirety is incorporated herein by reference. We claim the interests of U.S. Patent Application Publication No. 15 / 809427.

[0002] Description of research or development funded by the federal government. This invention is based on OD024576 and CA197, as granted by the National Institutes of Health in the United States. This invention was made with government support under 078. The United States Government has certain rights to this invention.

[0003] This disclosure relates to methods for treating cancer in general. In particular, this disclosure relates to one or more methods in the subject matter. Cancers including malignant solid tumors are treated with (1) immunomodulatory doses of targeted radiotherapy (TRT), for example Radioactive metal chelate compounds, radioactive halogenated compounds, radiolabeled antibodies, or solid tumor compounds Radioactive isotopes are differentially incorporated and retained by the fabric. (2) administered systemically to the body (radiosiotope), one or more immunostimulants, for example This method involves systemically administering one or more immune checkpoint inhibitors to the target area. To relate to. [Background technology]

[0004] Current cancer treatments generally involve systemic chemotherapy, which targets non-targeted small molecules or antibodies. Trophotic cytotoxic drugs preferentially enter cancer cells or interact with cancer cells through various mechanisms. They bind to (in the case of antibody-targeting drugs) and kill cancer cells. They are often used in combination with chemotherapy. External beam radiation therapy (xRT) induces breaks in the nuclear DNA double helix, resulting in cell cycle death. It kills cancer cells by dissecting the tumor. Unlike systemic chemotherapy, xRT dissects the tumor. It depends on the ability to accurately determine the site. Also, surgical removal of a tumor is necessary because residual tumor cells... The ability to examine and completely remove the tumor is also crucial for rapidly reconstructing it after surgery. And xRT is usually limited to the local treatment of malignant tumors, so disseminated or metastatic There are limitations to treating diseases. This is why chemotherapy is often used in conjunction with these other modes of treatment. This is the reason why systemic chemotherapy is used for many distant metastases, although brain metastases may be an exception. While it is possible to reach a certain level, for too many patients, the response is generally short-lived (several months). This can last from a month to several years, ultimately leading to tumor recurrence.

[0005] The body's innate immune system can also destroy cancer cells after they are recognized, so immunological methods can help with cancer It is rapidly gaining traction in the treatment paradigm. However, some cancer cells, and further In many cases, cancer stem cells initially try to evade immune surveillance and are relatively immunologically invisible. By staying the way they are, they evolve and ultimately acquire the ability to survive. [Gaipi [Immunotherapy 6:597-610, 2014].

[0006] One particular immunological technique that is being increasingly investigated is "in situ vaccination." In other words, it enhances tumor immunogenicity, generates tumor-infiltrating lymphocytes (TILs), and "vaccine" A strategy to induce a systemic antitumor immune response against disseminated tumors that have not been vaccinated. Yes. In situ vaccination reverses the microenvironment that makes the immune system tolerant to tumors. It simultaneously provides pro-inflammatory signals while promoting the release of tumor antigens in malignant solid tumors. Inject one or more drugs (or treat with them) [Pierce et al., Human Va Ccines&Immunotherapoeutics 11(8):1901-19 09,2015;Marabelle et al., Clin.Cancer Res.20(7) :1747-56, 2014; Morris et al., Cancer Res; 76(13); [3929-41, 2016].

[0007] Another completely different approach is systemic immunotherapy. Immunostimulants such as immune checkpoint inhibitors are injected locally into tumors. They are not administered intravenously, but rather circulate throughout the body. Such drugs are antitumor immunosuppressants. Treating tumors where an epidemic response exists but has been "exhausted" or is no longer effective. It can be used for this purpose. In the case of checkpoint inhibitors, tumor cells are treated with existing antitumor agents. Checkpoint ligands interact with checkpoint receptors on immune cells. Alternatively, it expresses other checkpoint molecules, causing inactivation of these cells. By blocking the interaction, systemically administered checkpoint inhibitors reduce fatigue in cancer patients. This activates the existing immune response, allowing the patient's own immune system to more effectively attack cancer cells. To promote.

[0008] Recent data from clinical trials and preclinical models demonstrate the potential of these methods. However, in this field, there is a great need for systemic immunotherapy methods that demonstrate improved systemic efficacy. It is said that...

[0009] Radiation hormesis is the natural preservation of ionizing RT at low doses, which is not activated without ionizing RT. This is a hypothesis from several decades ago that it may be beneficial to stimulate the activation of protective and repair mechanisms. [Cameron and Moulder,Med.Phys.25:1407,19 98] The pre-repair mechanism not only counteracts the harmful effects of ionizing RT, but also relates to RT exposure. It is assumed that it is sufficiently effective when stimulated to suppress a disease that does not exist. The abscopal effect is that xRT treatment of one tumor can affect another tumor outside the RT treatment area. This is a phenomenon reported in the 1950s that actually causes a reduction in the size of the ulcer. Although rare, This phenomenon is thought to depend on the activation of the immune system. In summary, hormesis and abscopa The effect is due to the interaction of the immune system by low doses (immunostimulative but non-cytotoxic) of RT. This supports the possibility of a stimulating effect. This is next for other immunological therapies such as systemic immunotherapy. It can be combined with other methods.

[0010] The inventors have found that if a single tumor is present, local xRT + in situ vaccine administration is recommended. A combination of species and / or systemic checkpoint inhibitor immunotherapy has shown significant improvement in mice. [Morris et al.] previously published that it is highly synergistic in treating established tumors. [Cancer Res;76(13);3929-41,2016]. However, I was surprised. In particular, the inventors have found that the combination of in situ vaccination and xRT is effective in treating a second non-irradiated tumor. We found that tumor growth was not suppressed in the presence of a ulcer. Clearly, non-irradiated tumors were suppressed by irradiated tumors. The inhibitory effect on the immunomodulatory effects of xRT and in situ vaccines (inventor) They exhibit what they call "associated immune tolerance."

[0011] If xRT is administered to the entire area of ​​the tumor, this associated immune tolerance can be overcome. This enables the effectiveness of in situ vaccination. However, xRT is used for multiple tumors. If present, especially if there are a significant number of tumors, or if the location of one or more tumors is precisely determined If not available, or if xRT cannot be delivered to all parts of the tumor. It cannot be effectively used in combination with in situ vaccination. Furthermore, administering xRT to all tumor sites in patients with metastatic disease leads to systemic immunosuppression. This is highly likely to occur and is not in line with the main objective of systemic immunotherapy.

[0012] Therefore, regardless of the number and anatomical location of the tumors, systemic immunotherapy can be combined with other treatments. Furthermore, an improved method is needed to deliver immunomodulatory doses of RT to all target tumors. be. [Overview of the project] [Problems that the invention aims to solve]

[0013] The inventors have found that certain alkylphosphocholine analogs are preferentially absorbed by malignant solid tumor cells. It was previously shown that it is incorporated and retained. The full text is incorporated herein by reference. In U.S. Patent Publication No. 2014 / 0030187, Weichert et al. described various 18-(p-iodine) is the main agent for the detection, localization, and treatment of malignant solid tumors. The use of an analog of nyl)octadecylphosphocholine (NM404; see Figure 1) is disclosed. The iodine portion is a radionuclide optimized for imaging, for example, iodine-124 ([ 124 If it is I]-NM404, this analogue is a positron emission tomography (POST) of solid tumors. Computed tomography (PET / CT) or single-photon emission computed tomography (S It can be used in PECT imaging. Alternatively, the iodine portion is iodine-125. Ta is iodine-131([ 125 I]-NM404 or [ 131 I]-NM404) etc. The analogue is optimized to deliver a therapeutic dose of RT to solid tumor cells that take up the analogue. If it is a radionuclide, this analog can be used to treat solid tumors.

[0014] Such analogues not only target a wide range of solid tumor types in vivo, but also tumor It is selectively retained in ulcer cells for a long period of time, and therefore has high potential as an RT agent. Furthermore, tumor uptake is limited to malignant cancers and does not occur in pre-malignant or benign lesions. stomach.

[0015] However, the radioactive iodine isotope used in previously disclosed alkylphosphocholine analogs Metal isotopes that have better properties for imaging and / or RT than the body. For example, as an imaging isotope, I-124 has insufficient positron emission ( Only about 24% of the radiation is positrons, and it is also often confused with gamma radiation (600 keV). This actually interferes with normal 511KeV PET detection. Certain positron-emitting metals It has good imaging characteristics. As another example, as an RT isotope, I-131 is different from other The energy generates other non-therapeutic releases, which are directed into adjacent normal tissues, including the bone marrow. Adding unwanted radiation dose measurements. The beta particle range of I-131 is also very long, and O2 This can cause toxicity. Some metal radiotherapy isotopes have cleaner emission levels. It offers a rhophilic and shorter pathway length, and therefore less potential toxicity.

[0016] The inventors of this invention have identified radioactive iodine isotopes (for example, the full text of which is incorporated herein by reference). Instead of chelated ions (see U.S. Patent Publication No. 2018 / 0022768), chelated ions are used. We have developed improved alkylphosphocholine analogs containing propellant metal isotopes. Since they contain the same skeleton as previously disclosed radioactive iodized compounds, these still pose a risk. It is selectively taken up and retained by tumor cells. However, chelated radioactive metal isotopes , the release has been improved for imaging and / or radiotherapy applications. The drug is present in the target area, regardless of whether the number and anatomical location of the tumors are known or not. It is less cytotoxic to all malignancies, but it is very effective in delivering immunomodulatory doses of ionizing RT. Suitable. [Means for solving the problem]

[0017] Therefore, in the first aspect, the disclosure relates to cancer including one or more malignant solid tumors in the subject. This method includes a method for treating (a) malignant solid tumor tissue. Targeted radiation of immunomodulatory doses that is (differentially) taken up and retained. The procedure includes the step of systemically administering (b) a radiotherapy (TRT) agent and one or more immunostimulants.

[0018] In some embodiments, one or more immunostimulants target one or more checkpoint molecules It is an immune checkpoint inhibitor with the ability to target specific immune points.

[0019] Examples of one or more immune checkpoint inhibitors include the following checkpoints Receptor molecules: A2AR (adenosine A2a receptor), BTLA (B and T lymphocyte attenuator (Dietary), CTLA4 (Cytotoxic T lymphocyte-associated protein 4), KIR (Killer Virus) (Hydrocyte immunoglobulin-like receptor), LAG3 (lymphocyte activating gene 3), PD-1 (prog Lamb cell death receptor 1), PD-L1 (programmed cell death ligand 1), CD40 (differentiation inhibitor) original group 40), CD27 (differentiation antigen group 27), CD28 (differentiation antigen group 28), CD137 ( differentiation antigen group 137), OX40 (CD134; differentiation antigen group 134), OX40L (OX4 0 Ligand; Differentiation antigen group 252), GITR (Glucocorticoid-induced tumor necrosis factor receptor) Body-related proteins), GITRL (glucocorticoid-induced tumor necrosis factor receptor-related proteins) protein ligand), ICOS (inducible T cell costimulatory) , ICOSL (inducible T cell costimulatory ligand), B7H3 (CD276; differentiation antigen group 27 6) B7H4 (VTCN1; V-set domain-containing T cell activation inhibitor 1), IDO (Indoleamine 2,3-dioxygenase), TIM-3 (T cell immunoglobulin) Main and mucin domain 3), Gal-9 (galectin-9), or VISTA ( Drugs capable of targeting one or more of the V-domain Ig suppressors involved in T cell activation are listed. It can be done.

[0020] In some embodiments, one or more immune checkpoint inhibitors are present in the following combination: It contains checkpoint molecular antibodies. In some such embodiments, one or more antiimmune antibodies. A checkpoint molecule antibody contains at least one monoclonal antibody.

[0021] In some embodiments, one or more immune checkpoint inhibitors include one or more immune checkpoint inhibitors. It contains one or more small molecules that have the ability to inhibit or block checkpoint molecules. Examples of small molecule checkpoint inhibitors include CA-170 and CA -327 is one example, and both of these target PD-L1.

[0022] In some embodiments, one or more anti-immune checkpoint molecular antibodies include anti-CTLA 4 antibodies, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG3 antibody, anti-KIR antibody, anti-A2A R antibody, and anti-BTLA antibody, anti-CD40 antibody, anti-CD27 antibody, anti-CD28 antibody, anti CD137 antibody, anti-OX40 antibody, anti-OX40L antibody, anti-GITR antibody, anti-GITRL anti- body, anti-ICOS antibody, anti-ICOSL antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-IDO antibody This includes an anti-TIM-3 antibody, an anti-Gal-9 antibody, or an anti-VISTA antibody.

[0023] In some embodiments, the TRT agent is a radioactive iodine isotope in which the iodine atom in MIBG is It is metaiodobenzylguanidine (MIBG).

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

[0025] In some embodiments, the TRT agent is a radioactive isotope of radium, such as Ra-223. And so on.

[0026] In some embodiments, the TRT agent is given by formula: [ka] It is a radioactive phospholipid ether metal chelate or a salt thereof having R1, where R1 is a metal atom It contains a chelated chelating agent, and the metal atoms are chelated for more than 6 hours but less than 30 days. It is an alpha, beta, or Auger-emitting metal isotope with a decay period, where a is 0 or 1. n is an integer between 12 and 30, m is 0 or 1, and Y is -H, -OH, or -COO H is -COOX, -OCOX, or -OX, where X is alkyl or aryl. R2 is -N + H3, -N + H2Z, -N + HZ2, or -N + It is Z3, and each Z is unique The compound is alkyl or alloalkyl, and b is 1 or 2. The metals that can be used are also... Examples of non-positional radioactive materials include Sc-47, 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.

[0027] In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododeca. 1,4,7-triacetic acid (DO3A) or one of its derivatives, 1,4,7-triazacy Chrononane-1,4-diacetic acid (NODA) or one of its derivatives, 1,4,7-tria Zacyclononane-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 the 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, 4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or one of its derivatives, 1,4,8,11-tetraazabicyclo[6.6.2]hexa Decane-4,11-diacetic acid (CB-TE2A) or one of its derivatives, diethylene triacetic acid Amine pentaacetic acid (DTPA), its diester, or one of its derivatives, 2-cyclohex Sildiethylenetriaminepentaacetic acid (CHX-A''-DTPA) or one of its derivatives, Deferoxamine (DFO) or one of its derivatives, 1,2-[[6-carboxypyrrh [Din-2-yl]methylamino]ethane (H2dedpa) or one of its derivatives, Selected from the group consisting of DADA or one of its derivatives, DADA has the following structure: [ka] It holds.

[0028] In some embodiments, a is 1 (aliphatic aryl-alkyl chain). Other embodiments In this state, a is 0 (aliphatic alkyl chain).

[0029] In some embodiments, m is 1 (acylphospholipid series). In such embodiments, n is an integer between 12 and 20. In some embodiments, Y is It is -OCOX, -COOX, or -OX.

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

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

[0032] In some embodiments, b is 1.

[0033] In some embodiments, n is 18.

[0034] In some embodiments, R2 is -N + Z3. In some such embodiments each Z is independently -CH2CH3 or -CH3. In some such embodiments each Z is -CH3.

[0035] In some embodiments, the chelating agent chelated to the metal atom is: [Chemical formula] JPEG0007839135000004.jpg20784JPEG0007839135000005.jpg25594JPEG0007839135000006.jpg229108JPEG0007839135000007.jpg5873

[0036] In some embodiments, the radiolabeled phospholipid ether metal chelate is one of the following compounds in which the selected compound is chelated to the metal atom: [Chemical formula] TIFF0007839135000009.tif250124TIFF0007839135000010.tif235124TIFF0007839135000011.tif255150TIFF0007839135000012.tif167124

[0037] In some embodiments, in the phospholipid ether metal chelate structure, a is 1, b is 1, m is 0, n is 18, and R2 is -N + (CH3)3. In some such embodiments, the phospholipid ether metal chelate is NM600 chelated to the metal atom, such as (but not limited to) Y-NM600 and the like. 90

[0038] In some embodiments, the TRT agent is given by formula: [ka] It is a radioactive halogenated phospholipid ether or a salt thereof having R1. It contains isotopes, where a is 0 or 1, n is an integer between 12 and 30, and m is 0 or 1 Y is composed of -H, -OH, -COOH, -COOX, -OX, and -OCOX. Selected from the group, where X is alkyl or arylalkyl and R2 is -N + H3, -N + H2Z, -N + HZ2 and -N + Selected from the group consisting of Z3, in the above equation Each Z is independently alkyl or aryl.

[0039] In some embodiments, radioactive halogen isotopes are 123 I, 124 I, 125 I, 131 I, 211 At, 76 Br, or 77 It is Br.

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

[0041] In some embodiments, n is 18.

[0042] In some embodiments, R2 is -N + (CH3)3 is used in some embodiments. In some such embodiments, a is 1, m is 0, and n is 18. Radioactive halogen isotopes are, 123 I, 124 I, 125 I, Taha 131 I (Radioactive halogenated phospholipid ethers are [ 123 I]-NM404, [ 124 I]-NM404, etc. 125 I]-NM404, etc. 131 I]-NM404, etc. 211 At]-NM404, 76 Br]-NM404, or [ 77 Br]-NM40 It is 4).

[0043] In some embodiments, a TRT agent, an immunostimulant, or both are administered intravenously. .

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

[0045] Examples of cancers that can be treated using this method as malignant solid tumors include, but are not limited to, the following: melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer Subcutaneous cancer, squamous cell carcinoma of the skin or head and neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, Breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms' tumor, Other examples include pediatric brain tumors.

[0046] In some embodiments, cancer is a tumor antigen that is not a checkpoint molecule. The treatment is administered without targeting the disease.

[0047] In some embodiments, the anti-GD2 antibody is not administered to the subjects.

[0048] In a second aspect, the Disclosure, as further described above, relates to one or more malignant properties in the subject matter. This includes the use of TRT agents and one or more immunostimulants to treat cancer, including solid tumors. .

[0049] In a third aspect, the Disclosure, as further described above, relates to one or more malignant properties in the subject matter. TRT agents and / or one or more for the manufacture of drugs for treating cancer, including solid tumors. This includes the use of immunostimulants.

[0050] Other objects, features, and advantages of the present invention are discussed in the specification, claims, and drawings. It will become clear afterwards.

[0051] This patent or application document includes at least one colored drawing. This one or more drawings A copy of the patent or application publication, including several color drawings, can be requested from the patent office for the necessary fees. You can get it by paying. [Brief explanation of the drawing]

[0052] [Figure 1] This figure shows the chemical structure of the main ingredient, 18-(p-iodophenyl)octadecylphosphocholine (NM404). [Figure 2A] This figure shows a graph demonstrating that xRT+IT-IC induces in situ tumor vaccination. More specifically, Figure 2A shows a tumor growth curve illustrating the synergistic effect between xRT and IT-hu14.18-IL2. 71% (22 / 31) of mice treated with xRT+IT-IC were disease-free. [Figure 2B] This figure shows another graph demonstrating that xRT+IT-IC induces in situ tumor vaccination. More specifically, Figure 2B shows the Kaplan-Meier survival curves illustrating the synergistic effect between xRT and IT-hu14.18-IL2. 71% (22 / 31) of mice treated with xRT+IT-IC were disease-free. [Figure 2C]This figure shows another graph demonstrating that xRT+IT-IC induces in situ tumor vaccination. More specifically, Figure 2C shows that 90% of the treated mice subsequently reject engraftment by B78 melanoma. [Figure 3] This figure shows a graph illustrating the associated immune tolerance. The response of the primary tumor is shown. The untreated distant tumor suppresses the response to xRT+IT-IC in a two-tumor B78 melanoma model. This suppression can be overcome by radiating the second tumor. [Figure 4] This figure shows a graph indicating that the associated immune tolerance is attributable to Tregs. The response of the primary tumor is shown. Untreated distant tumors suppressed the response to xRT+IT-IC in a 2-tumor B78 melanoma model, and this suppression can be overcome by depleting Tregs (using transgenic DEREG mice that express diphtheria toxin receptors in Tregs, and depleting Tregs by administering diphtheria toxin). [Figure 5] This figure shows an image illustrating the selective uptake of 124I-NM404 by B78 melanoma. Mice with B78 tumors of approximately 200 mm3 were intravenously administered IV 124I-NM404, and serial PET / CT scans were performed. This image at 71 hours shows selective uptake by the tumor and some residual background uptake by the heart and liver. [Figure 6] This figure shows a graph illustrating that in situ vaccination can be induced in the presence of residual levels of molecularly targeted radiotherapy (TRT). The combination treatment with xRT + IT-IC is equally effective with or without 3 μCi of 131I-NM404. This is approximately the same as the residual radioactivity of TRT that would be present when the inventors deliver xRT (day 0) followed by IT-IC (days 6-10), as described in Example 4. [Figure 7] This figure shows the changes in MRI images over time, illustrating tumor (T) growth up to 24 hours after injection of Gd-NM600 in mice with tumors. [Figure 8A] This figure shows tumor-specific suppression of the primary tumor response to the combination of local RT+IT-IC by distant untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice expressing the syngeneic disiaroganglioside (GD2+) with a primary flank tumor + / - secondary tumor on the opposite flank were treated, as shown, with xRT on day "1" only in the primary tumor, and with intratumor (IT) injection of 50 mcg of the anti-GD2 immune cytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2) on days 6-10. The mean volume of the primary tumor is shown in Figure 8A. More specifically, Figure 8A shows that in mice with a primary B78 melanoma tumor, the presence of an untreated secondary B78 tumor antagonized the primary tumor response to RT+IT-IC. The inventors describe this effect as "accompanying immune tolerance," that is, as the antagonistic effect of the untreated distant tumor on the local response of the treated tumor to xRT+IT-IC. [Figure 8B] Another figure shows tumor-specific suppression of the primary tumor response to the combination of local RT+IT-IC by distantly untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice expressing the syngeneic disialoganglioside (GD2+) with primary flank tumor + / - secondary tumor on the opposite flank were treated with xRT on day "1" for the primary tumor only, as shown, and with intratumor (IT) injection of 50 mcg of anti-GD2 immune cytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8B shows Kaplan-Meier survival curves for mice and replicate experiments. Almost all mice were euthanized due to progression of the primary tumor. [Figure 8C]This is another figure demonstrating tumor-specific suppression of the primary tumor response to the local RT+IT-IC combination by distantly untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice expressing the syngeneic disiaroganglioside (GD2+) with a primary flank tumor + / - secondary tumor on the contralateral flank were treated, as shown, with xRT on day "1" only for the primary tumor, and with intratumor (IT) injection of 50 mcg of the anti-GD2 immune cytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8C shows that in mice with a primary Panc02-GD2+ pancreatic tumor, the presence of an untreated Panc02 secondary tumor suppressed the response of the primary Panc02-GD2+ tumor to RT+IT-IC, regardless of the presence or absence of a secondary Panc02-GD2- tumor on the contralateral flank. [Figure 8D] This is another figure demonstrating tumor-specific suppression of the primary tumor response to the local RT+IT-IC combination by distantly untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice expressing the syngeneic disiaroganglioside (GD2+) with primary flank tumors + / - secondary tumors on the opposite flank were treated, as shown, with xRT on day "1" only for the primary tumor, and with intratumor (IT) injection of 50 mcg of the anti-GD2 immune cytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2), on days 6-10. More specifically, Figure 8D shows that in mice with primary B78 melanoma tumors, secondary B78 tumors suppressed the primary tumor response to RT+IT-IC, while secondary Panc02-GD2+ pancreatic tumors did not exert this effect. [Figure 8E]Another figure shows tumor-specific suppression of the primary tumor response to the combination of local RT+IT-IC by distantly untreated tumors in mouse melanoma and pancreatic tumor models. C57BL / 6 mice expressing the syngeneic disiaroganglioside (GD2+) with primary flank tumor + / - secondary tumor on the opposite flank were treated with xRT on day "1" only for the primary tumor, as shown, and with intratumor (IT) injection of 50 mcg of anti-GD2 immune cytokine (IC), hu14.18-IL2 (a fusion of anti-GD2 mAb and IL2) on days 6-10. More specifically, Figure 8E shows that in mice with primary Panc02-GD2+ tumors, secondary Panc02-GD2- tumors suppressed the primary tumor response to the combination of xRT and IT-hu14.18-IL2, but B78 secondary tumors did not. n = number of mice per group. NS = Not significant, *p<0.001. [Figure 9A-1] This figure shows immunohistochemical images (left and center) and graphs (right) demonstrating that associated immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9A shows immunohistochemistry (representative 400x magnification images are shown) of the tumor Treg marker, FoxP3, evaluated 6 days after xRT in mice with one tumor (Figure 9A, leftmost panel A1 and A2) or two tumors (Figure 9A, center panel A3 and A4). Mice either did not receive xRT or received xRT only for primary tumors. Primary tumors are shown in panels A1-A3 of Figure 9A, and secondary tumors are shown in panel A4 of Figure 9A. Small arrows indicate some FoxP3+ cells (brown nuclei = FoxP3+, blue = hematoxylin counterstaining). The graph on the right shows the blinded test for the number of FoxP3+ cells per 200x field of view, corresponding to the conditions shown in panels A1, A2, A3, and A4 of Figure 9A, respectively. [Figure 9A-2]This figure shows immunohistochemical images (left and center) and graphs (right) demonstrating that associated immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9A shows immunohistochemistry (representative 400x magnification images are shown) of the tumor Treg marker, FoxP3, evaluated 6 days after xRT in mice with one tumor (Figure 9A, leftmost panel A1 and A2) or two tumors (Figure 9A, center panel A3 and A4). Mice either did not receive xRT or received xRT only for primary tumors. Primary tumors are shown in panels A1-A3 of Figure 9A, and secondary tumors are shown in panel A4 of Figure 9A. Small arrows indicate some FoxP3+ cells (brown nuclei = FoxP3+, blue = hematoxylin counterstaining). The graph on the right shows the blinded test for the number of FoxP3+ cells per 200x field of view, corresponding to the conditions shown in panels A1, A2, A3, and A4 of Figure 9A, respectively. [Figure 9B] Another graph shows that concomitant immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9B shows that DEREG mice express the diphtheria toxin receptor under the control of a Treg-specific FoxP3 promoter, enabling specific depletion of Tregs upon IP injection of diphtheria toxin. DEREG mice with primary and secondary B78 melanoma tumors were treated with xRT+IT-IC to the primary tumor and IP injection of either diphtheria toxin or PBS (first replicate experiment shown). Concomitant immune tolerance was eliminated in these mice after Treg depletion, and the response to the primary tumor was improved (Figure 9B). n = number of mice per group. * *p<0.01, ***p<0.001. [Figure 9C]Another graph shows that concomitant immune tolerance is avoided by specific depletion of regulatory T cells (Tregs). More specifically, Figure 9C shows that DEREG mice express the diphtheria toxin receptor under the control of a Treg-specific FoxP3 promoter, enabling specific depletion of Tregs upon IP injection of diphtheria toxin. DEREG mice with primary and secondary B78 melanoma tumors were treated with xRT+IT-IC to the primary tumor and IP injection of either diphtheria toxin or PBS (first replicate experiment shown). Concomitant immune tolerance was eliminated in these mice after Treg depletion, and the response to the secondary tumor was improved (Figure 9C). n = number of mice per group. * *p<0.01, ***p<0.001. [Figure 10A] This figure shows a graph illustrating that the 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 treatment of the primary tumor with xRT + IT-IC. This was overcome by delivering 12 Gy of xRT to both the primary and secondary tumors, and IT-IC to the primary tumor, resulting in an improved primary tumor response from replicate experiments (the first replicate experiment is shown) (Figure 10A). n = number of mice per group. **p < 0.01, ***p < 0.001. [Figure 10B] This figure shows another graph demonstrating that the 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 treatment of the primary tumor with xRT + IT-IC. This was overcome by delivering 12 Gy of xRT to both the primary and secondary tumors, and IT-IC to the primary tumor, resulting in improved aggregate animal survival from repeated experiments (Figure 10B). n = number of mice per group. **p < 0.01, ***p < 0.001. [Figure 11A]This figure shows a graph indicating that low-dose xRT alone does not induce in situ vaccination, but when delivered to a distant tumor site in conjunction with 12 Gy + IT-IC treatment at the in situ vaccination site, it does indeed overcome the associated immune tolerance. More specifically, Figure 11A shows that in mice with only primary B78 tumors, 12 Gy + IT-IC induced in situ vaccination (as already shown) and resulted in complete tumor regression in most mice (4 / 6 in this experiment) and a memory immune response (Morris, Cancer Res, 2016). On the other hand, no animals showed complete tumor regression after IT-IC alone or low-dose (2 Gy) xRT + IT-IC (0 / 6 in both groups) p<0.05. [Figure 11B] Figure 11B shows another graph demonstrating that low-dose xRT alone does not induce in situ vaccination, but when delivered to a distant tumor site in conjunction with 12 Gy + IT-IC treatment at the in situ vaccination site, it does indeed overcome the associated immune tolerance. More specifically, Figure 11B shows that in mice with primary and secondary B78 melanoma tumors, low-dose xRT (2 Gy or 5 Gy) delivered to the secondary tumor is comparable to 12 Gy in its ability to overcome the associated immune tolerance in the primary tumor. [Figure 11C] Figure 11C shows another graph demonstrating that low-dose xRT alone does not induce in situ vaccination, but when delivered to distant tumor sites in conjunction with 12 Gy + IT-IC treatment at the in situ vaccination site, it does indeed overcome concomitant immune tolerance. More specifically, Figure 11C shows that in these same animals, overcoming concomitant immune tolerance by delivering low-dose xRT to secondary tumors rescues the systemic response to IT-IC immunotherapy. In this regard, when xRT is delivered to all tumor sites, IT-IC injection at the primary tumor elicits a systemic antitumor effect, making the secondary tumor response to 2 Gy or 5 Gy greater than the response to 12 Gy of xRT without IT-IC injection at the primary tumor. [Figure 12A]This figure shows PET images demonstrating that low-dose TRT and 131I-NM404 effectively deplete tumor-infiltrating FoxP3+ Tregs without causing systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical scenarios, it is impossible to deliver external beam radiation to all tumor sites, even at low doses, without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression. Here, we investigated whether systemic administration of TRT could specifically deplete tumor-infiltrating inhibitory immune cells (Tregs) without causing systemic immune cell depletion and leukopenia. More specifically, Figure 12A shows that a dose-measuring study in this B78 melanoma tumor model using positron-emitting 124I-NM404 confirms tumor-selective uptake of NM404. C57BL / 6 mice with B78 tumors were treated with 60 μCi of 131I-NM404. This radioactivity is approximately the same as the amount of 131I-NM404 required to deliver about 2 Gy of TRT to a B78 tumor. Peripheral blood and tumor samples were collected from untreated control mice (named C) and then at 8-day intervals (T1=d8, T2=d16, T3=d24, T4=d32). [Figure 12B] This figure shows a bar graph demonstrating that low-dose TRT and 131I-NM404 effectively deplete tumor-infiltrating FoxP3+ Tregs without causing systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical scenarios, it is impossible to deliver external beam radiation to all tumor sites, even at low doses, without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression. Here, we tested whether systemic administration of TRT could specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without causing systemic immune cell depletion and leukopenia. More specifically, Figure 12B shows that this dose of TRT did not cause significant systemic leukopenia. [Figure 12C]This figure shows another bar graph demonstrating that low-dose TRT and 131I-NM404 effectively deplete tumor-infiltrating FoxP3+ Tregs without causing systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical scenarios, it is impossible to deliver external beam radiation to all tumor sites, even at low doses, without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression. Here, we tested whether systemic administration of TRT could specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without causing systemic immune cell depletion and leukopenia. More specifically, Figure 12C shows that this dose of TRT did not have a significant effect on the level of tumor-infiltrating CD8+ effector T cells (ANOVA p=0.25). [Figure 12D] This figure shows another bar graph demonstrating that low-dose TRT and 131I-NM404 effectively deplete tumor-infiltrating FoxP3+ Tregs without causing systemic leukopenia or depletion of tumor-infiltrating CD8+ effector T cells. In most clinical scenarios, it is impossible to deliver external beam radiation to all tumor sites, even at low doses, without causing significant bone marrow depletion and leukopenia, which would result in immunosuppression. Here, we tested whether systemic administration of TRT could specifically deplete tumor-infiltrating suppressor immune cells (Tregs) without causing systemic immune cell depletion and leukopenia. More specifically, Figure 12D shows that tumor-infiltrating FoxP3+ Tregs were significantly depleted by this dose of TRT (ANOVA p=0.03; *p<0.05). [Figure 13A]This figure shows a graph demonstrating that low-dose TRT and 131I-NM404 effectively overcome concomitant immune tolerance and rescue the systemic antitumor effect of in situ vaccination. Considering the ability of low-dose 131I-NM404 TRT to deplete tumor-infiltrating Tregs without causing leukopenia in mice, we tested whether low-dose 131I-NM404 effectively overcomes concomitant immune tolerance. C57BL / 6 mice with two B78 tumors were treated with 60 mcCi of 131I-NM404 (NM404) on day 1, as shown. After one half-life (8 days), the animals were either administered 12 Gy of xRT to the primary tumor (in situ vaccine site) or not administered xRT. Control mice that did not receive 131I-NM404 were treated with the indicated doses (0, 2, or 12 Gy) for secondary tumors. On days 13–17, mice were administered daily IC IT injections to the primary tumors (in situ vaccine sites) as shown. More specifically, Figure 13A demonstrates the primary tumor response, showing that low-dose TRT administration effectively overcomes concomitant immune tolerance and rescues the systemic antitumor effect of in situ vaccination. [Figure 13B]This figure shows another graph demonstrating that low-dose TRT and 131I-NM404 effectively overcome concomitant immune tolerance and rescue the systemic antitumor effect of in situ vaccination. Taking into account the ability of low-dose 131I-NM404 TRT to deplete tumor-infiltrating Tregs without causing leukopenia in mice, we tested whether low-dose 131I-NM404 effectively overcomes concomitant immune tolerance. C57BL / 6 mice with two B78 tumors were treated with 60 mcCi of 131I-NM404 (NM404) on day 1, as shown. After one half-life (8 days), the animals were either administered 12 Gy of xRT to the primary tumor (in situ vaccine site) or not administered xRT. Control mice that did not receive 131I-NM404 were treated with the indicated doses (0, 2, or 12 Gy) for secondary tumors. On days 13–17, mice were administered daily IC IT injections to the primary tumor (in situ vaccine site) as indicated. More specifically, Figure 13B demonstrates the secondary tumor response, showing that low-dose TRT administration effectively overcomes concomitant immune tolerance and rescues the systemic antitumor effect of in situ vaccination. [Figure 14] This figure shows the chemical structure of an example alkylphosphocholine metal chelate (64Cu-NM600). Other metals may be used instead of 64Cu. [Figure 15] This figure shows PET / CT images from scans performed 48 hours after injection of 86Y-NM600 in two 1-tumor B78 mice. [Figure 16] This figure shows PET / CT images from scans performed 48 hours after injection of 86Y-NM600 in two 2-tumor B78 mice. [Figure 17] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into U87MG mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 18]This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into 4T1 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 19] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into HCT-116 mice. These images show tissue radioactivity calculated as the percentage of injected volume per g of tissue (%ID / g, scale shown on the far right). [Figure 20] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into A549 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 21] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into PC-3 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 22] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into HT-29 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 23] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 64Cu-NM600 into MiaPaca mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 24]This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 86Y-NM600 into 4T1 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 25] This figure shows PET / CT images from scans performed 3 hours (left panel), 24 hours (center panel), and 48 hours (right panel) after injection of 89Zr-NM600 into 4T1 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 26] This figure shows PET / CT images from scans performed 4 hours (left panel) and 1 day (right panel) after injection of 52Mn-NM600 into HT-29 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown on the far right). [Figure 27] This figure shows PET / CT images from scans performed 4 hours (left panel) and 1 day (right panel) after injection of 52Mn-NM600 into PC-3 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown to the right of each image). [Figure 28] This figure shows PET / CT images from scans performed 2 days (left panel), 3 days (second panel from the left), 5 days (second panel from the right), and 7 days (right panel) after injection of 52Mn-NM600 into HT-29 mice. These images show tissue radioactivity calculated as the percentage of injected volume / g of tissue (%ID / g, scale shown to the right of the image). [Figure 29] This figure shows PET / CT images from scans performed 2 days (left panel), 3 days (second panel from the left), 5 days (second panel from the right), and 7 days (right panel) after injection of 52Mn-NM600 into PC-3 mice. These images show tissue radioactivity calculated as injection volume / g tissue percentage (%ID / g, scale shown to the right of the image). [Figure 30]This figure shows a graph illustrating the PET quantitative range of the target data (chelate uptake as a function of time) for 4T1 tumor tissue from 4T1 mice injected with 86Y-NM600, 64Cu-NM600, and 89Zr-NM-600. [Figure 31] This figure shows a graph illustrating the PET quantitative range of the target data (chelate uptake as a function of time) for cardiac tissue of 4T1 mice injected with 86Y-NM600, 64Cu-NM600, and 89Zr-NM-600. [Figure 32] This figure shows a graph illustrating the PET quantification range of target data (chelate uptake as a function of time) for liver tissue of 4T1 mice injected with 86Y-NM600, 64Cu-NM600, and 89Zr-NM-600. [Figure 33] This figure shows a graph illustrating the PET quantitative range of target data (chelate uptake as a function of time) for the whole body of 4T1 mice injected with 86Y-NM600, 64Cu-NM600, and 89Zr-NM-600. [Figure 34] This figure shows bar graphs illustrating the ex vivo intracellular distribution of metal chelates in healthy and tumor tissues of 4T1 mice 48 hours (86Y-NM600, 64Cu-NM600, 89Zr-NM-600, and 177Lu-NM600) and 96 hours (177Lu-NM600) after injection. [Figure 35] This figure shows the chemical structure of an example alkylphosphocholine metal chelate (177Lu-NM600). Other metals may be used instead of 177Lu. [Figure 36] This figure shows audioradiographic images of three B78 mice taken 48 hours after injection of 90Y-NM600. The xenografted B78 tumor is visible as a large black dot in the lower right of each mouse image. [Figure 37] This figure shows audioradiography images of three B78 mice taken 96 hours after injection of 90Y-NM600. The xenografted B78 tumor is visible as a large black dot in the lower right corner of each mouse image. [Figure 38] This figure shows an audioradiography image of a B78 mouse taken 5 days after injection with 177Lu-NM600. The xenografted B78 tumor appears as two black dots at the bottom of the mouse. [Figure 39] This figure shows an audioradiography image of a B78 mouse taken 13 days after injection with 177Lu-NM600. The xenografted B78 tumor appears as two black dots at the bottom of the mouse. [Figure 40] This figure shows audioradiography images of MiaPaca mice taken 10 days after injection with 177Lu-NM600. The location of the xenografted MiaPaca tumor is indicated by arrows and dashed circles. [Figure 41] This figure shows audioradiography images of three 4T1 mice taken 48 hours after injection of 177Lu-NM600. The location of the xenografted 4T1 tumor is indicated by arrows and dashed circles. [Figure 42] This figure shows audioradiography images of three 4T1 mice taken 96 hours after injection of 177Lu-NM600. The location of the xenografted 4T1 tumor is indicated by a dashed circle. [Figure 43] This figure shows audioradiography images of three 4T1 mice taken 4 hours after injection of 90Y-NM600. The location of the xenografted 4T1 tumor is indicated by arrows and dashed circles. [Figure 44] This figure shows audioradiography images of three 4T1 mice taken 48 hours after injection of 90Y-NM600. The xenografted 4T1 tumor is visible as a large black dot in the lower right of each mouse image. [Figure 45] This figure shows audioradiography images of three 4T1 mice taken 96 hours after injection of 90Y-NM600. The xenografted 4T1 tumor is visible as a large black dot in the lower right of each mouse image. [Figure 46]This figure shows a graph illustrating the effects of 90Y-NM600 radiotherapy at two different doses (150 μCi and 300 μCi) in a B78 xenograft mouse model compared to a control (excipients only). Data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). [Figure 47] This figure shows a graph illustrating the effect of a single 500 μCi dose of 1 77 Lu-NM600 radiotherapy in a B78 xenograft mouse model compared to a control (excipients only). The data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). [Figure 48] This figure 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 a control (excipients only). The data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). [Figure 49] This figure shows a graph illustrating the effect of a single 500 μCi dose of 1 77 Lu-NM600 radiotherapy in a 4T1 xenograft mouse model compared to a control (excipients only). Data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). *P<0.05;**P<0.01;***P<0.001. [Figure 50] This figure shows a graph illustrating the effect of radiotherapy with two consecutive doses of 177Lu-NM600 (500 μCi and 250 μCi) in a 4T1 xenograft mouse model, compared to a control (excipients only). The data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). [Figure 51] This figure shows a graph illustrating the effect of 177 Lu-NM600 radiotherapy at two different doses (500 μCi and 250 μCi) in a 4T1 xenograft mouse model compared to a control (excipients only). The data are presented as a function of time, expressed as days after injection, with respect to measured tumor volume (mm3). [Figure 52]This figure shows a graph illustrating the effect of tumor volume on the comparison of the therapeutic effects of 90Y-NM600 and 131I-NM404 in conventional TRT. [Figure 53] This figure shows a bar graph comparing the average albumin-binding energies of three different metal chelate analogs of NM404, along with their amine analogues. For comparison, the I-NM404 binding energy is shown as a dotted line. [Figure 54] This figure shows a graph of tumor volume (mm3) as a function of time (days) in B78 melanoma flank tumor mice treated with the anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or the targeted radiotherapy (TRT) agent Y90-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi). Control mice were administered a vehicle (PBS) without anti-CTLA4 or TRT agents. After 18 days, combination treatment with 50 or 100 μCi of Y90-NM600 and anti-CTLA4 significantly reduced tumor growth (p<0.05 by ANOVA) compared to PBS, Y90-NM600 alone, or anti-CTLA4 alone. The 25 μCi Y90-NM600 combination treatment with anti-CTLA4 showed a moderate growth delay response showing a dose-response trend. [Figure 55] This figure shows a graph illustrating the overall animal survival rate of mice treated with a combination of TRT (50 μCi of Y90-NM600) and checkpoint blockade (anti-CTLA4) compared to mice treated with TRT alone, checkpoint blockade alone (anti-CTLA4), or a PBS vehicle. [Figure 56] This figure shows a graph illustrating the overall animal survival rate of mice administered with three different combinations of checkpoint blockade (anti-CTLA4) and TRT (Y90-NM600 at 25 μCi, 50 μCi, and 100 μCi). [Figure 57]This figure shows a graph of tumor volume (mm3) as a function of time (days) in B78 melanoma flank tumor mice treated with the anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or molecularly targeted radiotherapy (MTRT) agent 90Y-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi). Control mice were administered a vehicle (PBS) without anti-CTLA4 or MTRT agents. Combination treatment with 50 or 100 μCi of 90Y-NM600 and anti-CTLA4 significantly reduced tumor growth (p<0.05 by ANOVA) compared to PBS, 90Y-NM600 alone, or anti-CTLA4 alone. [Figure 58] This figure shows a graph of the overall animal survival rate of B78 melanoma flank tumor mice treated with an anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or molecularly targeted radiotherapy (MTRT) agent 90Y-NM600 at various doses (25 μCi, 50 μCi, or 100 μCi). Mice treated with a combination of MTRT (90Y-NM600 at 50 μCi or 90Y-NM600 at 100 μCi) and checkpoint blockade (anti-CTLA4) showed a significantly increased survival rate compared to the other groups. [Figure 59] This figure shows a graph of tumor volume (mm3) as a function of time (days) in NXS2 neuroblastoma tumor mice treated with the anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi of the molecularly targeted radiotherapy (MTRT) agent 90Y-NM600. Control mice were administered a vehicle (PBS) without anti-CTLA4 or TRT agent. Combination treatment with 90Y-NM600 MTRT and anti-CTLA4 significantly reduced tumor growth compared to PBS, 90Y-NM600 alone, or anti-CTLA4 alone. [Figure 60]This figure shows a graph of tumor volume (mm3) as a function of time (days) in mice with 4T1 breast cancer treated with the anti-CTLA4 immune checkpoint inhibitor (CTLA4) and / or 50 μCi of the molecularly targeted radiotherapy (MTRT) agent 90Y-NM600. Control mice were administered a vehicle (PBS) without anti-CTLA4 or TRT agent. Combination treatment with 90Y-NM600 MTRT and anti-CTLA4 significantly reduced tumor growth compared to PBS, 90Y-NM600 alone, or anti-CTLA4 alone. [Figure 61] This figure shows a graph of the tumor volume (mm3) of irradiated primary B78 tumors as a function of time (days) in B78 melanoma flank tumor mice having both primary and secondary (distant) tumors. Mice were treated with various combinations of EBRT (12 Gy, secondary tumors shielded) for primary tumors only, anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi of molecularly targeted radiotherapy (MTRT) agent 90Y-NM600. The combination treatment of 12 Gy EBRT, 90Y-NM600 MTRT, and anti-CTLA4 significantly reduced primary tumor growth compared to other groups. [Figure 62] This figure shows a graph of the tumor volume (mm3) of shielded secondary (distant) B78 tumors as a function of time (days) in B78 melanoma flank tumor mice having both primary and secondary tumors. Mice were treated with various combinations of EBRT (12 Gy, secondary tumors shielded) for primary tumors only, anti-CTLA4 immune checkpoint inhibitor (CTLA4), and / or 50 μCi of molecularly targeted radiotherapy (MTRT) agent 90Y-NM600. Combined treatment of EBRT for primary tumors, 90Y-NM600 MTRT, and anti-CTLA4 significantly reduced secondary tumor growth compared to other groups. [Modes for carrying out the invention]

[0053] I. Overview The specific methodologies, protocols, materials, and reagents described are subject to change. Therefore, it is understood that this disclosure is not limited to these terms. The terminology is used solely to describe specific embodiments and applies to non-provisional applications filed later. This is not intended to limit the scope of the present invention, which is limited solely by [the specified method].

[0054] In this specification and the appended claims, the singular forms "a", "an", and The definite article "the" includes references to plural nouns unless explicitly indicated by the context. Similarly, the terms "a" (or "an"), "one or more", and "at least one" are used in this context. The terms "comprising" and others may be used synonymously in the specification. The variations of these terms have a limited meaning when they appear in this specification and the claims. It has no taste. Therefore, the terms "comprising" and "incl" are used. The terms "uding" and "to possess" can be used synonymously.

[0055] Unless otherwise defined, all technical and scientific terms used herein This has the same meaning as it is generally understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described in the specification may not be used in the practice of the present invention or Methods and materials that can be used for testing, but are preferred, are described herein. All publications and patents specifically mentioned may be used in connection with the present invention. This includes describing and disclosing the chemical substances, equipment, statistical analyses, and methodologies reported in [the relevant document]. All references cited herein are incorporated by reference for any purpose whatsoever. The literature is interpreted as representing the level of art for those skilled in the art.

[0056] The technical terms used herein are for illustrative purposes only and apply to the overall context. This invention should not be construed as limiting. Unless otherwise specified, "a" and "an" refer to the present invention. "The" and "at least one" are used synonymously, meaning one or more. do.

[0057] This disclosure describes the compounds (including intermediates) described herein in a pharmaceutically acceptable form. It contains in one of its forms, and that form includes isomers (e.g., diastereomers and enantiomeric forms). This includes compounds such as tautomers, salts, solvates, polymorphs, and prodrugs. If the substance is optically active, the present invention relates to each of the enantiomers of the compound, as well as the enantiomeric This specifically includes Omar's racemic mixture. The term "compound" is used (occasionally, "salt" is explicitly stated). (Although it is mentioned) Whether explicitly stated or not, such forms Please understand that this includes either a part of it or all of it.

[0058] As used herein, "pharmaceutically acceptable" means that the compound or composition is pharmaceutically acceptable. Alternatively, the carrier may achieve the treatment described herein without excessively harmful side effects, taking into consideration the need for treatment. This means it is suitable for administration to the target for achieving the desired result.

[0059] The term "effective dose" in this specification refers to the effective dose used by researchers, veterinarians, physicians, or other clinicians. The amount of compound that elicits the desired biological or medical response from the target, tissue, or cell or This refers to the dosage.

[0060] In this specification, "pharmaceutically acceptable carrier" includes any dry powder or solvent. This includes dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retarders, and the like. A pharmaceutically acceptable carrier is a substance useful for administering the compound in the method of the present invention. Yes, there is. This substance is preferably non-toxic and may be a solid, liquid, or gaseous substance. In other respects, it is inert, pharmaceutically acceptable, and compatible with the compounds of the present invention. Examples of eel carriers are not limited to these, but include various lactoses, mannitols, Oils such as corn oil, buffers such as PBS, physiological saline, polyethylene glycol, Glycerin, polypropylene glycol, dimethyl sulfoxide, dimethylacetamide Amides such as albumin, proteins such as albumin, and surfactants such as Tween80, Monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin, and starch. Polysaccharides are one example.

[0061] The terms “administering” or “administering” in this specification mean “to treat or prevent The compound of the present invention is used in subjects who are suffering from or at risk of suffering from a disease or condition. This refers to providing a pharmaceutical composition.

[0062] In pharmacology, the route of administration is the path by which a drug is taken into the body. The route of administration is usually... They are classified according to the site to which the substance is applied. Common examples include oral administration and intravenous administration. Intravenous administration is one option. Furthermore, the route can be classified based on the location of the target of action. Yes, it is possible. The effects are local and enteral (systemic effects, but delivered through the digestive tract). or parenteral administration via the lungs by inhalation (systemic effects, but via routes other than the gastrointestinal tract) It may be delivered by [method].

[0063] In enteral administration, the desired effect is systemic (non-local), and the substance is administered via the gastrointestinal tract. In parenteral administration, the desired effect is systemic, and the substance is delivered via routes other than the gastrointestinal tract. It is administered.

[0064] Enteral administration may involve any part of the gastrointestinal tract and have a systemic effect. Examples include many drugs taken orally, in tablet, capsule, or drop form. Many drugs are administered via gastric feeding tube, duodenal feeding tube, or gastrostomy. Examples include the administration of enteral nutrition and the administration of various drugs in suppositories into the rectum. .

[0065] Examples of parenteral administration include intravenous administration (into a vein), such as many drugs, and central artery administration. Intra-arterial administration of nutritional methods (into the arteries), for example, vasodilators in the treatment of vasospasm and Thrombolytic agents for the treatment of thromboembolism: intraosseous injection (into bone marrow), intramuscular injection, intracerebral injection Administration (into the brain parenchyma), administration into the ventricle (into the ventricular system), intrathecal administration (injection into the spinal canal), Furthermore, subcutaneous (under the skin) administration can be mentioned. Among these, intraosseous injection is, Since the bone marrow is directly drained into the venous system, it is essentially indirect intravenous access. (Intraosseous injection) It is sometimes used in emergency medicine and pediatrics for administering drugs and intravenous fluids when intravenous access is difficult. It is sometimes used.

[0066] The following abbreviations are used in this disclosure: ADCC, antibody-dependent cell-mediated cytotoxicity; anti-CT L4, cytotoxic T lymphocyte-associated antigen 4 (C) found in cytotoxic T lymphocytes (CTLs). Antibodies targeting TLA4; B16, C57Bl / 6 mice with similar melanoma; B78, Transfection with GD2 synthase creates a B16 mutant that expresses GD2. ;D, Japan;Hu14.18-IL2, Primary immunotherapy used in the study disclosed in the examples Tokines (reactive against GD2); IC, immune cytokines (tumors bound to IL2) Reactive mAb fusion proteins; ICI, immune checkpoint inhibitors; IL-2, Taleikin 2; IT, intratumor; IV, intravenous; mAb, monoclonal antibody; MAHA Mouse anti-human antibody; NM404, used to refer to the phospholipid ether shown in Figure 1. Therefore, it is selectively taken up by most tumors and, in the studies disclosed in the examples, TRT Used to refer to NM600, the phospholipid ether shown in Figure 14. It can chelate with any metal, and this is also selectively removed by most tumors. Used in the investigations disclosed in the examples; NXS2, AJ mouse and Syngeneic neuroblastoma; transfected by Panc02-GD2 and GD2 synthase. For this purpose, C57Bl / 6 mice expressing GD2 are associated with pancreatic cancer; PLE, phospholipids Ether; RT, radiotherapy; TRT, targeted radiotherapy; W, week; 9464D-GD2 C57Bl / expresses GD2 for transfection by GD2 synthase. 6. Neuroblastomas of the same lineage as mice.

[0067] II. The present invention This disclosure relates to a method for treating any cancer that exists as one or more malignant solid tumors. The disclosed method combines two treatment steps, and through an unexpected synergistic effect, malignancy It shows a much improved effect against solid tumors. Specifically, in malignant solid tumor tissue... Therefore, the immunomodulatory dose is selectively incorporated and retained. If a patient is given a radioactive phospholipid metal chelate compound or a radioactive halogenated phospholipid compound... When administered, it provides additional treatment for at least one malignant solid tumor being treated with an immunostimulant. A set of one or more drugs that have the ability to stimulate specific immune cells, with or without xRT. Further immunomodulation can be achieved by systemically administering the substance (for example, by intravenous injection). It breaks.

[0068] Immunomodulatory doses of radiophospholipid metal chelates or radiohalogenated compounds are Tre It is highly likely to lower g levels (and other immunosuppressive factors), and xRT is one tumor When used for a ulcer, if one or more additional tumors are not irradiated, a decrease in the immune system (incidental) may occur. It prevents immune tolerance, but understanding the mechanism is not necessary for the practice of this invention, and this invention is not specific to Not limited to the mechanism of use.

[0069] A. Systemic immunotherapy: Examples of immunostimulants include immune checkpoint inhibitors. Methods of immune activation by directly administering immunomodulators to tumors (for example, in part of the example below) In contrast to the in situ vaccination-mediated intratumor immunity shown, systemic administration Donor immunotherapy is performed by administering immunostimulants systemically to the entire body of the target patient. It circulates throughout the body and stimulates the body's natural immune response.

[0070] Immune checkpoint inhibitors are not limited to such immunostimulants. T cells are characterized by the lymphocyte activation gene 3 (LAG-3) and programmed cell death protein 1 ( Several proteins, including PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA4). They express immunosuppressive receptors. These and other immune checkpoint molecules express specific non-immune receptors. It has been shown to regulate the T cell response to tumor antigens in the tumor microenvironment via repeated pathways has been.

[0071] More specifically, cancer growth is mediated in part by cancer-induced immunosuppression. Tumors can activate immunosuppressive checkpoint pathways to reduce the general immune response to the tumor. Therefore, blockade of the major immune checkpoint pathways <000089"4>can induce antitumor immunity promoted by the patient's own immune system.

[0072] CTLA4 was the first immune checkpoint molecule to be clinically targeted by administering a monoclonal antibody (anti-CLA4) that targets CTLA4. To date, the most promising immune checkpoint inhibitor strategies for cancer treatment involve the administration of monoclonal antibodies that target CTLA-4 and / or PD-1 / PD-L1 Other immune checkpoint inhibitor strategies are currently under development, and the combination methods of the present disclosure are not limited to targeting specific immune checkpoint pathways. A series of reviews covering checkpoint inhibitors and cancer immunotherapy were recently published in Volume 276 of Immunological Reviews. An introductory overview is, Sharpe, A.H., "Introduction to checkpoint

[0073] inhibitors and cancer immunotherapy", Imm unol Rev. 276 (March 4, 2017): 5-8 These reviews are incorporated herein by reference in their entirety. inhibitors and cancer immunotherapy", Im munol Rev. 276 (March 4, 2017): 5-8 These reviews are incorporated herein by reference in their entirety.

[0074] B. Immunomodulatory doses of radiolabeled phospholipid metal chelate compounds The radioactive phospholipid metal chelate compounds used are chelated to metal chelate compounds. The RT released by the metal isotope substantially enhances other tissue types in the released RT. To target malignant solid tumor tissue without any issues, a wide range of solid tumor cell types are selectively targeted. This should be the target. Radioactive metal isotopes included in radioactive phospholipid metal chelate compounds. It is known that it releases ionizing RT in a way that leads to immune activation of cells that take up the compound. Any radioactive metal isotope may be used. Examples include Lu-177, Y-90, Ho-166, Re-186, Re-188, C u-67, Au-199, Rh-105, Ra-223, Ac-225, Pb-212, Alternatively, Th-227 could be mentioned.

[0075] The immunomodulatory RT dose of radioactive phospholipid metal chelate compounds (in contrast to the injection dose) is harmful. The dose is far less than that conventionally used for radiotherapy for solid tumors. Specifically, the dose It does not remove desirable immune cells that are responsible for the immune-stimulating effect, (presumably immunosuppressive Tr (by reducing the level of other immunosuppressive cells or molecules) tumor microscopy It should be sufficient to stimulate a response in immune cells within the environment.

[0076] The appropriate immunomodulatory dose is determined after administering a “detection-enhancing” dose of the radiometal chelate compound. It can be calculated from the obtained image data. The detection-enhancing dose is completely different from the immunomodulatory dose. The radioactive metal isotopes incorporated into radioactive metal chelate compounds can be different. They may differ (but the rest of the compound structure should be the same). Radioactive metal isotopes used in the blotting and dose measurement calculations are obtained by conventional imaging methods. Any radioactive metal isotope known to emit RT in a form that can be immediately detected It is also acceptable. Examples of "conventional imaging methods" that are not limited to these include gamma-ray detection, P Examples include ET scanning and SPECT scanning. The limitations on the radioactive metal isotopes that can be used. Examples of non-compliant elements include Ga-66, Cu-64, Y-86, Co-55, Zr-89, and Sr -83, Mn-52, As-72, Sc-44, Ga-67, In-111, or Tc -99m is mentioned.

[0077] C.PLE analog metal chelate The disclosed structure utilizes an alkylphosphocholine (APC) support skeleton. If the drug retains tumor selectivity, as previously demonstrated with related radiohalogenated compounds, At the same time, they should possess formulation properties that make them suitable for injection. The structure chelates with radioactive metal isotopes to produce the final imaging or therapeutic agent. It contains a chelate portion.

[0078] D. Method for synthesizing example M-PLE analogues The proposed synthesis of compound 1 is shown below. The first step of the synthesis is Org Synt It is similar to what is described in h, 2008, 85, 10-14. Is the synthesis cyclene? Starting from there, this is converted to DO3A Tris-Bn ester. Next, this intermediate is converted to a base It is combined with NM404 in the presence of a Pd catalyst. Finally, the benzyl protecting group is removed in contact with water. Removed by pulverization. [ka]

[0079] The synthesis of Compound 2 is shown below. The synthesis starts from DO3A tris-Bn ester, and this is alkylated with 3-(bromo-prop-1-ynyl)-trimethylsilane. After alkylation, the trimethylsilyl group is removed, and the intermediate acetylene is coupled with NM404 by the Sonogashira reaction. The benzyl group is removed, and the triple bond is simultaneously hydrogenated in the last step of the synthesis. [Chemical formula]

[0080] Compounds 5 and 6 can be synthesized from the same precursors, DTPA dianhydride and 18-p-(3-hydroxyethyl-phenyl)-octadecylphosphocholine, as shown in the following scheme. [Chemical formula]

[0081] The NOTA-NM404 conjugate can be synthesized in a similar manner. One example is the following NOTA-NM404 conjugate 7. [Chemical formula] [[ID=4I]]

[0082] E. Dosage Forms and Administration Methods Any route of administration would be suitable for synergistic targeted RT. In one embodiment, the disclosed alkylphosphocholine analogs may be administered to a subject via intravenous injection. In another embodiment, the disclosed alkylphosphocholine analogs may be administered to a subject via any other suitable systemic delivery, such as parenteral administration, intranasal administration, sublingual administration, rectal administration, or transdermal administration. ​​​​​​​It can be administered to elephants.

[0083] In another embodiment, the disclosed alkylphosphocholine analog is used in the nasal system or mouth It may be administered to the subject, for example, by inhalation.

[0084] In another embodiment, the disclosed alkylphosphocholine analog is administered by intraperitoneal injection or It may be administered to the subject via IP injection.

[0085] In certain embodiments, the disclosed alkylphosphocholine analogs are pharmaceutically acceptable. It may be provided as a salt. However, other salts may be alkylphosphocholine analogs or This may be useful in preparing a pharmaceutically acceptable salt. This is not limited to, but for example, a solution of alkylphosphocholine analogs, Hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, cyanoacrylate Mix with a solution of a pharmaceutically acceptable acid such as oxalic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Examples of acid addition salts that can be formed by this process include acid addition salts.

[0086] If the disclosed alkylphosphocholine analog has at least one chiral center, These may exist as enantiomers accordingly. Disclosed alkylphosphocholic If an analog has two or more chiral centers, they will have diastereoisomers accordingly. Such isomers and mixtures of any proportion thereof are all within the scope of this disclosure. It is understood that it is enclosed within the enclosure.

[0087] This disclosure relates to one or more disclosed alkylphosphocholine analogs to pharmaceutically acceptable carriers. The method also includes using a pharmaceutical composition containing together with. Preferably, these compositions are tablets, Pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered-dose aerosols, or This includes parenteral administration, such as liquid sprays, drops, ampoules, auto-injectors, or suppositories, and nasal administration. Unit formulations for intraoral, sublingual, or rectal administration, or administration by inhalation or airflow. It is the shape of the form.

[0088] To prepare solid compositions such as tablets, the main active ingredient is placed in a pharmaceutically acceptable carrier. For example, conventional tablet ingredients such as corn starch, lactose, sucrose, and sol Bitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, It is mixed with rubber and other pharmaceutical diluents, such as water, to form the compound of the present invention, A solid pre-formulation composition containing a homogeneous mixture for its pharmaceutically acceptable salt. To form. When these preliminary formulation compositions are referred to as homogeneous, it means that the active ingredient is present throughout the composition. Because it is uniformly dispersed throughout, the composition is equally distributed in tablets, pills and capsules, etc. This means that it can be easily subdivided into effective unit dosage forms. This solid preliminary formulation composition is as follows: The above-mentioned unit dosage forms contain 0.1 to approximately 500 mg of the active ingredient of the present invention. Typical unit dosage forms are 1 to 100 mg, for example, 1, 2, 5, 10, 25, 50 mg. It contains 100 mg of the active ingredient. The tablets or pills of the novel composition offer the advantage of long-lasting action. It may be coated or otherwise formulated to provide a dosage that delivers the desired effect. For example, a tablet or pill may contain an internally administered component and an externally administered component, the latter This is the form of the outer covering on the former. The two components help to resist breakdown in the stomach, and inside Enteric coating helps the components on the side enter the duodenum without being damaged or delay their release. They can be separated by. A variety of materials are used for such enteric layers or enteric coatings. Such materials include numerous polymer acids, as well as polymer acids and shellac, cetylates. This includes mixtures of such materials as alcohol and cellulose acetate.

[0089] Liquids that may incorporate alkylphosphocholine analogs for oral or injectable administration. In terms of form, it can be an aqueous solution, a properly flavored syrup, an aqueous or oily suspension, and A flavored emulsion containing edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil. Examples include elixirs and similar pharmaceutical excipients. Examples of powders or anti-precipitation agents include tragacanth gum, acacia gum, alginate, and dextrose. Stran, sodium carboxymethylcellulose, methylcellulose, polyvinyl pyro Examples include synthetic and natural rubbers such as lidon or gelatin.

[0090] The disclosed alkylphosphocholine analogs include combinations with injectable carrier systems, pharmaceutical This is particularly useful when prescribed in an injectable form. The possible dosage forms and injection forms (i.e., parenteral dosage forms) are not limited to, Liposomes for injection or lipid bilayer vesicles containing phospholipids that encapsulate active pharmaceutical ingredients are examples. Injectable preparations include sterile preparations intended for parenteral use.

[0091] Five different classes of injectable drugs as defined by USP: emulsions, lipids, powders, Solutions and suspensions are available. Emulsion injections contain sterile pyrogens intended for parenteral administration. The emulsion contains preparations that do not contain [unclear]. Lipid complexes and powders for solution injection are non-effervescent. This is a sterile preparation intended for reconstitution to form a solution for oral use. For suspension injection. The powder is a sterile preparation intended for reconstitution to form a suspension for parenteral use. The lyophilized powder for liposome suspension injection is for parenteral use, with the formulation formed upon reconstitution. This is a sterile freeze-dried preparation intended for reconstitution within the lipid bilayer. Alternatively, a lipid biphosphate having phospholipids used to encapsulate an active drug substance in an aqueous space. Formulated to allow the incorporation of liposomes such as stratified vesicles. Freeze-dried for solution injection. The dried powder is produced by a method that involves removing water from a frozen product at extremely low pressure, and then By adding the liquid, a solution is produced that meets all the requirements for injection, freeze-dried ( This is a dosage form for solutions prepared by "freeze-drying". It is a lyophilized suspension for injection. The powder is a liquid preparation intended for parenteral use, containing a solid suspended in a suitable liquid medium. And it is a sterile suspension in which the drug intended to be a suspension is prepared by lyophilization. It meets all the requirements of the liquid. Solution injection is suitable for injection, suitable solvent or This includes a liquid preparation containing one or more active pharmaceutical ingredients dissolved in a mixture of mutually miscible solvents.

[0092] Concentrated solutions for injection, when combined with a suitable solvent, meet all the requirements for injection. Includes sterile preparations for parenteral use that produce a solution. The suspension injection contains insoluble particles and oil. A solid dispersed throughout the liquid phase, either dispersed throughout the aqueous phase or vice versa. Includes liquid preparations containing particles (suitable for injection). Suspension liposome injections are liposomes (Used to encapsulate active pharmaceutical ingredients either within a lipid bilayer or in an aqueous space) The oil phase is divided throughout the aqueous phase so that lipid bilayer vesicles (which typically contain phospholipids) are formed. It is a dispersed (suitable for injection) liquid preparation. The suspension sonicated injection solution has insoluble particles. It is a liquid preparation (suitable for injection) containing solid particles dispersed throughout the liquid phase. Furthermore, the product may be subjected to ultrasonic treatment when the gas is blown into the suspension, resulting in solid particles. Microspheres are formed by the offspring.

[0093] Parenteral carrier systems include one or more pharmaceutically suitable excipients, such as solvents and co-solvents, and solubilizers. Agents, wetting agents, suspending agents, thickeners, emulsifiers, chelating agents, buffering agents, pH adjusters, antioxidants, It contains reducing agents, antimicrobial preservatives, fillers, protective agents, isotonic agents, and special additives.

[0094] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. This is not intended. In fact, in addition to what is shown and described herein, the present invention Various modifications will become apparent to those skilled in the art from the above description and the following examples, and are attached to the Patent It falls within the scope of the request. [Examples]

[0095] Examples Introduction to the Examples These examples demonstrate how to leverage unexpectedly powerful synergistic effects in cancer treatment research. This demonstrates the possibility of combining two very different cutting-edge academic fields into one. These academic fields are 1 ) Systemically administered TRT, and 2) Locally administered antibody-mediated cancer immunotherapy or systemically administered This is a cancer immunotherapy. The data presented herein show that a powerful synergistic effect is observed in these methods. This suggests that it arises from combining them. In summary, these two strategies This means that the destroyed cancer cells can cause persistent residual metastasis to any type of solid tumor in any location. It functions as a powerful immunostimulant that generates tumor-specific T-cell immunity capable of eradicating sexually transmitted diseases. This method allows for the destruction of visible macroscopic tumors.

[0096] Our ongoing preclinical research shows that tumor-specific mAbs and IL2 (activate innate immune cells) The combination of (to transform) enhances antibody-dependent cell-mediated cytotoxicity (ADCC). This demonstrated [1, 2]. This has already translated into clinical benefits for children with neuroblastoma. This is a process [3]. Recent preclinical data suggest that the mAb-IL2 fusion protein is It shows a more potent antitumor efficacy when administered by intratumoral injection (IT) [4, 5]. In particular, the condition worsens when patients do not respond to these mAb / IL2 injections and are treated with local xRT alone. Large, continuously growing tumors can be completely eradicated when xRT is combined with mAb / IL2 therapy. This is possible. Most mice are cured, and T cells reject a similar attack by tumor cells. It develops memory [6]; the combined use of xRT+mAb / IL2 is powerful "in si This indicates that it is acting as an anti-cancer vaccine.

[0097] A key limitation is that these animals undergo xRT + mAb / IL2 for primary (first) tumors. If another macroscopic tumor is present after treatment, the second tumor will continue to grow, which is surprising. Furthermore, it suppresses the immune response and prevents the tumor from shrinking after the first treatment. The associated immune tolerance is partly due to the second tumor's suppressive regulatory T cells (Tregs). . Delivering RT alone to both tumors has minimal antitumor effect, but these Tregs To deplete it. Thus, when treating the first tumor with xRT + mAb / IL2, the second By adding RT to the tumor, this immune tolerance is circumvented, making it possible to eradicate both tumors. [7] These findings suggest that the use of in situ tumor vaccination in metastatic situations may be restricted. While this indicates a limitation, it also suggests RT's strong ability to overcome this limitation.

[0098] xRT generally allows all to be treated without the toxicity and immunosuppression to normal tissues that are prohibited. It cannot be delivered to all metastatic sites. However, xRT can be delivered to all macroscopic sites of the disease. If the drug is not delivered, the suppressive immune system can be left intact, which is the inventor's It can suppress the immunological response to local xRT + mAb / IL2 immunotherapy. Therefore, what is needed is to deliver RT to all tumor sites in cancer patients in a targeted manner. It is a means of delivery.

[0099] The inventors have demonstrated that systemically administered RT can target both primary and metastatic cancers. We developed a TRT excipient. One such TRT reagent is 131 I-NM40 4 is a phospholipid ether (PLE) analog administered intravenously (IV), with a value exceeding 60. This reagent exhibited nearly universal tumor targeting properties in in vivo cancer and cancer stem cell models. It is currently being clinically evaluated in multiple imaging and clinical trials.[8, 9] 131 I-NM404 systemic injections concentrate on all tumors, regardless of their anatomical location, and are effective. By severing the intratumor immunosuppressive pathway, it is possible to prevent the generation of a decisive tumor-eradicating immune response. It provides sufficient RT internally. A unique feature of this method is the nearly universal tumor of NM404. It possesses the ability to target tumors and deliver sublethal immunomodulatory doses of RT to all tumor sites. This is a capability that is generally impossible to achieve with xRT. The new thing about this is this The inventors' TRT agent immunomodulates all tumors, regardless of anatomical location, and also treats associated This can overcome tolerance, thereby enabling local xRT followed by tumor-specific mAb+ The long-term in situ tumor vaccination effect is achieved after IL2 injection. Yes, more and more tumor-specific mAbs are being approved for clinical use. Therefore, this combination strategy is effective against any tumor type that can be targeted by tumor-responsive mAbs. This also provides an extension of the method. Furthermore, this method applies to all in situ tumor vaccine injections. This can be easily generalized to species strategies.

[0100] In recent years, the inventors of this invention have found that 131 Iodine in I-NM404, a wide range of metal images Chelates capable of having MRI and PET and TRT radiotherapy portions. We found that it could be substituted with an agent. In these examples, 131 I-NM404 (and related Combination therapy of xRT + immunotherapy with related metal chelate analogs is effective for cancerous solid tumors (timu Systemic The inventors describe a method for evaluating the ability to initiate an immunomodulatory response. A similar method is used in P The combination of TRT delivered to LE analogs and other immunotherapies used for cancerous solid tumors. It can be used for various purposes. For example, in combination therapy, local in situ tumor vaccination is different from Quite different immunomodulation steps, namely immune checks Immunostimulants such as chlorophos inhibitors The inventors hereby show that systemic administration of ) may be used.

[0101] In short, the inventors have derived two seemingly separate fields of cancer treatment from the academic discipline of cancer treatment. This specification describes a therapeutic and research process that combines different methods into one integrated treatment. Disclosed in this document. The data presented in these examples demonstrates the synergistic effect of these two methods. This combination demonstrates that it can effectively eliminate malignant solid tumors and prevent tumor recurrence. vinegar.

[0102] In Example 1, the inventors obtained from the B78 GD2+ model as supporting evidence for this method. Present background data.

[0103] In Example 2, the inventors demonstrated the optimal in situ vaccine effect against primary tumors. The required dose of xRT, and the x for distant tumors necessary to prevent concomitant immune tolerance. This provides guidance for determining the minimum dose for RT (radiotherapy).

[0104] In Example 3, the inventors determined the necessity of xRT for metastasis as determined in Example 2. Close to the required dosage 131 Determine the dosage of I-NM404, and then, 131 I-NM40 This provides guidance for evaluating the effects of dose 4 on in vivo immune function. The guidance describes the use of the disclosed radiophospholipid metal chelate compounds as TRT agents. This can be similarly applied in the following cases.

[0105] In Example 4, the inventors used the data obtained from Examples 2 and 3 to create two or more In mice with tumors, locally treated tumors were destroyed, and all distant tumors were treated. To induce cell-mediated eradication, 131 I-NM404+local xRT+IT-mAb / Provides guidance for designing / testing / developing IL2 administration plans. TRT and x The critical issues of RT dose and timing are optimized for antitumor efficiency. However, such guidance also implies that the disclosed radioactive phospholipid metal chelate compounds are T The same can be applied when used as a rehydration therapy agent.

[0106] In Example 5, the inventors used a compound similar to a radioactive metal isotope chelates. This provides an example of a synthesis that can also find applications in the material.

[0107] In Example 6, the inventors replaced the iodine portion of NM404 (Gd-NM600) with Chelating agents and analogs having chelated metals used in solid tumor tissue The metal chelate is taken up (and can be imaged within the solid tumor) and thus revealed. This demonstrates proof of concept for use as a TRT agent.

[0108] In Examples 7, 8, 9, and 10, the inventors followed the guidance of Examples 1-4. This document provides information and specific data obtained from the empirical studies that have been conducted.

[0109] In Examples 11 and 12, the inventors demonstrated that the chelating agent and the chelated metal Further analogues in which the iodine portion of NM404 is replaced are found in the in vivo model. It is incorporated into various solid tumors, can be imaged within various solid tumors, and in various solid tumors It may be used therapeutically in TRT, and therefore the gold disclosed in the manner disclosed This demonstrates further proof of concept for using genus chelates as TRT agents.

[0110] In Example 13, how can dose measurement be used in combination with known radiosensitivity? The inventors have considered whether a person skilled in the art can optimize the treatment dose for any solid tumor. do.

[0111] In Example 14, the inventors performed the iodization described in Examples 1-4 and 7-10. Rather than a compound, the disclosed method uses alkylphosphocholine metal chelates. We will examine the differences and advantages of each.

[0112] In Examples 15 and 16, systemic immunotherapy was more effective than in situ vaccination. TRT in combination with [another drug] has been shown to be effective in treating solid tumors. It is administered systemically. The immunostimulant is an immune checkpoint blocker or inhibitor (in this case, anti-CTLA4). That's fine.

[0113] Example 1: Data supporting the background Sondel's lab found that tumor-specific mAbs + IL2 activate innate immune cells. It mediates ADCC in [2] and is clinically beneficial for children with neuroblastoma. 3] This study showed that in mice, intravenous administration of hu14.18-IL2 was effective against GD2 It was more potent than IV administration of mAb + IL2 [2, 10]. This was due to very small, recent Dramatic antitumor effect against established GD2+ tumors or very small microscopic metastases. This method can provide a solution and is clinically applicable to patients in remission but at high risk of relapse. This potentially explains bed use.[3] Measurable macroscopic tumors [i.e., about 50 mm] 3 G A more potent antitumor efficiency against D2+ tumors is achieved when IC is administered intratumorally rather than intravenously. This can be achieved if (IT-IC) is used [4, 5].

[0114] The inventors are now focusing on ways that will be beneficial in the context of much larger macroscopic tumors. Yes, it is there. It was established 5 weeks ago and is of medium size (200mm). 3 ) has a B78 melanoma tumor The mice did not respond to IV-IC, and their proliferation was slowed by IT-IC. However, the tumor continues to grow. These same 200mm 3 The tumor was also treated with 12 Gy xRT. It proliferates afterward. In contrast, when IT-IC and xRT are used in combination, tumors in 73% of animals The symptoms disappear, and the disease appears to be cured (Figures 2A and 2B). Next, these symptoms are Uss exhibits T cell-mediated rejection of reloading by the same tumor (Figure 2C). Therefore, IT- IC+xRT shows a synergistic effect, inducing tumors to become "in situ tumor vaccines." To lead [6].

[0115] To simulate clinical metastasis, the inventors administered B78 to the flank of mice on day 1. Then, two weeks later, the other flank is inoculated. At five weeks, the first tumor is 200 mm. 3 dea The second tumor was 50 mm 3 The inventors have shown that xRT+IT-IC treats the first tumor We predicted that destroying the tumor would trigger a T-cell response that would then destroy a second tumor. However, Even with the addition of IT-IC to xRT, 50mm 3 Tumors also 200mm 3 It is also substantially effective against tumors. This was not the case (Figure 3). This indicated a significant limitation to the treatment method provided by the inventors; in other words, Furthermore, when these mice receive xRT+IT-IC on the first tumor, another tumor is present. In this case, the second tumor triggers systemic tumor-specific associated immune tolerance, and either This also prevents tumor reduction. Importantly, the inventors have identified the first and second Local xRT (12 Gy) against the tumor simultaneously suppressed this tolerance effect, and in most mice... This shows that it induces an immune response that eradicates both tumors, leading to an IT-IC against the first tumor. The results were obtained (Figure 4) [7]. Treg depletion mAbs (not shown) or selective Treg depletion were enabled. Recent data using transgenic mice that can induce this immunotolerance (Figure 4) [7] shows that This shows that the effects are partly mediated by regulatory T cells (Tregs). RT to the two tumors partially depletes these Tregs, and irradiation to both tumors is tolerant. This potentially explains ways to avoid the effect.[7]

[0116] Local xRT for both the first and second tumors avoids tolerance, but clinical metastatic disease The disease is often present in several locations. All macroscopic metastatic diseases inhibit immune tolerance. Therefore, xRT+IT-IC allows for the effective eradication of all tumor sites. RT is required. However, 12 Gy of xRT will be delivered to all diseased sites. This involves "systemic R," which is characterized by major dose-dependent (potentially lethal) toxicity and severe systemic immunosuppression. It can sometimes be close to "T".

[0117] Previously, the Weichert lab delivered RT to all systemic tumor sites, and simultaneously Minimize "off-target" RT in normal tissues (especially bone marrow and immune tissues). Therefore, we pioneered the development of TRT.

[0118] Based on the finding that tumor cells contain excess phospholipid ethers (PLEs)

[11] , The inventors have developed over 30 radioactive compounds with the aim of identifying analogs that selectively target tumors. They synthesized a propellant iodized PLE analog

[12] . One of these was NM404 This includes brain metastases and cancer stem cells, and over 70 in the surveyed, regardless of anatomical site. Not only did it show nearly universal tumor uptake in all but three of the vivo models, but it also showed tumor Once invaded, these cells were selectively retained for a long period of time.[8] These diagnostic and therapeutic (dia) peutic)PLE analogs are said to avoid pre-malignant and inflammatory lesions. It is unique in this respect. Surface membrane lipid rafts that are overexpressed in cancer cells compared to normal cells are NM4 It serves as an entry point for PLE, including O4, into cancer and cancer stem cells.[8] Radioactive iodine Substrates NM404 (I-124 and I-131) were used in the five Phase 1 and Phase 2 PE trials. Evaluated in T imaging trials and three Phase 1 TRT radiotherapy trials, respectively. It exhibits similar tumor uptake and retention characteristics in more than a dozen human cancer types.[8] Excellent tumor uptake in cancer models (B78 GD2+ mouse melanoma) related to these examples. Miha, 124 This was confirmed by I-NM404 PET imaging (Figure 5).

[0119] Example 2: Determination of xRT dosage The inventors' data suggest these four hypotheses: (1) To treat one tumor The xRT dose used for this purpose caused moderate direct in vivo tumor death, which was mediated by the immune system. (2) IT mA The strong T cell response induced by the addition of IT-IC rather than b is observed in the presence of IL2. mAbs that bind to tumor cells that have been injected promote enhanced antigen presentation and induction of adaptive immunity. (3) If a second tumor is present, immunosuppressive cells present in the second tumor [For example, Treg cells and possibly myeloid-derived suppressor cells (MDSCs), etc.] Due to the tolerance primarily induced by its action, xRT+IT-IC for the first tumor It produces virtually no antitumor effect; this tolerance is due to Treg depletion (Figure 4). (4) To avoid tolerance Therefore, the required RT dose for the second tumor is the same as for the first tumor, which received the "in situ vaccine". The xRT dose may be far lower than the dose required to achieve this.

[14]

[0120] Optimization of xRT dose for primary ("in situ vaccine") tumor sites.

[0121] Our in vivo study of xRT+IT-IC showed that 12 Gy of 12 Gy was used against the first tumor. We have focused on the number of doses administered. This is the in situ vaccine effect of xRT+IT-IC. However, our in vivo research demonstrates that mice with functional Fas-L are required. Data linked to in vitro RT showed dose-dependent functional improvement of Fas in B78 tumor cells. Our data showing that preregulation is induced (around the peak >12Gy) Based on (6). The inventors conducted an in vivo pilot study before selecting a dose of 12 Gy. The procedure was performed. It was found that high doses (16 Gy) or increased fractional flank RT were toxic (dermatitis, ulcers). The patient presented with a tumor and hind limb edema, and did not show improvement in the tumor response. (The inventors) For the in vivo study, they selected xRT with a single fraction of 12 Gy. However, when the inventors proceeded to clinical interpretation, they aimed to ensure the in situ vaccine effect was safe and effective. To effectively induce the local xRT effect, it is necessary to better understand the mechanism and dose conditions of the local xRT effect. That would be beneficial.

[0122] Our mouse data (Figures 2A, 2B, and 2C) shows that even with 12 Gy xRT... Even if tumor reduction is not achieved by this alone, a powerful 12Gy xRT+IT-IC may be effective. It can demonstrate that it can induce a vaccine effect; it only slows down progressive growth. Yes. The inventors have found that in si is as powerful as when using a lower dose of RT. We thought that the tu vaccine would be effective. To test this, the inventors conducted approximately 200 mm 3 In mice with B78 tumors, a series of xRT doses (4-16) were treated as a single irradiation. Gy) and the subsequent standard IT-IC administration plan of the inventors (50 mc on days 6-10) The inventors evaluated which xRT dose (g / day) is most effective when used in combination with IT-IC. Determining whether it will result in effective tumor eradication and T-cell memory. Doses below 12 Gy are more toxic. If the effect is minimal and comparable efficacy is observed, such low doses are considered to be "i" in Examples 3 and 4. This would be a better target for the inventors' xRT dose to the "n situ vaccine" site. Similar methods may be used to optimize drug administration for specific targets or subjects.

[0123] To prevent tolerance from hindering "in situ vaccination" in distant tumors Optimization of RT dose.

[0124] By treating both the first and second tumors with 12 Gy (Figure 3), the first tumor IT-IC for this condition can induce a robust response that eradicates both tumors. Our objective is to provide xRT+IT-IC to a single tumor while simultaneously improving tolerance. To avoid this, use the minimum necessary RT dose at the metastasis site, The goal is to enable the achievement of the vaccine effect. The inventors believe that xRT itself is particularly broad We recognize that if the condition extends to a wide range, it can be myelosuppressive. Therefore, The inventors track TRT in Examples 3 and 4, even if it is targeted. TRT provides some systemic delivery of RT. TRT minimizes systemic immunosuppression. To achieve this, the inventors have found a way to avoid causing systemic RT-induced overall immunosuppression. Administering low doses of TRT (transcatheter resuscitation therapy) as needed to effectively suppress tumor-induced immune tolerance. This is what we hope for. Therefore, when used in combination with IT-IC for the first tumor, A higher xRT dose allows it to function as an in situ vaccine. Therefore, it is best to select xRT with the minimum dose required to deliver to the distant tumor.

[0125] As an example of an optimization experiment, 200mm 3 The first B78 tumor and approximately 50 mm 3 The In mice with two B78 tumors, on day 0 (approximately 5 weeks after transplantation of the first B78 tumor), 1 2 Gy of xRT is administered to the first tumor. Subsequently, on days 6 to 10, the inventors' target A preliminary IT-IC administration plan will be implemented. Different doses of xRT will be administered to separate groups of mice. It is administered to the tumor. 3 Gy of systemic xRT prevented immunosuppression in a myeloma model. Based on data from B. Johnson's laboratory demonstrating that this is possible (15), the inventors This involves adding doses of 0, 1, 5, and 8 Gy (to the currently known effective dose of 12 Gy). (and) evaluate. The inventors have found that a dose of substantially less than 12 Gy to the second tumor is effective in preventing the onset of symptoms. We are trying to determine if a total dose of 12 Gy is as effective as eliminating disease tolerance. Ku.

[0126] If the inventors select a limit dose for xRT that loses beneficial effects, the limit dose can be improved. To optimize, perform the following analysis. For example, 5Gy is as effective as 12Gy. However, if 1 Gy is not much different from 0 Gy, the inventors of this invention use 12 Gy + IT- In these two tumor models, IC is administered to the first tumor, comparing 2, 3, and 4 Gy. This eliminates tolerance and identifies the minimum effective RT dose required to achieve efficacy.

[0127] Next, we conducted a replicated study and found that the dose to the first tumor was not 12 Gy, but rather a single tumor model. If this is the minimum effective dose (tested in Example 2 above), then this minimum effective dose to the second tumor To verify whether the dose is still sufficient for in situ vaccination. In short, Example 2 The study found that the minimum dose of xRT for the first and second tumors was 12 Gy for both. The inventors will optimize the product without losing the effectiveness they have demonstrated.

[0128] xRT doses required for the first and second tumors in mice with tumors other than B78 The research begins.

[0129] Our mouse studies demonstrate a greater potential for clinical generalization. We will begin the analysis of RT+IT-IC in an additional model of GD2+ tumor. hu14.18-IL2 in AJ mice with GD2+NXS2 neuroblastoma The inventors have published an IT-IC with an IC [5]. In addition, the inventors have published GD2+9464 D-GD2 neuroblastoma, and GD2 is produced by insertion of the GD2 synthase gene. In C57BL / 6 mice exhibiting Panc02-GD2 pancreatic cancer, this same IC The IT-IC is being evaluated in Example 2. Regarding Example 2, for each model, the inventors have evaluated i To maintain the effectiveness of the n situ vaccine, the minimum amount of primary and secondary tumors required Determine the effective xRT dose.

[0130] Example 3: 131 Determination of I-NM404 dosage and in C57BL / 6 mice Evaluation of immunosuppressive dosimetry using TRT and the effects of TRT on immunosuppression. 131 I-NM404 was found in >95% of tumor lines (human and mouse) in vitro. It showed selective uptake, with little uptake by non-malignant cells, and similar tumors were observed in vivo. Surgery specificity was observed. This included selective in vivo uptake by B78 tumors. (Figure 5). In preliminary dose measurement studies, the inventors 124 I-NM404 to C57BL / 6 mice were administered the drug and TRT exposure was determined by serial PET / CT imaging (as shown in Figure 5). This study characterized the temporal changes in dew. Monte Carlo dosimetry calculations based on this research [16-18] This allows for the delivery of approximately 3 Gy to the established B78 tumor over a 4-week decay period. to approximately 60 μCi 131It was shown that I-NM404 is necessary. These four weeks Subsequently, the remaining TRT dose to the B78 tumor will be less than 0.25 Gy. The inventors of this invention have developed xR Data obtained from two tumor models is replicated using T (Figure 3), but all sites of the distant disease To enable the effective elimination of tumor-induced tolerance, targeted 13 1 Use the minimum possible dose of I-NM404 TRT. However, use all doses within a few minutes. Unlike xRT, which is performed after delivery, TRT involves the biological half-life and physical properties of the target isotope. Both of the effective half-lives ( 131 For I, the dose is accumulated over time according to the 8th day (t1 / 2). The inventors are seeking the initial TRT effect at distant tumor sites to eradicate immune tolerance. However, the inventors have found that administering IT-IC can treat ADCC and in situ vaccines. When inducing an antitumor effect, we want to minimize the immunosuppressive effect of TRT. It is essential for completely destroying the tumor in that area.

[0131] Using dose measurement calculations obtained from preliminary data from the inventors, a dose of 3 μCi 131 The present invention states that I-NM404 should deliver an amount equivalent to approximately 0.2 Gy to the tumor site. The inventors estimated that the dose assumed by the inventors should not be immunosuppressive, but rather affect lymphocytes. The mediated tumor destruction should not be hindered. As mentioned above, this is the first 131 I-NM4 The inventors estimated that a dose of 60 μCi would still not be delivered even 28 days later. In this way, the inventors have created a single 200 mm 3 mice with B78 tumor The group was evaluated. On day 0, all mice were administered 12 Gy of xRT to their tumors, and on day 6 On day 10, all mice were administered 50 mcg / day of IT-IC. One group was On day 0, 3 μCi 131 I-NM404 was also administered (approximately 0.2 Gy). Figure 6 shows: 131 The group that received I-NM404, 131 Similar to the group that did not receive I-NM404. This demonstrated tumor eradication, and this low-dose "residual" TRT in the tumor was RT+IT-I This demonstrates that it does not prevent immune-mediated disruption by the C in situ vaccine. The inventors stated that on the 22nd day, the initial dose was 60 μCi. 131 I-NM404 TRT Using it, it effectively inhibits the immune tolerance-induced effects of distant tumors, and furthermore, the primary tumor xRT on day 0 and IT-IC on days 6-10 (28 days after TRT) It functions as a situ vaccine, and then induces an adaptive response that eradicates all tumors. Let's assume that...

[0132] The experiment outlined in this embodiment optimizes the dose correlation tested in Figure 6. In the tumor B78 model, the inventors of this invention, 131 I-NM404 TRT dose range Test to interfere with the desired in situ vaccine effect (and thereby the first This can lead to undesirable systemic immunosuppression (which can delay or prevent the eradication of the tumor). Select the best TRT dose. This is important for Example 4. It is a remote disease. In mice having [specific characteristic], at the time of initiation of IT-IC to the first tumor, residual radiation from TRT was detected. This is because it is possible to confirm that the performance has decreased to a value smaller than this. The inventors Furthermore, the rate of TRT response after various TRT doses was evaluated to determine the "TRT dose that impedes tolerance." After administering "[product name]" to animals with multiple tumors, the first tumor was treated with RT+IT-IC. But it can induce in situ vaccine efficacy and eradicate both primary and distant tumors. To achieve this, choose the optimal waiting period.

[0133] Related studies have shown that the dose of TRT administered as monotherapy is optimal for a single B78 tumor. We will also focus on what is most beneficial for slowing, reducing, or eradicating tumors. The most beneficial TRT dose for eliminating tumors is actually (from TRT alone) complete tumor destruction. This would be substantially less than the TRT dose required to induce the condition.

[0134] Finally, if the effects of various optimized doses of TRT are determined in a single tumor model, then the inventors The goal is to evaluate the immune response of these subjects' serum samples to the human IgG component of IC. Therefore, we evaluate the slight immunosuppressive effect of TRT. The inventors found that immunocompetent mice The levels of mouse anti-human antibodies (MAHA) that can be easily quantified after treatment with these humanized ICs are readily detectable. (19) The inventors have shown that TRT generates the immune response of mice. As a means of determining the dose that is seen to cause a detectable dose-dependent decrease in intensity Using this, these mice receive overall immunosuppression due to systemic administration of RT from this TRT. The inhibitory effect is measured. The inventors determine the low T required to inhibit tumor-induced immune tolerance. The RT dose will likely cause minimal systemic immunosuppression.

[0135] Example 4: In mice with 2 or more tumors 131 I-NM404 + local xRT + I Development of an optimal T-mAb / IL2 administration plan A trial of the efficacy of TRT+RT+IT-IC in a 2-tumor B78 model.

[0136] The dose and timing information obtained from the studies outlined in Examples 2 and 3 is the present invention. To optimize the dosage and timing of TRT required for efficacy in the researchers' two tumor models Provides the necessary information. For the C57BL / 6 mouse, B78 on the left (L) and right (R) sides. The injections are administered simultaneously to the abdomen. Each tumor will grow to approximately 50 mm after two weeks. 3 Approximately 200mm after 5 weeks 3 This should be the case. In the dose measurement calculation of Example 3, 60 μC was used to approach the RT of 3 Gy. If the inventors believe that TRT i needs to be delivered to the second tumor (to inhibit immune tolerance) (To stop it), our external beam xRT research shows that this dose minimizes the slowdown of tumor growth. It is predicted that it should have an effect. The inventors observed that at the 2-week mark, different groups of We would plan to treat the stool with 30, 60, or 90 μCi (when the tumor is about 50 m m 3 (If this is the case). After 3 weeks, the tumor was approximately 200 mm. 3 It should be like that at that point; The inventors administered xRT (the dose determined as outlined in Example 2) followed by 6 days Later (approximately 28 days after TRT), the tumor in the left flank was administered five times daily by injection of IT-IC, i It induces the effect of the n situ vaccine. Control mice have tolerance from distant tumors. Anticipating the lack of an in situ vaccine, TRT was not administered, and xRT and IT- Only IC is administered to the left flank. In the other group, both are administered due to the in situ vaccine effect. To predict tumor eradication, local xRT was administered to both tumors, and IT-IC was administered to the left flank. In another group, TRT+IT-IC will be administered, anticipating an incomplete vaccine effect. Local xRT will not be administered.

[0137] The follow-up experiment involved various doses of TRT and TRT followed by local xR on the primary tumor (left flank). Further evaluation of the timing variation between T+IT-IC. The reading information is (A) original (B) Eradication of primary tumors; (C) Eradication of secondary tumors; and (D) ELISA analysis of MAHA response. This results in systemic immunosuppression. The inventors' objective is (measured by the MAHA response) (r) To eradicate both tumors in most subjects while minimizing systemic immunosuppression. To add a local xRT+IT-IC administration plan that can be used, specific target and disease models The goal is to determine the optimal TRT dose and timing using Dell.

[0138] Optimization of TRT+xRT+IT-IC in mice with three or more B78 tumors.

[0139] This section of Example 4 describes the relevant clinical situation, namely, the in situ vaccine site. They have injectable tumors that can be used, but each one causes tumor-induced immune tolerance. These studies are most similar to patients with multiple possible distant metastases. The first part of Example 4 (above) will reproduce the conditions that were found to be most effective. The important difference is that each of these subjects is located on the left and right flanks and the left and right scapular flanks. The patient had four separate tumors. TRT was used in the study outlined in the first section of Example 4. Administered at the dose and timing deemed most effective, xRT+IT-IC Subsequently, it is administered only to the left flank lesion. TRT is administered to the three sites where xRT was not administered. The objective here is to effectively eliminate tumor-induced immune tolerance caused by The TRT dose and timing issues that enable the most effective in situ vaccines The choice is made. The measure of effectiveness is the removal of all four tumors in the majority of subjects. Yes. Testing TRT dose and timing modifications to find the most effective optimized dosing time. Create a plan. Such a treatment plan is available at the clinic for patients with multiple distant metastases. And applications can be found. All of these multiple remote transitions are irradiated by an external beam. It is not possible to do this directly, but it can be used in combination with local xRT + IT-IC at the "in situ vaccine" site. And it can be irradiated by TRT.

[0140] Example 5: Synthesis of metal-chelated NM600 In this example, the inventors present a phospholipid chelate, Gd-NM600, as one example. The synthesis scheme used to synthesize it is shown. It incorporates various radioactive isotopes. Analogues can also be synthesized using a similar method, and the radioactive isotope in question can be used instead of Gd. It is possible.

[0141] Scheme for synthesizing Gd-NM600 (the disclosed radioactive metal isotope is Gd) (It can be used as a substitute.) [ka]

[0142] Example 6: Proof of concept using in vivo imaging In this example, the inventors successfully used Gd-NM600 as an MRI contrast agent. The image shows in vivo MRI imaging of the tumor. The data is skeletal phospholipid and chelate. This document demonstrates that the agent is taken up and retained by solid tumors, and various methods disclosed herein This demonstrates that such chelates incorporating radioactive metals exhibit similar properties.

[0143] Demonstration of concept for tumor uptake of Gd-NM404 agent, for in vivo imaging in the flank. We scanned nude mice with a thymic defect and a xenograft containing an A549 tumor (non-small cell lung cancer). Gd-NM600 (2.7 mg) was delivered by tail vein injection. Mice were anesthetized, Scans are performed before contrast agent administration and 1, 4, 24, 48, and 72 hours after contrast agent delivery. The imaging was performed using a 4.7T Varian preclinical MR with a right-angle phase volume coil. The procedure was performed using a scanner. The following pulse sequence parameters were used: Repetition time (TR) = 206 min 0 seconds, echo interval = 9 milliseconds, echo train length = 2, effective echo time (TE) = 9 milliseconds Seconds, average tens, field of view 40 x 40 mm 2 192 x 192 matrix, 1 mm thick each. Using a fast spin echo scan with 10 sections, T1-weighted images were acquired at all acquisition times. The image was retrieved.

[0144] As shown in Figure 7, MRI imaging of tumors increased significantly within 24 hours after injection. I was forced.

[0145] These results indicate the differential properties of alkylphosphocholine analogs. ) Incorporation and retention are maintained with respect to the metal-chelated analogues disclosed herein. It is demonstrated that the metal chelates disclosed are used in clinical treatment and imaging. It can be easily applied to chemical applications.

[0146] Example 7: xRT dose required for optimal in situ vaccine efficacy against primary tumors and to determine the minimum dose of xRT to distant tumors necessary to prevent associated immune tolerance. Experiment As a follow-up study for Examples 1-4, various tests were conducted on mice with one or two tumors. Dose titration experiments were conducted to evaluate the xRT dose. The first objective was to evaluate the xRT dose of a patient with one tumor. In mice, synergistic effects were observed, and the tumor-reactive mAbs bound to IT-IC and IL2 were also present. This involves testing the xRT dose necessary to promote "in situ vaccines." Initial experiments showed that 12 Gy of RT alone could eradicate established B78 melanoma tumors. Neither of them proliferated or regressed (complete regression was 0%), while 12Gy+IT-IC This results in complete regression of most B78 tumors in mice with one tumor (66 This confirmed the inventors' previous findings that %). On the other hand, 2Gy+IT-IC is IT - Slows tumor progression compared to IC alone (average tumor size at day 32 was, respectively) 472mm 3 and 1214mm 3 However, none of the mice became disease-free (0% complete regression). ).

[0147] In the inventors' "two-tumor model," one "primary" tumor was treated with xRT+IT-IC. The treatment of tumors applies to both treated primary tumors and untreated "secondary" tumors. The inventors have previously shown that it is ineffective. In fact, in these two tumor models, The inventors have found that the presence of a second tumor negates the efficacy of IT-IC injection after xRT. The inventors observed that this phenomenon is called "associated immune tolerance" (CIT), and that it is less likely to occur. At least a portion of them originate from regulatory T cells (Tregs) of distant (non-irradiated) secondary tumors, and It circulates throughout the body and re-accums in the primary tumor treated with xRT / IT-IC injection. It was proven. These Tregs returning to primary tumors are the desirable "in situ vaccine" efficacy. It is believed that this will hinder the results.

[0148] The inventors now deliver 12 Gy of xRT to both primary and secondary tumors. This confirms our previous findings that CIT can be overcome by [doing something]. Furthermore, considering that Tregs are quite sensitive to RT, they overcome CIT and primary tumors In situ vaccination in a ulcer (primary tumor treated with 12 Gy + IT-IC) To rescue the response, lower doses of RT can be delivered to secondary tumors. The inventors hypothesized that there might be a 2 A dose of xRT of Gy or 5 Gy dulls CIT, and primary IC with 12 Gy + IT-IC We observed that it was comparable to 12 Gy in its ability to rescue the response to tumor treatment. Their important experiment was repeated twice, and (as hypothesized) to prevent CIT by administering to distant tumors. The dose of xRT that must be administered is 1 for the purpose of producing an in situ vaccine effect. This suggests that the dose at the primary tumor site where T-IC was injected was far lower than the required dose. .

[0149] This supports the comprehensive hypothesis of the inventors in this disclosure, and in animals with multiple tumors The inventors used the targeted radiotherapy drug (TRT) NM600 to treat all parts of the disease. Because it can deliver relatively low doses of RT, this can be used for local xRT and in one tumor area. When used in combination with IT-IC injection at the in situ vaccine site, CIT can be overcome. This suggests that...

[0150] Example 8: As determined above, close to the required dose of xRT for metastasis. 131 I - Determine the NM404 dosage, and then... 131 I-NM404 dose in vivo immune function Experiments to evaluate the effects Based on the preliminary data from Examples 1-4 above, these concepts are applied in vivo using TRT. Research has been conducted to move to testing. Dosimetry studies have been conducted in patients with one or two B78 tumors. Mice that perform (the inventors' best in situ vaccine method and overcome the hurdles of CIT) This was performed using a tumor model (used by the inventors to demonstrate the findings) of approximately 2 Gy This is necessary to get closer to xRT. 131 This was done to estimate the amount of I-NM404. .

[0151] Next, an equivalent dose of approximately 2 Gy 131 I-NM404 is a tumor lymphocyte (especially T To determine whether it has a desirable effect on (reg), two separate methods are used. This has been pursued. First, the inventors demonstrated NM404 uptake comparable to that of B78 tumors. This dose was administered to mice with radiosensitive lymphoma tumors. 131 Administer I-NM404 Following this, the inventors also demonstrated a substantial reduction / deceleration of B78 tumors and circulating lymphocytes. Under conditions that do not cause obvious depletion of cells, powerful reduction / dose-dependent suppression of lymphoid tumors. The system was described (measured by peripheral whole blood count). These data are typical of lymphoid cells. This is consistent with the fact that they are far more sensitive to low doses of RT than typical solid tumor cells. Furthermore, selective uptake of TRT in tumors is associated with intratumoral lymphopenia without systemic lymphopenia. These studies also suggest that it may be possible to deplete lymphoid cells. To identify and monitor the effects of TRT on lymphoid cells within tumors in lymphoid tumors such as these This suggests that it could serve as an in vivo biological "dosimeter."

[0152] The second method involves administering these same doses to mice with B78 tumors. 131 I-NM404 This involved treating the animals. Next, these animals were sacrificed at half-life intervals (8 days) and then irradiated. After sufficiently delaying sex breakdown, effector T cells and T cells are identified by immunohistochemistry. The tumor was stained for the presence of reg. Interestingly, in this initial experiment... 131 I-NM Animals administered 404 did not show systemic lymphopenia at any point in time (peripheral whole blood cells) (Depending on the number) TRT administration showed a decrease in intratumor FoxP3+ Tregs within two half-lives. At this half-life point, the inventors also observed a decrease in tumor effector CD8+ T cells. However, importantly, at the subsequent 3rd and 4th half-lives, the inventors found that either Compared to untreated baseline and second half-life levels, intratumoral CD8+ effect We observed an increase in T cells, but a further decrease in intratumor Treg levels. The findings also suggest that the effectiveness of in situ vaccines can be restored in animals with multiple tumors. Therefore, it may be feasible to overcome Treg-mediated CIT using TRT. This supports the inventors' hypothesis.

[0153] Finally, in order to characterize the immunological effects of TRT on immune cells within tumors, the inventors The mouse having B78 131 Treatment with I-NM404, pretreatment and subsequent half-life Tumor tissue was collected at intervals of 8 days. Next, these tissues were subjected to an immunoassay panel. Gene expression was analyzed by RT-PCR. The results showed that TRT treatment alone was effective in reducing immunity. Expression of disease-susceptible tumor cell markers and prominent expression of genes normally expressed only by immune cells It was shown that the gene caused a change, with a decrease in expression followed by a rebound overexpression. It shows clear changes over time.

[0154] Example 9: Using the data obtained from Examples 5 and 6, mice with two or more tumors were used. Okeru 131 We developed an administration plan of I-NM404 + local xRT + IT-mAb / IL2. Experiments to induce T cell-mediated eradication of all distant tumors This embodiment describes the treatment of an animal having tumors in at least two locations. Our strategy is to enhance antitumor immune activity in all tumor sites by using CIT xRT and in situ vaccine site combined with systemic TRT for suppression Including the use of local IT-IC. Critical dose and timing of TRT and xRT. The problem is optimized for anti-tumor efficiency.

[0155] Using the data compiled in Examples 7 and 8, we created mice with two distinct B78 tumors. The study was conducted in mice to determine the estimated systemic needs. 131 I-NM404 dose administered Then, xRT and in situ local immunotherapy were administered to the vaccine site. With proper control, this dose 131 I-NM404 was used in mice with two tumors. As required, it was thought to weaken CIT. Moreover, Mau had one tumor. In this case, this TRT dose is assumed to provide the desired local in situ vaccine effect. (Sea urchin) did not appear to interfere. C without suppressing the in situ vaccine effect. To maximize the desired effect of blocking IT, further testing and experimental variables are needed. Some modifications are underway. Further details regarding these experiments can be found in Example 10 below. It will be disclosed.

[0156] Example 10: Data obtained from mice with 2 or more tumors. Local xRT+IT- due to distantly untreated tumors in mouse melanoma and pancreatic tumor models Tumor-specific suppression of primary tumor response to IC combinations.

[0157] C57BL / 6 with GD2+ primary flank tumor + / - secondary tumor in the contralateral flank. Mice were treated with xRT on day "1" and then on day 6, with only the primary tumor as shown. On the 10th day, the patient was treated with 50 mcg of anti-GD2 IC and hu14.18-IL2 IT injection. I placed it down.

[0158] In mice with primary B78 melanoma tumors, the presence of untreated secondary B78 tumors is x It antagonized the primary tumor response to RT+IT-IC (Figure 8A). The inventors of this invention developed The use refers to "associated immune tolerance," that is, the local response of the treated tumor to xRT+IT-IC. This is explained as an antagonistic effect on untreated distant tumors. (See Kaplan-Meier survival curve.) We obtained results from our mice and repeated experiments (Figure 8B). Almost all mice showed progression of the primary tumor. They were euthanized for that reason.

[0159] In mice with a primary Panc02-GD2+ pancreatic tumor, secondary Panc02-GD Regardless of the presence or absence of c02-GD2 tumors, the presence of untreated Panc02 secondary tumors indicates that The response of primary Panc02-GD2+ tumors to xRT+IT-IC was suppressed (Figure 8). C). In mice with primary B78 melanoma tumors, secondary B78 tumors were xRT+IT- The primary tumor response to IC was suppressed, but secondary Panc02-GD2+ pancreatic tumors It did not exert any effect (Figure 8D). In mice with primary Panc02-GD2+ tumors... Secondary Panc02-GD2-tumor is xRT and IT-hu14.18-IL2 The combination therapy suppressed the primary tumor response, but did not suppress B78-derived tumors (Figure 8E). ).

[0160] The associated immune tolerance is avoided by the specific depletion of regulatory T cells (Tregs).

[0161] In mice with one or two tumors, Treg tumors examined on day 6 after xRT were found to be Treg tumors. Immunohistochemical images of the maker and FoxP3 were obtained (Figure 9A). The mice received xRT. They either did not receive xRT or received xRT only for the primary tumor. DEREG mice are T Under the control of the reg-specific FoxP3 promoter, the diphtheria toxin receptor is expressed, and diphtheria This allows for the simultaneous IP injection of rhia toxin and the specific depletion of Treg cells (Figures 9B and 9C). DEREG mice with primary and secondary B78 melanoma tumors were subjected to xR to the primary tumor. Treatment involved T+IT-IC and IP injection of either diphtheria toxin or PBS. This immune tolerance was eliminated in these mice after Treg depletion, and primary (Figure 9) B) and secondary (Figure 9C) tumor response improvement.

[0162] The associated immune tolerance is overcome by delivering xRT to both tumor sites.

[0163] In mice with primary and secondary B78 tumors, secondary tumors were treated with xRT+IT-I It suppresses the primary tumor response to primary tumor treatment with C. This is equivalent to 12 Gy xRT. By delivering this to both primary and secondary tumors, and IT-IC to the primary tumor... This was overcome, and as a result, the primary tumor response improved in repeated experiments (Figure 10A), and in total animals The survival rate (aggregate animal survival) improved (Figure 10B). ).

[0164] Low-dose xRT alone does not induce in situ vaccination, but in situ When delivered to a distant tumor site along with 12 Gy + IT-IC treatment at the tu vaccine site, Actually overcome the associated immune tolerance.

[0165] In mice with only primary B78 tumors, 12 Gy + IT-IC was administered in situ. Vaccination induces complete tumor regression in most mice (as already shown) (Figure 11A) and leads to a memory immune response (Morris, Cancer Res, 2 016). On the other hand, complete treatment with IT-IC alone or low-dose (2Gy) x RT + IT-IC. No animals showed tumor regression (0 / 6 in both groups) p<0.05.

[0166] In mice with primary and secondary B78 melanoma tumors, the delivery of low levels of the drug to the secondary tumors was observed. The dose xRT (2 Gy or 5 Gy) has the ability to overcome the immune tolerance associated with primary tumors. The force is equivalent to 12 Gy (Figure 11B). In these same animals, low-dose xRT was continued. Overcoming the associated immune tolerance by delivering to the tumor is the purpose of IT-IC immunotherapy. It is clear that this provides relief for systemic responses to the law (Figure 11C). In this regard, RT When delivered to all tumor sites, IT-IC injections of primary tumors have a systemic antitumor effect. This causes a secondary tumor response to 2 Gy or 5 Gy, and IT-IC of the primary tumor. The response should be greater than the response to 12 Gy of RT in the case without injection.

[0167] Low-dose TRT and 131 I-NM404 is used without systemic leukopenia or in tumor invasion. Effectively target tumor-infiltrating FoxP3+ Tregs without depleting moist CD8+ effector T cells. To deplete it.

[0168] In most clinical scenarios, significant bone marrow depletion and leukemia will result in immunosuppression. Delivering external beam radiation to all tumor sites without causing globopenia, even at low doses. Even if such a thing existed, it would be impossible to achieve. Here, the inventors administered TRT systemically to the whole body's immune system. Without causing cell depletion and leukopenia, tumor infiltration-suppressing immune cells (Tre We tested whether g) could be specifically depleted. This B78 melanoma tumor model Dosimetry studies in the area involve positron emission. 124 This was performed using I-NM404, NM4 Selective uptake of tumor-specific 04 was confirmed (Figure 12A). C57BL / 6 with B78 tumor. The mouse was 60 μCi 131 The treatment was performed with I-NM404. This radioactivity was approximately 2 Gy of T The amount needed to deliver RT to B78 tumors 131 The amount is almost the same as that of I-NM404. Peripheral blood and tumor samples were collected from untreated control mice (C), and then at 8-day intervals (T1=d8 , T2 = d16, T3 = d24, T4 = d32) were collected. The TRT of this dose did not cause significant systemic leukopenia (Figure 12B) and had no significant effect on the level of tumor-infiltrating CD8 effector T cells (Figure 12C). However, tumor-infiltrating FoxP3+Tre g was significantly depleted by this dose of TRT (Figure 12D).

[0169] Low-dose TRT and 131 I-NM404 effectively overcome the accompanying immune tolerance and rescue the systemic antitumor effect of in situ vaccination.

[0170] Low dose 131 Considering the ability of I-NM404 TRT to deplete tumor-infiltrating Tregs without causing leukopenia in mice, the inventors tested whether low-dose 131 I-NM404 effectively overcomes the accompanying immune tolerance. C57BL / 6 mice bearing two B78 tumors were treated with 60 μCi of I-NM40 131 4 as shown on day 1 (NM404). After one half-life (8 days), the animals were given 12 Gy of xRT to the primary tumor (in situ vaccine site) or no xRT. Control mice not treated with I-NM404 were treated with the indicated (0, 2, or 12 13 1 Gy) treatment for metastatic tumors as shown. On days 13 - 17, the mice were given daily IC IT injections to the primary tumor (in situ vaccine site) as shown. The primary tumor (Figure 13A) and metastatic tumor (Figure 13B) responses indicate that the administration of low-dose TRT effectively overcomes the accompanying immune tolerance and rescues the systemic antitumor effect of in situ vaccination. This To demonstrate.

[0171] References cited in Examples 1 to 4 and 7 to 10: [1] Hank JA, Robinson RR, Surfus J, Muelle r BM, Reisfeld RA, Cheung N--K and Sondel PM. Augmentation of antibody dependent ce ll mediated cytotoxicity following in vi vo therapy with recombinant Interleukin- 2. Cancer Res. 50:5234-9. 1990. [2] Neal ZC, Yang JC, Rakhmilevich AL, Buh toiarov I, Lum HE, Imboden M, Hank JA, Lode HN, Reisfeld RA, Gillies SD, Sondel PM. Enha nced activity of hu14.18‐IL2 IC against the murine NXS2 neuroblastoma when combi ned with IL2 therapy. Clin Cancer Res. 200 4 Jul 15;10(14):4839‐47. [3] Yu AL, Gilman AL, Ozkaynak MF, London WB, Kreissman S, Chen H, Smith M, Anderson B , Villablanca J, Matthay KK, Shimada H, Grup p SA, Seeger R, Reynolds CP, Buxton A, Reisf eld RA, Gillies SD, Cohn SL, Maris JM, Sonde l PM.Anti‐GD2 antibody with GM‐CSF,inter leukin‐2,and isotretinoin for neuroblast oma.N Engl J.Med.2010 Sep 30;363(14):132 4‐34. [4] Johnson EE,Yamane BH,Lum HD,Buhtoia rov IN,Rakhmilevich AL,Mahvi DM,Gillies SD,Sondel,PM.Radiofrequency Ablation Com bined with KS‐IL2 IC(EMD 273066)Results in an Enhanced Anti-tumor Effect Against Murine Colon Adenocarcinoma.Clin Cancer Res.2009 Aug 1;15(15):4875-84. [5] Yang RK,Kalogriopoulos NA,Rakhmilev ich AL,Ranheim EA,Seo S,Kim KM,Alderson KL,Gan J,Reisfeld RA,Gillies SD,Hank JA, Sondel PM.Intratumoral hu14.18‐IL2(IC)In duces Local and Systemic Antitumor Effec ts that Involve Both Activated T‐ and NK cells as well as Enhanced IC Retention. J Immunol.2012 Sep 1;189(5):2656‐64. [6] Morris ZS,Emily I.Guy EI,Francis DM ,Gressett MM,Carmichael LL,Yang RK,Armst rong EA,Huang S,Navid F,Gillies SD,Korma n A,Hank JA,Rakhmilevich AL,Harari PM,So ndel PM.Combining Local Radiation and tu mor‐specific antibody or IC to elicit in situ tumor vaccination.Cancer Research, e-pub ahead of print,2016. [7] Morris ZS,G.E.,Francis DM,Gressett MM,Armstrong EA,Huan S,Gillies SD,Korman AJ,Hank JA,Rakhmilevich AL,Harari PM,an d Sondel PM.,IC augments local and absco pal response to radiation and CTLA‐4 che ckpoint inhibition in a murine melanoma model.Am.Soc.Therapeutic Radiation Oncol ogy.Abstract accepted Oct.2015(and selec ted as the meeting’s winning abstract in the basic--‐translational science categ ory). [8] Weichert JP,Clark PA,Kandela IK,Vac caro AM,Clarke W,Longino MA,Pinchuk AN,F arhoud M,Swanson KI,Floberg JM,Grudzinsk i J,Titz B,Traynor AM,Chen HE,Hall LT,Pa zoles CJ,Pickhardt PJ,Kuo JS.Alkylphosph ocholine Analogs for Broad Spectrum Canc er Imaging and Therapy.Science Translati onal Medicine 6,240ra75,1‐10.2014. [9] Morris ZS,JP Weichert,J Sakera,EA A rmstrong,A Besemer,B Bednarz,R Kimple,PM Harari.Therapeutic combination of radio labeled NM404 with external beam radiati on in head and neck cancer model systems .Radiotherapy and Oncology.J.Radiation O ncology,DOI:10.1016.2015.

[10] Lode HN,Xiang R,Dreier T,Varki NM, Gillies SD,Reisfeld RA.Natural killer ce ll-mediated eradication of neuroblastoma metastases to bone marrow by targeted i nterleukin‐2 therapy.Blood 91(5),1706‐17 15.1998.

[11] Snyder F,Wood R.Alkyl and alk‐1‐en yl ethers of glycerol in lipids from nor mal and neoplastic human tissues.Cancer Res 29,251‐257.1969.

[12] Pinchuk AN,Rampy MA,Longino MA,Ski nner RW,Gross MD,Weichert JP,Counsell RE ,Synthesis and structure‐activity relati onship effects on the tumor avidity of r adioiodinated phospholipid ether analogu es.J Med Chem 49,2155‐ 2165.2006.

[13] Swanson KI,Clark PA,Pinchuk AN,Lon gino MA,Farhoud M,Weichert JP,Kuo JS.Ini tial Studies on Novel Cancer‐Selective A lkylphosphocholine Analogs CLR1501 and C LR1502 for Fluorescence‐guided Neurosurg ery.Neurosurgery.76(2):115‐123.2015.

[14] Filatenkov A,Baker J,Mueller AM,Ke nkel J,Ahn GO,Dutt S,Zhang N,Kohrt H,Jen sen K,Dejbakhsh‐Jones S,Shizuru JA,Negri n RN,Engleman EG,Strober S.Ablative Tumo r Radiation Can Change the Tumor Immune Cell Microenvironment to Induce Durable Complete Remissions.Clin Cancer Res.21:3 727‐39.2015.

[15] Jing W,Gershan JA,Weber J,Tlomak D ,McOlash L,Sabatos‐Peyton C,Johnson BD.C ombined immune checkpoint protein blocka de and low dose whole body irradiation a s immunotherapy for myeloma.J Immunother Cancer.3:2. 2015.

[16] Bednarz B.,Besemer A.,Yang Y.A Mon te Carlo‐Based Small Animal Dosimetry Pl atform for Pre‐Clinical Trials:Proof of Concept.Med.Phys.39,3899.2012.

[17] Besemer et al.Towards Personalized Dosimetry Using Diapeutic Radiopharmace uticals.Med.Phys.40,382.2013.

[18] Besemer A.and Bednarz B.Validation of a patient‐specific Monte Carlo targe ted radionuclide therapy dosimetry platf orm.Med.Phys.41,303.2014.

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

[0172] [[ID=1第十七条]] Example 11: In vivo uptake of multiple NM600 metal chelates in mice xenografted with eight different solid tumor types, demonstrated by PET imaging In this example, the inventors show differential uptake of four different metal-chelated NM600s in various solid tumors in vivo, as demonstrated by PET / CT imaging of such tumors. These data provide further support for using metal-chelated alkylphosphocholine analogs as TRT agents to abrogate tumor-induced immune tolerance. The structure of NM600 is shown in Figure 14 as an exemplary species chelated with Cu ([Cu-NM600]); however, any metal can 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( 00 of different uptake in different solid tumors in vivo, as demonstrated by PET / CT imaging of such tumors. These data provide further support for using metal-chelated alkylphosphocholine analogs as TRT agents to abrogate tumor-induced immune tolerance. The structure of NM600 is shown in Figure 14 as an exemplary species chelated with Cu ([Cu-NM600]); however, any metal can be readily chelated to NM600. 发性免疫寛容を排除するためのTRT剤として金属キレート化されたアルキルホスホコリ ン類似体を使用するためのさらなる裏付けを提供する。NM600の構造は、 64 Cuで キレート化された例示的な種( 64 Cu-NM600)として図14に示される;しかし 、どんな金属もNM600に容易にキレート化することができる。

[0173] Specifically, eight different solid tumor cell lines (B78 (melanoma), U87MG (glioblastoma), breast cancer), 4T1 (breast cancer), HCT-116 (colorectal cancer), A549 (lung cancer), PC-3( It should be noted that there may be some inaccuracies or unclear parts in the original text, especially in the Japanese part which might need further clarification for a more precise translation. One of the following (prostate cancer), HT-29 (colorectal adenocarcinoma), or MiaPaca (pancreatic cancer) Each mouse was xenotransplanted. Each xenotransplanted mouse contained tumor cells. The cell suspension was inoculated into the subcutaneous tissue of one or both sides of the flank of the mouse. Xenograft Once the sore reaches its maximum size, each mouse receives a dose between 150 and 300 μCi. 64 C u, 89 Zr, 86 Y, or 52 Mn-labeled NM600 is administered by lateral tail vein injection. Therefore, it was injected. After the uptake period, PET imaging was performed using Inveon micro The procedure was performed using PET / CT. Immediately before each scan, the mice were given isoflurane (2%). The patient was anesthetized and placed prone on the scanner. 40 to 80 million matched events were scanned over time. Static PET scans were taken 3, 12, 24, and 48 hours after injection of the radiotrace. The image was reconstructed using the OSEM3D / MAP reconstruction algorithm.

[0174] Figure 15 shows 86 Obtained 48 hours after injection of Y-NM600 in 1-tumor B78 mice. The image shown is; Figure 16 is 86 Post-injection of two tumor B78 mice injected with Y-NM600 The images obtained over 48 hours are shown; Figure 17 is, 64 U87MG injected with Cu-NM600 Images obtained at 3, 24, and 48 hours after injection in mice are shown; Figure 18 shows, 64 cu- N Images obtained at 3, 24, and 48 hours post-injection of 4T1 mice injected with M600 are shown. Figure 19 shows, 64 HCT-116 mice injected with Cu-NM600 were 3 and 24 hours post-injection. The images obtained over 48 hours are shown; Figure 20 shows,64 A54 injected with Cu-NM600 Images obtained 3, 24, and 48 hours after injection in 9 mice are shown; Figure 21 shows, 64 Cu- Images obtained 3, 24, and 48 hours after injection of NM600 in PC-3 mice. Figure 22 shows: 64 HT-29 mice injected with Cu-NM600 were 3 and 24 hours post-injection. The images obtained over 48 hours are shown in Figure 23. 64 Mia was injected with Cu-NM600. Images obtained at 3, 24, and 48 hours after injection in Paca mice are shown; Figure 24 is 86 Images obtained 3, 24, and 48 hours after injection of Y-NM600 in 4T1 mice. Figure 25 shows: 89 3 and 24 minutes after injection of Zr-NM600 in 4T1 mice The images obtained over 48 hours are shown.

[0175] 52 PET images of HT-29 and PC3 mice injected with Mn-NM600. This was observed 4 hours after injection and on day 1 (Figure 26 for HT-29; Figure 27 for PC3). Furthermore, data obtained on days 2, 3, 5, and 7 after injection (Figure 28 regarding HT-29; PC- Regarding point 3, see Figure 29).

[0176] As can be seen in Figures 15-29, the scanned mice were concentrated in the xenografted tumors. A PET / CT three-dimensional volume rendering showing the cumulative absorbed dose distribution was generated. These results show the difference between metal-chelated NM600 and xenografted solid tumor tissue. Differential uptake has been confirmed and is being released in the disclosed therapeutic methods. The feasibility of using NM600 analogs incorporating injection metal isotopes is demonstrated.

[0177] Quantitative region of interest analysis of images involves manually tracing the contours of target tumors and other organs. Therefore, the procedure was carried out. Quantitative data was expressed as the percentage of injection volume per gram of tissue (%ID / g). This was shown. Exemplary data shows that 4T1 tumor tissue increases its uptake over time. Although it effectively retained all three NM600 chelates tested, 86 Y-NM600 , 64 Cu-NM600 and 89 Zr-NM600 (see Figure 30), healthy heart (Figure 3) 1) The liver (Figure 32) and all tissues (Figure 33) all undergo uptake / retention over time. This indicates a significant decline.

[0178] Ex vivo in vivo distribution analysis was performed after the final time-series PET scan in mice. The animals were anesthetized, tissue was collected, wet weight was measured, and counted using an automated gamma counter (Wiza (rd 2480, Perkin Elmer). Exemplary in vivo distribution data are from tumor tissue (4 Significant uptake and retention at T1) are shown for different NM-600 chelates. 86 Y-NM600, 64 Cu-NM600, 89 Zr-NM600 and 177 Lu- NM600 (see Figure 34).

[0179] In summary, these results indicate that the metal chelates of the disclosed invention are effective in the therapeutic methods of the disclosed invention. It demonstrates that it can be easily used in stepping stones.

[0180] Example 12: Two different NM600 metal cells were used to treat multiple solid tumor types in xenograft mice. Demonstration of antitumor activity by rate and tumor autoradiography In this embodiment, three different solid tumor models were used, and alkylphosphocholine metals were used. The inventors have shown that rate analogs can be effectively used to promote conventional TRT. These results demonstrate that using metal chelates in the TRT step of the treatment method disclosed herein is effective. Further demonstrate the potential for its use.

[0181] Subcutaneous flank xenografts of B78, MiaPaca, and 4T1 were used in mice as described above. It was then introduced into the system. Subsequently, mice were given a therapeutic dose (250-500 μCi). 90 Y-NM60 0, 177 Lu-NM600 or a control solution was administered by lateral tail vein injection.

[0182] Planar 2D phosphor images of drug distribution in the body are generated using a cyclone phosphoimager (Cyclo Images were acquired using a Perkin Elmer Phosphorimager. The mice were anesthetized and placed in direct contact with a fluorescent plate in a supine position, and then the mice were subjected to 15 minutes of sanitation. Leave it as is for 30 minutes; then read the plate with a phosphoimager. Various images were recorded between 4 and 96 hours after dose injection. The resulting autoradiography... - The images show rapid and selective uptake of the chelate in all solid tumor tissue types tested. Demonstrates both absorption and long-term retention (see Figures 40, 41, 42, 43, 44, and 45).

[0183] Tumor response was evaluated by comparing tumor growth in treated mice and control mice. The volume was calculated by measuring the length and width of the tumor with calipers and using the formula for the volume of an ellipsoid. Determined by calculation. Mouse body weight was also recorded. Humane endpoint: tumor Volume > 2500m³ 3Alternatively, it was defined as a significant weight loss of less than 13g.

[0184] As can be seen in Figures 46, 47, 48, 49, 50, and 51, these results are two The tested NM600 chelate showed statistically significant in vivo therapeutic effects compared to the control. It has been shown to be effective, resulting in twice the dose in 4T1 xenografts. 177 Lu-NM For 600, the average tumor volume decreased (see Figure 50), and the single dose 177 Lu-NM60 MiaPaca, 4T1 or B78 xenografts administered with 0 (Figures 47, 48, and 4 (See 9) or single dose 90 B78 or 4T1 xenotransplant with Y-NM600 administration Reduce the proliferation of the fragments (see Figures 46 and 51) to near zero or slow down their proliferation rate. To demonstrate that it is possible.

[0185] These results indicate that TRT can be delivered using the disclosed alkylphosphocholine metal chelate. This further demonstrates the effectiveness of efficiently treating various types of solid tumors.

[0186] Example 13: Radiation dose measurement for predicting TRT response in a wide range of solid tumor types Combination of radiation sensitivity indices In this embodiment, the inventors demonstrate the TRT method disclosed for various solid tumor types. We will consider the factors for determining the appropriate chelate dosage for each patient.

[0187] Estimation of tumor absorbed dose Administered 177 Lu / 90 Is the amount of Y-NM600 immunostimulant or cytotoxic? Whether or not this is the case depends on the tumor absorbed dose. 64 Cu / 86 Y-NM600 is a therapeutic metal. 177 Lu / 90 Each can be used as a substitute for imaging with the Y-NM600. By utilizing the diagnostic and therapeutic (diapeutic) characteristics of the NM600, tumor dose measurement can be promoted. It's settled. Finally, 64 Cu / 86 In vivo body sculpting using Y-NM600 PET / CT Quantitatively measure the internal distribution and limit the dose to the organs and 177 Lu / 90 Y-NM600 We estimated radiation dose measurements that could help identify the potential tumor efficacy of TRT.

[0188] The general concepts are as follows: (1) within the tumor 64 Cu / 86 Y-NM600 concentration (2) This is quantified over time using long-term PET / CT imaging. 64 Cu / 86 The concentration of Y-NM600 is attenuated and corrected. 64 Cu / 86 Y-NM600 and 177 Lu / 90 (3) In the tumor, to explain the difference in attenuation rate between Y-NM600 177 Lu / 90 YN (4) Radiation We model and quantify the accumulation of sex nuclide decay within tumors.

[0189] Steps (1) through (3) can be performed using a medical image processing software package. It is possible, but step (4) requires high-performance radiation dose measurement software. INDA / EXM (Stabin, Sparks and Crowe 2005) is, Uses a format developed by the American Society of Nuclear Medicine's Medical Internal Dose (MIRD) Committee. 510(k) is approved dose measurement and estimation software (Bolch et al., 2009). MIRD's method involves detecting radiation emitted from within the organ itself or from another source organ. To estimate the average absorbed dose received by a tissue or organ due to the MIRD formula. It is a simple shape.

number

number

number

number

number

[0190] When calculating tumor dose measurement, OLINDA / EXM is used as part of step (1). The volume of isolated unit-density spheres was estimated from the generated tumor region of interest (ROI). The tumor is modeled as follows. The concentration of NM600 (%ID / g) within the tumor is determined at each time point. Therefore, attenuation correction was applied. Next, the concentration was integrated over all time using trapezoidal integrals. The cumulative radioactivity was calculated using `mk`.

[0191] Table 1 shows the results of radiation dose measurements for many cell lines. Using this information, tumor Absorbed doses for radiation therapy research aimed at either eradicating or stimulating the immune system It can be estimated. [Table 1]

[0192] Radiation sensitivity index for predicting dose response

[0193] Intrinsic radiosensitivity is a crucial factor underlying the response to radiotherapy, and it is important to consider the cancer type. What we know in advance is how it will respond to radiation from TRT. It can be helpful in measuring. However, there is no routine method for evaluating it in tumors, so radiation Radiation sensitivity is determined by clonality testing, specifically the survival rate after irradiation with 2 Gy (SF2) (0-1 It is measured as (between). The relative radiosensitivity of cancer cell phenotypes is such that radiosensitivity is very high. From those with low radiosensitivity (pancreas, colorectal, glioma, and breast) to those with high radiosensitivity (phosphorus) This extends to patoma. Cancers are classified or ranked according to their radiosensitivity index. This is possible (Table 2).

[0194] The inventors have developed a method for treating highly radiosensitive tumors such as lymphoma, as well as gliomas, breast tumors, and pancreatic tumors. In highly radioresistant tumors such as visceral or colorectal tumors, APC metal chelates are used. If these drugs can demonstrate good tumor uptake and growth inhibition, then they can be used in vivo. If these drugs can target tumors, then they could be used to create a SF between lymphoma and glioma. This means it is effective against any tumor with a binary count (0.3-0.82). This is possible. In that case, the amount of radiation required to eradicate glioma tumor cells is Higher than the radiation dose required to treat more radiosensitive lymphoma cells. It is also expected that this will happen.

[0195] The inventors have now investigated the tumor selectivity and therapeutic response in all tumor cell lines listed in Table 2. We have in vivo imaging to confirm the response (tumor growth suppression) data. Therefore, it is necessary to administer APC chelate multiple times to induce sufficient cancer cell death. It is possible. By using quantitative imaging in conjunction with radiation dose measurement calculations, The inventors have developed a method, as disclosed herein, to kill cancer cells (high dose) or to eliminate immunosuppressants. It is possible to estimate the tumor-absorbed dose required to stimulate the disease system (low dose).

[0196] Estimated dose measurements for various cancer cell lines (Table 1) and their respective radiosensitivity indices ( By combining the information in Table 2), the establishment of the dose response landscape for NM600 is supported. This can be achieved by understanding the tumor targeting properties and efficacy of NM600 within a series of cell lines. Estimate the absorbed tumor dose and potential efficacy of cell lines with similar radiosensitivity indices. It is possible to do so. Furthermore, the therapeutic dose will result in the desired outcome of tumor eradication or immune stimulation. Depending on the result (as disclosed herein), it may be increased or decreased linearly according to Table 1. [Table 2] 1 Taghian, Alphonse et al., “In vivo radiation s sensitivity of glioblastoma multiforme.''I International Journal of Radiation Oncolo gy * Biology * Physics 32.1(1995):99-104. 2 Ramsay, J., R. Ward, and NMBleehen. "Radio sensitivity testing of human malignant g liomas.” International Journal of Radiati on Oncology * Biology * Physics 24.4(1992):6 75-680. 3 Fertil, B., and EPMalaise. "Intrinsic ra diosensitivity of human cell lines is co rrelated with radioresponsiveness of hum an analysis of 101 published surv ival curves.」International Journal of Ra diation Oncology * Biology * Physics 11.9( 1985):1699-1707. 4 Wollin,Michaelら,「Radio sensitivity of human prostate cancer and malignant mela noma cell lines.」Radiotherapy and Oncolo gy 15.3(1989):285-293. 5 Kodym、Elisabethら,「The small-molecule C DK inhibitor,SNS-032,enhances cellular r adiosensitivity in quiescent and hypoxic non-small cell lung cancer cells.」Lung Cancer 66.1(2009):37-47. 6 Unkel,Steffen,Claus Belka,およびKirsten L auber.「On the analysis of clonogenic sur vival data:Statistical alternatives to t he linear-quadratic model.」Radiation Onc ology 11.1(2016):11. 7 EP Malaise,Patrick J.Deschavanne,and B ernard Fertil.「Intrinsic radiosensitivit y of human cells.”Advances in radiation Biology 15(2016):37-70. 8 Siles, E. et al., “Relationship between p53 st atus and radiosensitivity in human tumou r cell lines.”British journal of cancer 73.5(1996):581-588.

[0197] References cited in Example 13: Bolch, W. E., K. F. Eckerman, G. Sgouros, and S. .R.Thomas.2009.“MIRD Pamphlet No.21:A Ge neuralized Schema for Radiopharmaceutical Dosimetry--Standardization of Nomenclat ure.” Journal of Nuclear Medicine 50(3): 477-84.doi:10.2967 / jnumed.108.056036.

[0198] Stabin, MG, RB Sparks, and E Crowe.2005. “OLINDA / EXM:The Second-Generation Person al Computer Software for Internal Dose A ssessment in Nuclear Medicine.” J Nucl M ed 46(6):1023-27.

[0199] Example 14: Radioactive iodized compounds, as exemplified in Examples 1-4 and 7-10. Advantages and differences when using alkylphosphocholine metal chelates instead of [the other method]. In this embodiment, the inventors used radioactive iodized compounds (Examples 1-4 and 7-10). We will consider the advantages of using APC metal chelates instead of the example compounds. The inventors also describe the placement of the metal chelate intended to be used in the TRT step of the disclosed method. We will also consider the factors that a person skilled in the art should consider when optimizing the dosage.

[0200] Chelates are a wide variety of stable or radioactive metal ions used for imaging and therapeutic purposes. It enables the use of a wide variety of alpha, beta, auger, and gamma. And it can be combined with a positron emitter, but iodine has one positron (I-124), one Beta (I-131), one gamma (I-123), and one auger (I-12 5) Limited to isotopes.

[0201] Metal isotopes are more diagnostically and therapeutically useful than I-131 and I-124. (Cally) is effective.

[0202] Lu-177 has low levels of high-energy gamma, making it suitable for SPECT imaging and linear imaging. It is preferable for quantitative measurement. However, its beta energy is slightly smaller than that of I-131. Therefore, it is ideal for treating small tumors.

[0203] I-131 and Lu-177 are equivalent in terms of the "horsepower" of their therapeutic effect, but Lu- The contribution of gamma emissions from 177 to the overall dose is remarkably small. In the case of Y-90, cancer The contribution of radiation dose from the emission is very small.

[0204] Compared to I-131, as seen in Figure 52 and discussed further below, Y-90 is, TRT is more effective than I-131 in killing cancer cells.

[0205] The Medical Internal Radiation Dose (MIRD) Committee has determined the internal radiation dose from administered radiopharmaceuticals. We are developing standard methods, models, assumptions, and mathematical schemas for evaluating this MI. The RD method simplifies the problem of evaluating radiation doses for many different radionuclides. Therefore, it is a widely used 510(k) approved software, OLINDA / EXM1 It is implemented in OLINDA / EX, along with many of its standard anthropomorphic phantoms. M has a Spheres Model that can be used to estimate tumor dose. The Spheres Model uses units for various tumor volumes (0.01 to 6,000 g). This assumes that the radiopharmaceutical is uniformly distributed within the density sphere.

[0206] Using this standard model, the inventors have found that the radiation dose normalized by the administered radioactivity is related to Then, Y-90 and I-131 were compared. The results of this comparison were used to determine the tumor volume between 1 and 100 g. This is shown in Figure 52. The ratio of Y-90 to I-131 reaches 4 at 4g of tumor, and 10g of tumor. Up to 0g, it remains between 4.0 and 4.2, and on an mCi / mCi basis, Y-90 is up to 10g. In tumors of this size, it is 3.6 to 4.1 times more cytotoxic than I-131, and in large tumors exceeding 10g... It should be noted that this strongly suggests approximately 4.1 times more effective in Kisa tumors.

[0207] Different pharmacokinetic properties Unlike iodized analogs, APC chelates are too large to bind to known albumin in plasma. Because it doesn't fit in a pocket, it exhibits different in vivo pharmacokinetic and biodistribution profiles. (See Figure 53). When the binding energy is low, the proportion of free molecules in the plasma increases, This allows for more rapid tumor uptake. Some APC chelates are transmitted via the renal system. Although it is removed by the hepatobiliary system, the iodized analog is removed by the hepatobiliary system. APC chelates are tumors. It also accumulates in sores and is removed from the blood more quickly than iodized analogs. For faster blood purification, This is directly linked to bone marrow reduction and off-target toxicity of therapeutic radiopharmaceuticals.

[0208] These differences in PK and in vivo distribution profiles result in various dose-limiting organ toxicity And ultimately leads to usefulness. Due to hematological toxicity, dose-limiting toxicity, the kidneys or liver. Moving to this location would increase the usefulness of radioactive metal chelates for TRT.

[0209] Furthermore, the pharmacokinetic profile of APC chelates is influenced by the structure of the chelate (e.g., chelate It can be easily manipulated by slight changes in electric charge. The range of selectable chelating agents is expanding. It is large. The faster the clearance from normal tissue, the better the image contrast and therapeutic win. The dosage is improved, and as a result, the maximum permissible dose increases.

[0210] APC chelates have different physicochemical properties than their iodized analogs. Since they are much more water-soluble, surfactants are used to make them suitable for intravenous injection. No agent is needed. APC chelates are based on ionic bonding of metals to chelates. In contrast, iodized compounds form covalent bonds with these support molecules. In vivo deiodization Chelation is very common with alkyl iodides, but chelation is extremely stable in vivo. There is a tendency for this to be the case.

[0211] When deiodination occurs, free iodide rapidly accumulates in the thyroid gland, and its subsequent excretion half-life is In contrast to this, free radioactive metals are usually eliminated from the body much more quickly. It will be detoxified.

[0212] The in vivo distribution of APC chelates in the body may vary considerably depending on the metal ions present. Therefore, both metals and chelates contribute to the tumor targeting properties of APCs. Chelates do not target tumors. Tumor targeting is due to the cumulative properties of APC carriers, chelates. Types of chelates (linear chelates undergo rapid renal removal, while macrocyclic chelates undergo hepatic biliary excretion) It depends on the chelate structure and the metal ion. Even slight changes in the chelate structure can have a unique effect in vivo. This results in significant variations in sexuality. Simple isotopic changes alter tumor targeting by more than 50%. It is possible.

[0213] Radioactive APC-metal chelates can be easily released under simple conditions with nearly quantitative (>98%) yield. While radioactive labeling is possible, the radioactive iodination yield of iodized analogs is much lower (generally). Approximately 50% for I-131 and 60% for I-124. Furthermore, high specific activity chelates This can be achieved using [a specific method]. Synthesis can be performed using any nuclear [material] without the need for sophisticated ventilation equipment or training. This can be performed in pharmacy using a radiolabeling kit. Radioactive iodination involves radioactivity during the labeling reaction. Due to the volatility of iodine, the procedure must be carried out in a fume hood equipped with a wastewater monitoring system.

[0214] Contrast agents do not necessarily create better therapeutic drugs, and vice versa.

[0215] Even if tumor uptake is successful using contrast agents, this does not necessarily mean that treatment will be clearer. It cannot be assumed that this means it is white. In addition, the therapeutic agent needs to have long-term tumor retention compared to normal tissue, and bone marrow exposure Iodine must be rapidly removed from the blood to reduce its associated toxicity. The chemical analogs remain in the bloodstream for extended periods, leading to dose-limiting myelotoxicity. In contrast, our inventors' As mentioned above, APC chelates have low albumin binding in plasma, It is most likely to exhibit rapid blood clearance dynamics.

[0216] Finally, compared to iodine-131, the short path lengths of metallic beta and alpha emitters are Due to its physical properties, there is no concern about exposure to healthcare workers or family members after injection. 131 Patients receiving treatment are often shielded with lead before being released from the hospital before being discharged. They must be confined to the designated room for a period of time (up to one week). Patients who receive injections of beta-releasing APC chelate will no longer need to remain hospitalized. cormorant.

[0217] Example 15: Administration of an anti-CLA4 immune checkpoint inhibitor with Y90-NM600 TRT, delivered in combination with other drugs, synergistically inhibits cancer in an in vivo melanoma model. In this embodiment, the inventors demonstrate the effectiveness of the disclosed combined method. Here, In vitro immunization involves systemic administration of immune checkpoint inhibitors (anti-CTLA4 antibodies). Thus, the TRT was used in the previous example. 90 Y-NM600 Chelate for the whole body This is done by administering the drug.

[0218] As mentioned above, subcutaneous flank xenografts of B78 melanoma were transplanted into male C57BL / 6 mice. The mice were then randomized to receive various doses (25 μCi, 50 μCi, or 1 μCi). 00μCi) 90 The patient was treated with Y-NM600 (day 1), and an anti-CTLA4 antibody (immunotherapy) was administered. There were cases with and without (2 on days 4, 7, and 11) 00 μg) (n=6 for each experimental group). Both drugs were administered via lateral tail vein injection (same It was administered intravenously. This included control groups that received PBS treatment only and anti-CTLA4 only. The tumors were measured twice a week with calipers, and the animals' survival was monitored for 60 days.

[0219] As shown in Figure 54, three combination therapies (three different doses of anti-CTLA4+) 90 Y-NM600) is a single therapy (three different doses of anti-CTLA4 or 90 YN Compared to M600 only or PBS control, it showed substantial suppression of tumor growth. (18 days later) , 50 or 100 μCi 90 The combination therapy using Y-NM600 and anti-CTLA4 is P BS, 90 Compared to Y-NM600 alone or anti-CTLA4 alone, tumor growth was significantly reduced. (P < 0.05 by ANOVA) was reduced. 25 μCi using anti-CTLA-4 90 Y - The NM600 combination treatment group showed a moderate growth delay response that indicated a dose-response trend.

[0220] As shown in Figure 55, 50 μCi combined with anti-CTLA4 90 Y-NM60 Mice treated with 0 showed significant differences compared to mice treated with TRT alone or PBS vehicle. It showed a high overall survival rate (p<0.05). Compared to anti-CTLA4 monotherapy, the combination therapy (t The log rank of reatmnent was p=0.06.

[0221] As shown in Figure 56, all three combined treatments significantly improved survival. This is significant. In addition, non-combined control arms (PBS, 50 μCi TRT only, 100 μCi TRT only) Compared to 0 / 24 for full responders (and anti-CTLA4 only), 50 and a therapeutic dose of 100 μCi 90 With the Y-NM600, using a TRT+CTLA4 combined arm Of these, 6 out of 12 (50%) were complete responders.

[0222] These results suggest that molecularly targeted radiotherapy and immune checkpoint inhibitors (ICIs) are effective. The results indicate the possibility of treatment by combining any drug that causes the condition. The combination of TRT and ICI produces a synergistic effect compared to treatment with each drug alone. In addition to showing significant tumor regression, this combination therapy generates immunological memory and tumor recurrence. It may ultimately provide a powerful in situ cancer vaccine effect that prevents the development of cancer.

[0223] Example 16: To enhance the effectiveness of systemic checkpoint inhibition in a metastatic cancer model The use of molecularly targeted radiotherapy In the follow-up investigation of Example 15, the previous example used 90 Y-NM600 Kirei Systemic administration of immune checkpoint inhibitors and TRTs is performed by administering them systemically. The inventors have provided significantly expanded supporting data demonstrating the effectiveness of the combined method of this disclosure. It provides efficacy in mouse melanoma, neuroblastoma, and breast cancer models, as well as disseminated This has been demonstrated in multiple tumor melanoma models with "cold" tumors.

[0224] The clinical trial involved a subset of patients treated with immune checkpoint inhibitors (ICIs). This indicates that patients experience persistent complete remission (CR) at all disease sites. However, ICIs are generated by low levels of T cell infiltration and / or a small number of mutations. It is generally effective in patients with immunologically "cold" tumors characterized by neoantigens. There is no such method. In this embodiment, the combined method of the present disclosure is used to suppress the immune response of such tumors. The inventors demonstrate that it enhances the response in aggressive, "hot" tumors. More specifically The inventors have identified systemic lymphocyte depletion as a counterproductive factor in generating an antitumor immunotherapy response. To deliver low doses of immunostimulant radiation to all parts of the disease without consequently causing harm. Combining systemic molecular targeted radiotherapy (MTRT) with systemic intracorporeal injection (ICI) can be possible. This enhanced the effectiveness of systemic ICIs.

[0225] method: For tumor uptake studies of MTRT, flank tumors (B78 melanoma and Panc02) For each of the following (n=3), 1-2 x 10 units in 100 μL PBS 6 Cells C57BL / 6 This was established by injecting using the IACUC protocol approved in the US. B78 tumor and Both Panc02 tumors exhibit low to moderate immunogenicity, slow growth, and radioresistance. Since it is a tumor strain, this profile can be used in MTRT research. Due to the slow rate of decay in the MTRT, the combination of radioresistance and low immunogenicity was used. This allows for testing the synergistic improvement in efficacy between MTRT and ICI.

[0226] After the tumor is well established, approximately 5 weeks after the injection, the animals are given IV. 86 Y-NM600 The patient was treated with a dose, and sequential PET / CT images were collected 1, 2, and 3 days after MTRT injection. PET uptake values ​​are used in areas of background radioactivity, including the heart and liver. A comparison was made. A paired t-test was performed to compare background organs and tumor sites. 86 Y - We tested for a significant difference in NM600 uptake.

[0227] 90 Y-NM600 and / or ICI are immunosuppressive T in B78 melanoma flank tumors. To demonstrate the ability to reduce the reg cell population, the inventors have shown that B78 melanoma flank tumors A model (n=4 for each group) was generated. MTRT (50 μCi), anti-CTLA4 (4 Day 1, Day 7, Day 10 (200 μg), MTRT and CTLA4, and PBS plastic The sevogen control group was the group treated by the inventors. The effect of the treatment on the tumor immune cell population was determined by irradiation or Tumor tissue was collected on days 1, 7, and 14 after delivery of saline placebo for histological purposes. The remaining tumor samples were examined by freezing a portion and saving another portion for quantitative PCR. The samples were prepared for mRNA and RT-PCR analysis. Quantitative RT-PCR was used for immunosensitivity analysis. Evaluate changes in tumor cell expression of markers (e.g., Fas, MHC-I, and PD-L1). It was used to determine value.

[0228] For efficacy studies, a bilateral abdominal tumor model of B78 melanoma was developed in C57BL / 6 mice. Generated. The tumor was 80-120 mm. 3 Once the tumors proliferated, they were randomized to the next treatment group: Anti-CTLA-4 only in 200 μg IP on days 4, 7, and 10, and on day 1 90 Y-NM60 0 IV (50 μCi), and whole-body irradiation (EBRT) of anti-CTLA4, 12 Gy and Anti-CTLA4, 12 Gy EBRT + 50 μCi 90 Y-NM600, 12Gy EB RT+50μCi 90 Y-NM600 and anti-CTLA4. Tumor measurements twice a week for 30 days. The procedure was performed over a period of time, with survival rates tracked up to 60 days, and the euthanasia endpoint being a tumor with a diameter of 15 mm. It was a burden.

[0229] Mice that achieved complete remission from treatment received 2x10⁶ doses 90 days after MTRT. 6 B78 or 1x1 0 6 Panc02 cells were reloaded onto the contralateral flank, and then again on day 120. Reloading 02 (for B78 only) and B16 melanoma to induce tumor-specific immune memory responses. We tested it.

[0230] result: Our MTRT agent, 90 Selective acquisition of the Y-NM600 is B78 and Pa This was confirmed in both nc02 tumor models. In B78 melanoma, 90 Tumors of Y-NM600 Due to uptake, most of the drug was in the blood pool as expected after the initial injection, but the injection Within the next 48 hours, most of the drug was retained in tumors or excretory organs (liver, kidneys). This was demonstrated. The gamma count of tissue sections collected on day 48 was found to be the same as that of the PET image. Check the uptake values ​​of the radioactive material. In tumor tissue, the radioactivity count is high and increases over time. In the bone marrow cavity, the value is low and decreases over time. Monte was conducted as a collaborative research attempt. Carlo dosimetry indicates that when the experimental dose of 50 μCi, as determined by the inventors, is delivered, it is approximately 2-3 Gy This indicates that it is delivered throughout the lifespan of the MTRT agent. Panc02 PET scan in pancreatic cancer Studies of tissue infiltration and biodistribution also showed that tumor tissue compared to bone marrow tissue after 72 hours. 9 0 Increased capture and retention of the Y-NM600 were demonstrated.

[0231] To study the therapeutic effect on tumor immune cell populations, tumor cells were studied at various points in time after radiation therapy. Textile samples were collected. MTRT treatment (50 μCi) 90 On the 14th day after the Y-NM600, The combination of MTRT and anti-CTLA4 reduces the CD4 / FoxP3 and CD8 / in tumor tissue. The ratio of effector T cells to immunosuppressive T cells is determined by the FoxP3 infiltration. The inventors found that the width increases. Quantitative PCR (qPCR) study of gene expression. This includes genes that are part of the stimulating factors for the interferon gene pathway (STING). This showed an increase in inflammatory gene expression. All of these were Mx1, which is downstream of STING activation. IFNα, IFNβ, and PDL1 levels were upregulated compared to the PBS control. .

[0232] Next, the inventors established a single B78 R flank tumor in mice, which was approximately 80 m m 3 Once they reach that point, randomly assign them 25, 50, and 100 uCi of MTR on day 1. Each group received anti-CTLA4 on days 4, 7, and 10 after T-dose treatment. Each group that did not receive the treatment, as well as each group that received only PBS and anti-CTLA4 as a control. It was divided into two categories. The combined use of MTRT and anti-CTLA4 at dose levels of 50 and 100 uCi was also considered. Compared to the other group, tumor growth was significantly slower (Figure 57) and survival rate (Figure 58) was greatly improved. The inventors have found that this has been demonstrated. In an MTRT of 25uCi, there is an intermediate response. Furthermore, mice that showed complete remission to the treatment were only included in the combination treatment group. 66%, 33%, and 25uCi of animals in the MTRT dose groups. The figure was 16%. All mice that were in complete remission 60 days after MTRT injection were contralateral ventral. When B78 cells are loaded onto the area, there is a 100% rejection rate compared to the naive control, thus the present invention This demonstrates that it was possible to generate a treatment-related immune memory response in the subjects.

[0233] Since then, this study has been replicated and has shown a similar trend, with survival rates in both studies being similar to those in Logran. A test showed that Mau was treated with MTRT (50, 100 uCi) and anti-CTLA4. This showed that the overall survival rate of the group was significantly improved compared to other groups.

[0234] Next, the inventors applied this study to neuroblastoma (NXS2) and breast cancer (4T1) similarly. This was extended to the mouse model. As can be seen in Figure 59 (NXS2) and Figure 60 (4T1), The combined use of CTLA4 and MTRT also resulted in a significant reduction in tumor growth (in fact, a reduction in tumor volume). It was the only group that showed (small).

[0235] Next, the inventors expanded this research to show that the response rate was improved in mice with multiple large tumors. The inventors demonstrated that it is effective. The inventors established a study of MTRT treatment in two tumor mouse models. The goal was to treat mice with multiple large tumors, which involved multiple parts This corresponds to patients with a massive metastatic disease in the periphery. In this experiment, EBRT was used with an immune checkpoint. The current clinical paradigm of combining injection blockade with delivery to a single site is more effective than MT We investigated whether RT could improve performance.

[0236] We established two tumor models: Panc02 and B78 melanoma. First, with B78 melanoma, Conventional immunosensitization EBRT (12 Gy) is administered to the primary site of the disease (blocking secondary sites). In combination with CTLA4, radiation therapy alone, MTRT and anti-CTLA4, or to the primary site. Compared to combined treatment with EBRT and combined treatment with MTRT and anti-CTLA4 at all sites The tumor growth curves showed that the triple-drug combination treatment was superior to the other groups in primary tumors (Figure 61) and This demonstrates improved tumor regression in both secondary tumors (Figure 62). Furthermore, survival rates are higher. Compared to the group receiving two combination therapy, the group receiving three combination therapy (p<0.01) showed a significant improvement. The combination therapy resulted in a 40% complete remission rate (compared to 16% complete remission with MTRT + anti-CTLA4, and other combinations). The group yielded 0%, and all responsive animals were against B78 or related B16 melanoma. It possessed tumor-specific immunological memory.

[0237] Finally, we developed a mouse model with two macroscopically visible distant tumors and disseminated microscopic metastases. It is used to treat advanced, multi-site "cold" cancers (i.e., cancers that do not trigger a strong immune response). Therefore, we simulated multi-site tumors that are highly resistant to checkpoint inhibition.

[0238] To form a large primary tumor, 2 × 10¹⁶ cells were placed in one flank of the mouse. 6 Individual B78 Black Irodactyl tumor cells were injected. Twelve days later, the mice were reversed to form small secondary tumors. 5 × 10 on the flank 5 Individual B78 melanoma tumor cells were injected. 17 days later (Day 1) To create disseminated metastasis, 2 × 10⁶ mice were given 2 × 10⁶ mice. 5 Individual B16 melanoma cells intravenously I got an injection.

[0239] Mice were exposed to various single or combined treatments: PBS control injection; MTRT on day 1. 50μCi IV; ICI on days 4, 7, and 10, anti-CTLA4 / PD1; day 1 In situ vaccine (IS), 12 Gy local RT + anti-GD2 mA on days 6-10. Intratumoral injection of b and IL2. The single and combination treatments tested were PBS, MTRT, ICI, IS, MTRT+ICI, MTRT+IS, ICI+IS, and MTRT+I The diagnosis was S+ICI. From day 60, tumor growth in mice and animal survival were monitored. On day 90, mice without tumors were reloaded with B78.

[0240] On day 90, less than 20% of ICI mice were still alive, but MTRT+IS and IC Approximately half of the I+IS mice survived (MTRT+IS mice had a slightly higher survival rate). Surprisingly, 100% of the MTRT+IS mice survived (compared to the survival rate of all other groups). The survival rate was zero. In particular, 83% of these mice were found to be tumor-free and immune. The patient showed complete remission (CR) with epidemic cellular memory (i.e., cured), but the rest remained uncontrolled. The tumor remained intact.

[0241] Furthermore, the inventors have identified neuroblastoma (NXS2, 9464D), rhabdomyosarcoma (M3-9- M), including high-grade glioma, Lewis lung cancer, and head and neck cancer (MOC-2), Uptake and dose delivery in various other cancers were also confirmed. Furthermore, toxicity analysis was conducted, and radiation-induced myelotoxicity (measured in serum leukocytes or lymphocytes) was detected. This was not observed with the inventors' therapeutic radiation dose of 50 μCi (tumor dose of 2-3 Gy). Furthermore, the inventors administered both an external beam and 90Y-NM600 at various doses to mice. Irradiated tissue, and tissue images stained with IHC and tissue were collected for mRNA analysis by PCR. The data obtained from these studies were obtained using 50 μCi in an 90Y-NM600 interface. This shows upregulation of the feron signaling pathway and increased PDL1 expression. Furthermore, the present invention demonstrates that tumor-infiltrating regulatory T cells are reduced by molecularly targeted radiotherapy. They discovered it.

[0242] In short, the inventors' findings from this study are that low doses of NM600 MTRT are effective. When combined with checkpoint blockade, it can enhance the abscopal response of tumors. This suggests that, in particular, NM600 MTRT radiotherapy delivery agents are usually immunosuppressed. It shows the ability to improve the response of "cold" tumors that do not respond only to citrate blockade. Furthermore, A relatively low MTRT dose of 50 μCi (2.5 Gy tumor dose) is used for systemic lymphocytes. It is sufficient to achieve an immunostimulatory effect that enhances the effectiveness of ICI without depletion. MTRT In addition to single-site EBRT and checkpoint blockade, this is used for both local and distant tumors. Superior tumor response and cure rates can be achieved in specific areas. Our results indicate that MT RT has great potential to improve the therapeutic effects of immunotherapy treatments in patients. This indicates that.

[0243] Conclusion of the Examples These examples include targeted systemic delivery of radiotherapy and immune checkpoint inhibitors. This study presents an anti-cancer strategy based on a synergistic and widely applicable combination of systemic delivery of immunostimulants. The disclosed metal chelated and radioactive alkyl phosphocholine analogs are Because it can target cancer in virtually any tissue, mAbs target immune checkpoints. Alternatively, systemic administration of small molecules (immune checkpoint inhibitors) is effective for virtually any type of cancer. It has been shown to be useful (tumor-reactive mAbs are approved or nearly approved). (Used in clinical trials for various cancer tissue types). Therefore, two different combination strategies are used in clinical trials. Transitioning to this approach would give it broad applications for virtually all high-risk cancers.

[0244] Other embodiments and uses of the present invention are disclosed herein and in the Specified Invention and This is obvious to those skilled in the art when considering practice. All academic journal citations and U.S. / foreign patents and All references cited herein for any reason, including patent applications, are provided by reference. The present invention is more specifically and fully incorporated herein. Not limited to specific reagents, formulations, reaction conditions, etc., but including any other claims that fall within the scope of the following claims. It is understood that these modified forms should be included.

Claims

1. The use of (a) a molecularly targeted radiotherapy (TRT) agent in an immunomodulatory dose that is selectively taken up and retained by malignant solid tumor tissue, and (b) one or more immunostimulants comprising one or more anti-immune checkpoint molecular antibodies or one or more small molecule immune checkpoint inhibitors that can stimulate specific immune cells in the tumor microenvironment, in the manufacture of a drug for the treatment of metastatic cancer in a subject, The TRT agent is 90 Y-NM600, and the immunostimulant is selected from the group consisting of anti-CTLA4 antibody, anti-PD-1 antibody, and anti-PD-L1 antibody.

2. The use according to claim 1, wherein the immunostimulant is an anti-CTLA4 antibody.

3. The use according to claim 1 or 2, wherein the TRT agent; immunostimulant; or both; is administered intravenously.

4. The use according to any one of claims 1 to 3, wherein the subject is a human.

5. The use according to any one of claims 1 to 4, wherein the cancer to be treated is selected from the group consisting of melanoma, neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, liver cancer, subcutaneous cancer, squamous cell carcinoma of the skin or head and neck, intestinal cancer, retinoblastoma, cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, Wilms' tumor, and pediatric brain tumors.

6. The use according to any one of claims 1 to 5, wherein cancer is treated without administering antibodies against tumor antigens that are not checkpoint molecules.

7. The use according to any one of claims 1 to 6, wherein cancer is treated without administering an anti-GD2 antibody to the subject.

8. The use according to any one of claims 1 to 7, wherein the immunomodulatory dose is a low dose or a radiotherapy dose that is less than cytotoxic.

9. The use according to any one of claims 1 to 8, wherein the immunomodulatory dose is 2 Gy to 5 Gy.

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