Intratumoral alpha-emitter irradiation in combination with immune checkpoint modulators

Abstract

1. A substance that modulates an immune checkpoint for use as a medicament for the treatment of a tumor in a patient, wherein the administration pattern of the medicament comprises administering a therapeutically effective amount of the substance to the tumor in one or more sessions, and implanting seeds (204) carrying radium-224 into the tumor for intratumoral alpha-emitter radiotherapy less than two weeks after administering the substance.

Description

[Technical field]

[0001] The present invention relates generally to tumor therapy, and in particular to the combination of intratumoral alpha-emitter irradiation with immune checkpoint modulators. [Background technology]

[0002] Cancer is the leading cause of death in many countries around the world. Accordingly, enormous amounts of resources are spent on the treatment of cancer, and a wide variety of such treatments have been proposed. One type of tumor treatment is tumor ablation, which kills tumor cells in situ. In addition to killing cells in situ, tumor ablation can induce antitumor immune responses that result in the elimination of remaining and distant tumor cells. This occurs through the spread of tumor antigens and danger signals released from dead and / or dying tumor cells. Tumor antigens are captured by antigen-presenting cells (APCs), such as dendritic cells (DCs), and then presented to T cells via the cross-presentation pathway, as described, for example, in Nakayama, Masafumi. "Antigen presentation by MHC-dressed cells." Frontiers in immunology 5 (2015): 672. Several ablation methods have been proposed, such as high or low temperature, microwave, laser, electrical, photodynamic, chemical (e.g., using reactive oxygen species (ROS)) and radioactive ablation, which may be applied externally (e.g., external beam radiation therapy) or internally (e.g., brachytherapy), and may include different types of irradiation, such as alpha, beta and photon irradiation. A discussion of these methods can be found, for example, in Keisari, Yona. "Tumor abolition and antitumor immunostimulation by physico-chemical tumor ablation." Front Biosci 22 (2017): 310-347. The ablation method used for any particular patient is generally selected according to the type of tumor, its location, size, stage and / or other parameters of the tumor.

[0003] Another type of tumor treatment, called immunotherapy, involves the promotion of the patient's immune response against tumor cells. Many immunotherapy methods have been proposed, such as immune checkpoint inhibitors, Toll-like receptor (TLR) agonists (e.g., CpG), local gene therapy, cytokine therapy, antibodies against certain protein targets, CAR-T cell therapy, dendritic cell vaccines, adoptive transfer of tumor-infiltrating lymphocytes, inhibition of immune suppressive cells, and oncolytic virus therapy. These methods are discussed, for example, in Papaioannou, Nikos E., et al. "Harnessing the immune system to improve cancer therapy." Annals of translational medicine 4.14 (2016). In general, the particular method used for each patient is selected according to the type of tumor or its stage. A number of combinations of the above types of therapy have been tested in preclinical and clinical trials, for example as described in Table 1 of Aznar, M. Angela, et al. "Intratumoral delivery of immunotherapy act locally, think globally." The Journal of Immunology 198.1 (2017): 31-39. In the case of treatment with immune checkpoint inhibitors, for example, the response rate of the treatment is relatively low (about 20%). Patients who receive the treatment not only rarely respond, but also suffer from significant adverse effects. Extensive efforts have been made to find treatments that can promote the response rate of immune checkpoint inhibitors, but there has been no significant success to date.

[0004] The paper, "Randomized Phase-II Trial of Nivolumab with Stereotactic Body Radiotherapy Versus Nivolumab Alone in Metastactic Head and Neck Squamous Cell Carcinoma," by Sean McBride et al., Journal of Clinical Oncology, vol. 39, issue 1, pages 30-38, describes a trial in which the addition of stereotactic body radiotherapy to nivolumab did not result in any improvement. Summary of the Invention

[0005] An aspect of some embodiments of the present invention relates to tumor treatment based on synergy between immune checkpoint modulators and intratumoral alpha-emitter radiotherapy. The term intratumoral refers herein to a treatment in which an alpha-emitter radionuclide is implanted in a seed within the tumor at one or more initial locations, and the alpha-emitter radionuclide or its daughter radionuclide migrates to other locations in the tumor where alpha-radiative decay occurs. The migration of the radionuclide from the seed may be by diffusion or by decay, where the radionuclide on the seed initiates multiple chains of radioactive decays. The various options and alternatives set out in the following description and claims can be used alternatively or together in any suitable combination, unless the options are specifically contradictory. Thus, according to one embodiment of the present invention, there is provided a substance that modulates an immune checkpoint for use as a medicament for the treatment of a tumour in a patient, wherein the administration pattern of the medicament comprises administering a therapeutically effective amount of said substance to the tumour in one or more sessions, and implanting seeds carrying radium-224 into the tumour for intratumoural alpha emitter radiotherapy less than two weeks after administering said substance.

[0006] The pharmaceutical administration pattern may include starting administration of the substance less than 5 days after implantation of the seed. The pharmaceutical administration pattern may include starting administration of the substance at least 12 hours after implantation of the seed. The pharmaceutical administration pattern may include starting administration of the substance at least 72 hours after implantation of the seed. The pharmaceutical administration pattern may include starting administration of the substance at least 12 hours before implantation of the seed. The pharmaceutical administration pattern may include starting administration of the substance at least 72 hours before implantation of the seed. The substance may include an antiangiogenic agent. The substance may include a checkpoint inhibitor. The substance may include a small molecule inhibitor. The substance may include nivolumab, pembrolizumab, cemiplimab, toripalimab, or sintilimab. The substance may include atezolizumab, avelumab, or durvalumab. In some embodiments, the agents include ipilimumab, leratolimab, LY3321367, tiragolumab, epacadostat, and / or enoblituzumab.

[0007] The material may include HLX23 or ORIC-533. Alternatively or additionally, the material may include monalizumab, pexidartinib, and / or lacnotuzumab. The material may include pepinemab. The material may include enapotamab. The material may include taborimab or cudarolimab. The material may include vopratelimab, sotigalimab, or elotuzumab. In some embodiments, the seed comprises a support having a length of at least 1 millimeter; and radium-224 atoms bound to the support such that when the seed is implanted in a tumor, no more than 20% of the radium-224 atoms leave the support in 24 hours in the tumor without decay, but at least 5% of the daughter radionuclides of the radium-224 atoms leave the support upon decay. There is further provided in accordance with one embodiment of the present invention a method of treating a patient having a tumor comprising: treating the tumor with intracellular alpha-emitter radiotherapy; and administering to the patient an agent that modulates an immune checkpoint within two weeks of initiating treatment of the tumor with intratumoral alpha-emitter radiotherapy.

[0008] The step of administering the agent may comprise administering an immune checkpoint inhibitor and / or an immune checkpoint bispecific antibody. In some embodiments, the step of administering the agent comprises administering an immune checkpoint internalizing molecule. In some embodiments, the step of administering the agent comprises administering a LAG3 checkpoint inhibitor. In some embodiments, the step of administering the agent comprises administering a PD-1 checkpoint inhibitor. In some embodiments, the step of administering the agent comprises administering a PDL-1 checkpoint inhibitor. In some embodiments, the step of administering the agent comprises administering a CTLA4 checkpoint inhibitor.

[0009] In some embodiments, administering the substance comprises administering a small molecule inhibitor. In some embodiments, administering the substance comprises administering a costimulatory molecule. In some embodiments, administering the substance comprises administering nivolumab or pembrolizumab. In some embodiments, administering the substance comprises administering atezolizumab, avelumab, or durvalumab. In some embodiments, administering the substance comprises administering ipilimumab or tremelimumab. In some embodiments, administering the substance comprises administering relatolimab. In some embodiments, administering the substance comprises administering tebotelimab. In some embodiments, administering the substance comprises administering TSR-022. In some embodiments, administering the substance comprises administering etidilimab or tiragolumab. In some embodiments, the step of administering the substance comprises administering enoblituzumab, pomalidomide, beruzosertib, and / or celecoxib. In some embodiments, the step of administering the substance comprises administering vemurafenib. In some embodiments, the step of administering the substance comprises administering vorinostat. In some embodiments, the step of administering the substance comprises administering sorafenib or sunitinib. In some embodiments, the step of administering the substance comprises administering tavolimab. In some embodiments, the step of administering the substance comprises administering elotuzumab. In some embodiments, the step of administering the substance comprises administering the substance at least 72 hours after the initiation of treatment of the tumor with intratumoral alpha emitter radiotherapy. In some embodiments, the step of administering the substance comprises administering the substance less than 2 weeks after the initiation of treatment of the tumor with intratumoral alpha emitter radiotherapy. In some embodiments, administering the agent comprises administering the agent less than 144 hours after initiation of treatment of the tumor with intratumoral alpha emitter radiotherapy. In some embodiments, administering the agent comprises administering an immune checkpoint blockade.

[0010] Further provided is a kit for treatment of a patient according to one embodiment of the invention, comprising at least one source having alpha emitting atoms mounted thereon for at least partial introduction into a body of a subject, at least one immune checkpoint modulator; and a package comprising the at least one source and the at least one immune checkpoint modulator.

[0011] Further provided is an alpha emitting device designed for use in alpha emitter radiotherapy treatment of a tumor in a patient according to an embodiment of the invention, wherein the alpha emitter radiotherapy treatment pattern comprises treating the tumor with the alpha emitter device, followed by administration of a therapeutically effective amount of an immune checkpoint modulator in one or more sessions less than six weeks after initiation of alpha emitter radiotherapy. The alpha emitter radiotherapy treatment pattern may comprise treating the tumor with the alpha emitter device, followed by administration of a therapeutically effective amount of an immune checkpoint modulator in one or more sessions less than two weeks after initiation of alpha emitter radiotherapy.

[0012] There is further provided an alpha-emitting device designed for use in alpha-emitter radiotherapy treatment of a tumor-bearing population that has been treated with a therapeutically effective amount of an immune checkpoint modulator in one or more sessions less than six weeks after initiation of alpha-emitter radiotherapy, in accordance with an embodiment of the present invention.

[0013] The device may include a support having a length of at least 1 millimeter; and radium-224 atoms bound to the support such that when the device is implanted into a tumor, no more than 20% of the radium-224 atoms leave the support and enter the tumor within 24 hours without decay, but upon decay, at least 5% of the daughter radionuclides of the radium-224 atoms leave the support upon decay. [Brief description of the drawings]

[0014] [Figure 1] 1 is a flow chart of a treatment method according to one embodiment of the present invention. [Diagram 2] 1 is a schematic diagram of a kit for the combined use of alpha emitter irradiation and immune checkpoint modulators, according to one embodiment of the present invention. [Diagram 3] 1 is a graph showing the results of an experiment testing the effect of combining alpha emitter irradiation with anti-PD-1 on mouse squamous cell carcinoma tumor growth, according to one embodiment of the present invention. [Figure 4] 1 is a graph showing the results of an experiment testing the effect of combining alpha-emitter irradiation with anti-PD-1 on mouse pancreatic tumor growth in accordance with one embodiment of the present invention. [Diagram 5] 1 is a graph showing the results of an experiment examining the effect of alpha-emitter irradiation on dendritic (DC) cell activation in mouse squamous cell carcinoma mouse tumors. [Figure 6A-6C] 1 is a dot plot showing the results of an experiment testing the effect of alpha-emitter radiation in combination with anti-PD-1 on CD3+, CD8+, and granzyme BT cells, respectively, in mouse squamous cell carcinoma mouse tumors according to one embodiment of the present invention. [Figure 6D] FIG. 1 shows CD3 T cell density in squamous cell carcinoma tumor tissues after treatment with a-PD1 versus DaRT together with a-PD1, according to an embodiment of the present invention. [Figure 7] 1 is a graph showing the results of an experiment testing the effect of alpha-emitter irradiation in combination with anti-PD-1 on immune myeloid derived suppressive cells (MDSC) in mouse spleens according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] An aspect of some embodiments of the present invention relates to a combination tumor treatment comprising alpha emitter irradiation in a patient's tumor and applying a therapeutically effective treatment of one or more immune checkpoint regulators to the patient within a time frame to achieve an interaction between the alpha emitter irradiation and one or more immune checkpoint regulators. In some embodiments, the one or more immune checkpoint regulators are administered to the patient within 4 weeks or even 2 weeks before or after the start of the alpha emitter irradiation treatment. Applicant has found that the combination of alpha emitter irradiation followed by specific tumor ablation applying specific immunotherapy of immune checkpoint regulators has a substantially greater therapeutic effect than each of the treatments separately. Conventional radiation therapy can damage the patient's innate immune system, for example by damaging immune organs or by expanding immune suppressive cells such as MDSCs, thus preventing the benefits of immune checkpoint regulators, but applicant has found that alpha emitter irradiation has a positive effect when applied together with immune checkpoint regulators, reducing the immune suppressive population.

[0016] According to experiments performed by the applicant, after alpha-emitter irradiation, dendritic cells (DCs) are activated in tumors within about one week after alpha-emitter irradiation. Due to this discovery, the applicant determined that it may be advantageous to start immune checkpoint regulator treatment within two weeks of the start of alpha-emitter irradiation treatment. The applicant also showed that with this sequencing during treatment, the combination treatment resulted in synergistic effects on T cell infiltration into tumors, a well-documented criterion for responsiveness to immune checkpoint regulators. The applicant also found that T cell function, as represented by granzyme B secretion, was elevated only in the combination treatment. Furthermore, the systemic MDSC population was reduced in the combination treatment compared to aPD-1 monotherapy.

[0017] It should be noted that in the applicant's experiments, the applicant found that 7 days after the initiation of DaRT treatment, there was a massive destruction of cells in the tumor, including T cells. Also, at this stage, negative regulatory cells entered the tumor. However, after another 7 days, 14-16 days after the initiation of DaRT treatment, there was an unexpected increase in T cells and T cell function. Furthermore, at this stage, negative regulatory cells showed a decrease. This unexpected change did not occur when treated with immune checkpoint regulators alone.

[0018] Applicant believes that alpha-emitter irradiation stimulates effector T cells to respond to checkpoint regulation by allowing spatial and temporal coexistence of immune cells and dead / dying cells in the tumor microenvironment after alpha-emitter insertion. Intratumoral Diffusing alpha-emitter Radiation Therapy (DaRT) utilizes alpha-emitting atoms for the treatment of tumors, optionally using a source coated with radioactive atoms. The alpha-emitting atoms are released locally in a sustained manner both in time and space. That is, the atoms gradually diffuse in the tumor. At the initial time point, most of the radioactivity is concentrated near the source. Over time, this distribution changes such that some of the atoms move from positions close to the source to more distant positions in the tumor. In addition to the change in the distance traveled by the radioactive atoms over time, for each time point, there is a different distribution of activity as a function of distance from the source. That is, for each given time point and distance traveled, there is a different amount of activity along the line between the source and the maximum distance traveled, where points closer to the source generally have higher activity, allowing for non-uniform and non-instantaneous destruction of tumor tissue.

[0019] Immune checkpoint regulators, particularly immune checkpoint inhibitors, inhibit the negative regulation of cytotoxic T cells, allowing them to properly recognize and kill tumor cells. For T cells to identify tumor cells, an activation event must first occur, which depends on the interaction between T cells and activated antigen-presenting cells (APCs). This interaction can occur in lymph nodes or in the tumor itself. In general, immune checkpoint inhibitors do not function in the absence of T cell clones that have been preactivated by APCs to recognize the presence of a specific tumor antigen. If T cells have been preactivated by APCs specifically against tumor antigens, T lymphocytes can infiltrate tumors that present this antigen, which can condition the successful action of immune checkpoint inhibitors. Such a process can also occur in metastatic cells.

[0020] Tumor cell killing by DaRT leads to the induction of specific anti-tumor immune responses. This process involves local inflammatory reactions, recruitment of APCs such as dendritic cells and macrophages, and their activation by tumor antigens released from or presented on dying cells, and by damage-associated molecular patterns (DAMPs), eat-me signals, and cytokines present in the tumor microenvironment. Antigen-loaded and activated APCs present tumor antigens to T cells for their specific activation. Because DaRT-induced DNA damage is thought to be complex and the release of radioactive atoms from the seeds is gradual, the applicant believes that DaRT-mediated in situ tumor destruction and robust inflammation will result in stronger, long-term systemic and specific adaptive immune responses against a wide range of tumor antigens. Furthermore, the applicant found that DaRT reduced the amount of suppressive immune cells such as MDSCs, which impair T cell function. In contrast to other tumor ablation therapies, DaRT does not immediately cause complete destruction of the tumor microenvironment, but rather causes it gradually. This may allow the coexistence of APCs and T cells with dying / dead cells, allowing the interactions necessary to activate cytotoxic T cells whose function is then enhanced with checkpoint inhibitors.Also, due to the short-range effect of DaRT, important immune organs such as lymph nodes and bone marrow, as well as tertiary lymphoid structures in close proximity to the tumor, remain intact, supporting local and systemic immune responses.

[0021] Treatment 1 is a flow chart of a therapeutic method 100 according to an embodiment of the present invention. Following identification of a tumor in a patient (102), the therapeutic method 100 begins with initiating an alpha emitter radiation treatment (also referred to herein as alpha emitter radiation therapy) (104), for example, by implantation of an alpha emitter radiation source into the tumor. A limited period of time after initiating the alpha emitter radiation treatment (106), a therapeutically effective immune checkpoint modulator is administered to the patient in one or more sessions (108).

[0022] In some embodiments, after the alpha emitter radiation treatment is completed, the effectiveness of the treatment is evaluated (110). In some embodiments, after the evaluation, surgery (112) is used to remove the remaining primary tumor. In some embodiments, the surgery is performed at least one week or even at least 14 days after the start or completion of the radiation treatment. While surgery to remove the cancerous tumor is generally performed as soon as possible, applicants have found that it is better to wait after applying the combined immune checkpoint modulation and alpha emitter radiation therapy treatment so that the treatment can take effect and only then remove the tumor. Alternatively, surgery is performed at any other suitable time, perhaps before the alpha emitter radiation therapy, or not at all if deemed unnecessary or infeasible. Additionally or additionally, evaluation (110) is not performed. In some embodiments, the treatment method 100 further comprises the step of providing supportive treatment (114).

[0023] Tumor type The therapeutic method 100 may be used in the treatment of any tumor type, including cancerous tumors, benign neoplasms, in situ neoplasms (pre-malignant), malignant neoplasms (cancer), and neoplasms of uncertain or unknown behavior. In some embodiments, the method of Figure 1 is used to treat relatively solid tumors, such as breast cancer, renal cancer, pancreatic cancer, skin cancer, head and neck cancer, colorectal cancer, ovarian cancer, bladder cancer, brain cancer, vulvar cancer, and prostate cancer. In other embodiments, the method of Figure 1 is used to treat non-solid tumors. The method of Figure 1 may be used for both primary and secondary tumors.

[0024] Exemplary tumors that may be treated by the method of FIG. 1 include, but are not limited to, tumors of the digestive tract (colon cancer, rectal cancer, colorectal cancer, colorectal adenocarcinoma, hereditary nonpolyposis 1, hereditary nonpolyposis 2, hereditary nonpolyposis 3, hereditary nonpolyposis 6; colorectal cancer, hereditary nonpolyposis 7, small intestine and / or large intestine cancer, esophageal cancer, calluses with esophageal cancer, gastric cancer, pancreatic cancer, pancreatic endocrine tumors). , endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, biliary tract tumors, prostate cancer, prostate adenocarcinoma, kidney cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cell tumor, immature teratoma of the ovary, uterus, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental trophoblastic tumor, epithelial adult tumor, ovarian cancer, serous ovarian cancer, ovarian sex cord tumor, cervical cancer, small cell and non-small cell lung cancer, nasopharynx, breast cancer (e.g., ductal carcinoma, invasive ductal carcinoma; breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer 1, breast cancer 3;breast cancer-ovarian cancer), squamous cell carcinoma (e.g., in the head and neck), vulvar cancer, neurogenic tumor, astrocytoma, ganglioneuroblastoma, neuroblastoma, glioma, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, malignant brain tumor (tumor), various other cancers (e.g., bronchogenic large cell, ductal, epidermoid, large cell, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinous cell, transitional cell, anaplastic, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastic), fibroblastoma, giant cell tumor, glial tumor, glioblastoma (e.g., polymorphic, astrocytoma), glioma hepatoma hepatoma), heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), adrenal tumor, insulinoma, pancreatic islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, lymphosarcoma, melanoma, breast tumor, mast cell tumor, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, neural tissue glial tumor, neural tissue neural tumor, schwannoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma , osteosarcoma (e.g., Ewing), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, histiocytic, Jensen, osteogenic, reticular cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratomas, testicular tumors, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, solid lymphoma, liposarcoma, Lynch familial cancer syndrome type II (lynch These include Turcot's syndrome with rhabdoid tumor family syndrome II, male germ cell tumors, medullary thyroid, multiple meningiomas, endocrine neoplasms myxosarcoma, paraganglioma, familial nonchromaffin, pilomatricoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial rhabdoid tumor, soft tissue sarcoma, and glioblastoma;

[0025] In some embodiments, the method of Figure 1 is applied to tumors known to be substantially affected by alpha emitter irradiation alone, hi other embodiments, the method of Figure 1 is applied to tumors of a type that are not substantially affected by alpha emitter irradiation alone, e.g., do not decrease in size at all, or do not decrease in size by more than 5% or 10%.

[0026] Experiments performed by applicants show that tumors that do not show enhanced T cell infiltration following alpha-emitter radiation treatment or immune checkpoint modulator treatment alone dramatically enhance T cell infiltration into the tumor when targeted with a combination of immune checkpoint modulator and alpha-emitter radiation treatment according to the method of Figure 1. This T cell infiltration also correlates with a reduction in tumor size, supporting a functional T cell response.

[0027] Immune checkpoint regulators Immune checkpoint modulators can be small molecules, antibodies (also known as blockers) or any other type of drug that results in modulation of immune checkpoint pathways. In some embodiments, the immune checkpoint modulator comprises an immune checkpoint inhibitor, which inhibits the function of one or more molecules in a cell, such as an immune checkpoint molecule. The immune checkpoint inhibitor can be an antibody or a small molecule. Table 1 lists various molecules that can be targeted by an immune checkpoint inhibitor, along with the specific corresponding immune checkpoint inhibitor.

[0028] [Table 1-1] [Table 1-2]

[0029] In other embodiments, an immune checkpoint modulator comprises an immune checkpoint bispecific antibody, such as any of the following: a.Anti-CTLA-4 + OX40 (ATOR-1015) b. Anti-PDL1+Lag3 (MGD013, FS118) c. Anti-PDL1 + TGF beta receptor (M7824) d. Anti-PDL1+TIGIT e. Anti-PDL1+4-1BB (INBRX-105, ATG-101) f. Anti-PD1+CTLA4 (MGD019) g. Anti-PD-1+TIM-3 (RO7121661) h. Anti-CD47-PD-L1 (PF-0725876) i. Anti-PD-L1 + Claudin 18.2 (Q-1802) j.ImmTAC (IMC-F106C, IMCgp100-teventafsp) k.Anti-PD-1+LAG3 (RO7247669, tebotelimab) l.Anti-PD-1+CD137(IBI319) m.Anti-CD137+HER-2(PRS-343) n.Anti-PD-1 + ICOS (izularimab) o.Anti-PD-L1+CD27(CDX-527)

[0030] In yet other embodiments, the immune checkpoint modulator comprises a molecule that inhibits immune checkpoint expression, such as any of the following: ATR inhibitors (e.g., beruzosertib) b. Cox-2 inhibitors (e.g., celecoxib) c.BRAF inhibitors (e.g., vemurafenib, dabrafenib, encorafenib) d. MEK inhibitors (e.g., trametinib, binimetinib, selumetinib, and cobimetinib) e. PI3K inhibitors (e.g., idelalisib, alpelisib, taselisib, pictilisib, duvelisib, copanlisib, gedatolisib, apitolisib, dactolisib) f. HDAC inhibitors (entinistat, vorinostat, mocetinostat, panobinostat, ACY-241) g.DNMT inhibitors (decitabine, guadecitabine, azacitidine) h. Bromodomain inhibitors (JQ1, I-BET151) i.RTK inhibitors (cediranib, semaxinib)

[0031] In yet other embodiments, the immune checkpoint modulator comprises a molecule that inhibits immune checkpoint expression and a.IMiDs (e.g., pomalidomide, lenalidomide, breferamid, thalidomide, iveldimide, apremilast) b.TK inhibitors (sorafenib, dasatinib, sunitinib, nilotinib, gefitinib, erlotinib (eriotinib), bosutinib, lapatinib, pazopanib, regorafenib, lestaurtinib, imatinib) and other antiangiogenic drugs that prevent the development of blood vessels.

[0032] In other embodiments, the immune checkpoint modulator in combination with alpha emitter irradiation comprises a molecule that internalizes an immune checkpoint. Such molecules can include, for example, ARB-272572 and / or ARB-276309. In other embodiments, the immune checkpoint regulator comprises an immune co-stimulatory molecule. Because these molecules act on immune pathways relevant to those affected by immune checkpoint inhibitors, Applicant has concluded that using these molecules with DaRT, such as one or more of the following, will lead to similar results by positive regulation of these pathways, via stimulation rather than removal of negative regulation: A) OX40 (tavolimab, kudarolimab, GSK3174998, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, INBRX-106, PF-0451860) B) ICOS (GSK3359609, JTX-2011 / bopratelimab, MEDI-570, KY104) C) CD137 / 4-1BB (PF-05082566) D) SLAM (elotuzumab) E) CD40 (APX005M (sotigalimb), SEA-CD40, CDX-1140, MP0317) The administered immune checkpoint modulators may comprise a single drug or a combination of multiple different drugs as described above, which may be administered together or in separate sessions.

[0033] Route of Administration In some embodiments, administration of the immune checkpoint modulator to tumors and / or metastases is by systemic administration, for example orally or by intravenous (IV) injection or infusion, hi some embodiments, delivery of the immune checkpoint modulator uses a suitable method of targeted delivery.

[0034] Alternatively, or in addition, the immune checkpoint modulator is administered in situ directly to one or more identified tumors (108). In this alternative, the immune checkpoint modulator may be administered by intratumoral injection. In some embodiments, the immune checkpoint modulator is administered from seeds carrying an alpha-emitter radionuclide, but preferably, the immune checkpoint modulator is administered separately from the seeds to achieve broader coverage of tumors affected by the immune checkpoint modulator. Prior to administering the immune checkpoint modulator to a patient, the size of the tumor, tumors, and / or metastases may be estimated and the amount of immune checkpoint modulator to be administered may be selected accordingly.

[0035] timing In some embodiments, the checkpoint modulator is administered to the patient in a single session (108). Alternatively, the immune checkpoint modulator is administered in multiple sessions, perhaps at least three, at least seven, or even at least twelve sessions (108). The separate sessions may be separated from each other by at least 4 hours, 8 hours, 24 hours, 48 ​​hours, or even at least 72 hours. The multiple sessions may use the same immune checkpoint modulator. Alternatively, the different sessions use different immune checkpoint modulators. For embodiments in which the immune checkpoint modulator is administered in multiple sessions (108), the following paragraphs relate to the first session of administration unless otherwise stated.

[0036] One embodiment found to provide particularly promising results involved the first immune checkpoint inhibitor administration session 1-2 days after alpha emitter radiotherapy source insertion, and continuation of treatment for approximately 2 weeks. In a first type of embodiment, the timing of immune checkpoint regulator therapy is selected such that alpha-emitter irradiation is applied initially without the immune checkpoint regulator, when alpha-emitter irradiation is most effective, so that T cells are not induced by the immune checkpoint regulator to infiltrate the destruction area of ​​the alpha-emitter irradiation.

[0037] In this first type of embodiment, administration of the immune checkpoint modulator begins after implantation of the alpha emitter (104) for a limited buffer period (106). The buffer period may be selected to allow the immune checkpoint modulator to take effect after a predetermined percentage of the alpha emitter particles on the seeds have undergone decay. The predetermined percentage may be at least 10%, at least 20%, at least 30% or even at least 50%. The buffer period (106) between the alpha emitter radiotherapy induction (104) and the start of the immune checkpoint modulation treatment (108) is selected to allow the alpha emitter radiotherapy induction to take effect before administering the immune checkpoint modulator. For example, when the alpha emitter radiotherapy induction is induced by the insertion of an alpha emitter source, the limited period (106) is selected to be a suitable buffer period to allow upregulation of MHC1 expression at the tumor cell membrane, cytokine and DAMP secretion, and activation of APCs for the specific type of killing effect of alpha emitter radiotherapy on tumor cells. Alternatively, or in addition, the length of the period (106) is selected to be sufficient to allow time for the killed tumor cells to activate immune cells.

[0038] The buffer period (106) between implantation of the alpha emitter source (104) and the first session of administration of the immune checkpoint modulator (108) may be at least 6 hours, at least 9 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 96 hours, or even at least 120 hours. The buffer period (106) may be less than one month, less than three weeks, less than two weeks, less than ten days, less than one week, less than 120 hours, less than 96 hours, less than 72 hours, or even less than 48 hours, so that the effect of the alpha emitter radiotherapy is already activating immune cells when the immune checkpoint inhibitor is applied. The buffer period may be short enough so that the promotion of infiltration of T cells into the tumor takes effect before the tumor has a chance to recover and regenerate large scale malignant cells. In some embodiments, the limited period of time (106) is less than 30 hours, or even less than 20 hours, for example in tumors that respond more rapidly to alpha emitter radiotherapy.

[0039] In a second type of embodiment, the immune checkpoint modulator is administered after most of the radionuclides of the implanted seeds have undergone radioactive decay, which occurs within about two weeks. In a third type of embodiment, the immune checkpoint modulator is administered prior to the implantation of alpha-emitting seeds, so that the immune checkpoint modulator acts substantially throughout the duration of the radiation therapy. This type of embodiment is used, for example, in tumors of particularly malignant type, where it is best to start attacking malignant cells as soon as possible, without the delay of waiting for alpha-emitter radiotherapy seeds. The immune checkpoint modulator may be administered in this type of embodiment at least 6 hours, at least 12 hours, at least 24 hours, or even at least 48 hours, at least 72 hours, at least 96 hours, or even at least one week before the start of the alpha-emitter radiation treatment. Alternatively, the immune checkpoint modulator is administered a short time before the implantation of the alpha-emitting seeds. For example, the immune checkpoint modulator is administered less than 72 hours, less than 48 hours, less than 24 hours, less than 12 hours, or even less than 6 hours before the implantation of the seeds according to this alternative.

[0040] In some embodiments, one or more parameters of the tumor are monitored after implantation and / or activation of the alpha emitter seeds to determine the most suitable time to apply immune checkpoint modulator therapy. Monitoring may include imaging the tumor using an appropriate modality (e.g., X-ray, ultrasound, PET-CT, MRI, CT) to identify when the tumor begins to change due to activation of the alpha emitter radiotherapy. Alternatively, monitoring may include performing blood tests to identify the level of the characteristic. However, it should be noted that in some embodiments, the immune checkpoint modulator is administered before the effects of the alpha emitter radiotherapy are detectable.

[0041] Alpha emitter radiation Alpha emitter radiation treatment may be performed by insertion of seeds carrying radioactive atoms, such as radium 224 or radium 223, that release alpha-emitting atoms inside the tumor. The alpha-emitting atoms may be bound to the seeds such that the atoms do not leave the seeds, but do leave the seeds upon radionuclide decay that produces daughter radionuclides. The seeds may emit daughter radionuclide atoms at a rate of at least 0.1%, 0.5%, or even at least 1% of the number of radionuclide atoms that were bound to the seeds when originally used per 24 hours. In some embodiments, the daughter radionuclide atoms are slowly released from the seeds at a rate of less than 25%, less than 10%, less than 5%, or even less than 3% of the radionuclide atoms bound to the seeds per 24 hours.

[0042] Instead of binding alpha-emitting atoms to the seeds such that the atoms do not exit the seeds without radionuclide decay, the alpha-emitting atoms are bound to the seeds such that the atoms controllably exit the seeds at a rate of at least 0.1% per 24 hours by means other than radionuclide decay, as described, for example, in PCT Publication WO 2019 / 193464, entitled "controlled release of radionuclides," which is incorporated herein by reference. In some embodiments, the alpha emitter irradiation comprises diffuse alpha emitter radiotherapy (DaRT). Alpha emitter radiotherapy may be administered using any of the methods and / or devices described in U.S. Patent No. 8,834,837, U.S. Patent Application No. 2009 / 0136422, U.S. Provisional Patent Application No. 62 / 913,184, filed October 10, 2019, and / or PCT Publication No. WO2018 / 207105, which are incorporated herein by reference.

[0043] The alpha emitter radiation treatment may be initiated (104) by inserting into the tumor one or more seeds that contain alpha emitting atoms on the outer surface of the seed. Alternatively, alpha emitter radiotherapy is initiated by activating previously inserted seeds carrying alpha emitting atoms. In accordance with this alternative, the seeds may be inserted into the patient with a bioabsorbable coating that prevents alpha radiation and / or daughter radionuclides from leaving the seed. The bioabsorbable coating may include polylactic acid (PLA), polyglycolide (PGA) or copolymers of PLA and PGA adapted to achieve a desired resorption rate of the coating. Alternatively, or in addition, the coating includes copolylactic / glycolic acid (PLGA). The polymer of the coating may have a molecular weight in the range of 5,000 to 100,000. The material of the coating dissolves in the patient through any of the methods known in the art, such as one or more of ultrasonic energy, reaction with body temperature and / or reaction with bodily fluids. Further discussion of bioabsorbable polymers that may be used in accordance with embodiments of the present invention, after adjustment to a desired resorption rate, is described in U.S. Patent 8,821,364 and U.S. Patent Application 2002 / 0055667, which are incorporated herein by reference. In some embodiments, initiation (108) involves applying a stimulus that dissolves the coating to allow alpha irradiation and / or daughter radionuclides to exit the seed. In other embodiments, initiation (108) is accomplished by dissolving the coating upon contact with tumor tissue without further physician initiation.

[0044] Alpha emitter radiotherapy may be administered to the patient for at least 24 hours, at least 5 days, at least 10 days, or even at least 14 days. Spreading out the destruction of tumor cells over such a period allows time for the immune checkpoint modulator to help the patient's immune system adapt and participate in the destruction of the remainder of the tumor and / or metastases. In some embodiments, the seeds are removed from the patient after a designated treatment period. For example, the seeds may be removed during surgery for tumor removal. Alternatively, the seeds are not removed. In some embodiments, the seeds comprise a biodegradable material.

[0045] Further Actions Providing supportive treatment (114), in some embodiments, includes one or more treatments to counteract undesirable side effects of radiation therapy and / or immune checkpoint inhibitors. The supportive treatment may include one or more treatments to counteract accelerated tissue repair induced by alpha emitter radiation therapy, which supports remaining tumor cells and promotes tumor recurrence. Alternatively, or in addition, the supportive treatment includes one or more anti-inflammatory treatments to downregulate inflammation following tissue damage caused by radiation therapy and / or immune checkpoint inhibitors. In some embodiments, the supportive treatment includes one or more treatments to prevent DNA repair, to impede the patient's body's attempts to repair the DNA of tumor cells damaged by radiation therapy. In other embodiments, the supportive treatment includes one or more treatments to stimulate pathogen attack.

[0046] In some embodiments, the supportive treatment comprises one or more immunostimulants, such as immune enhancers, cytokines, RIG-1 agonists, STING agonists, and / or TLR agonists. In some embodiments, supportive treatment includes treatment that has two or even three of the above-listed effects. In some embodiments, the supportive treatment includes any of the treatments known in the art for supportive immune modulation, for example, inhibition of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) and / or Treg inhibitors (e.g., cyclophosphamide) and / or activation of the TLR pathway (TLR agonists). MDSC inhibitors include, for example, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors such as epacadostat, TGFb inhibitors such as galunisertib, PDE5 inhibitors such as sildenafil, and / or Cox2 inhibitors such as etodolac.

[0047] Alternatively, or in addition, supportive treatment may include administering one or more pattern recognition receptors and / or agonists, such as TLR7, 8 (e.g., MEDI9197, imiquimod), TLR9 (e.g., MGN1703, SD-101, TLR4, GSK1795091, G100, GLA-SE), TLR3 (e.g., Poly-ICLC) and / or STING (e.g., MIW815). Alternatively or in addition, the further treatment comprises administering a type of DNA repair inhibitor that has been shown to enhance, not affect, or only minimally impede the immune response induced by alpha-emitter radiotherapy. In some embodiments, the administered DNA repair inhibitor includes an ATR inhibitor, such as beruzosertib, AZD6738, and / or NU6027. Alternatively or in addition, the DNA repair inhibitor includes an ATM / ATR inhibitor, such as KU-55933, KU-60019, and / or EPT-46464, a DNA-PK inhibitor (e.g., 6-nitroveratraldehyde, NU7441), a Wee1 inhibitor (e.g., adavosertib), an Hsp90 inhibitor (e.g., tanespimycin), and / or a PARP inhibitor (e.g., olaparib, talazoparib).

[0048] Alternatively, or in addition, the further treatment comprises an anti-angiogenic factor of a type known to augment, not affect, or only minimally impede the immune response induced by alpha emitter radiotherapy and / or immune checkpoint inhibition, hi some embodiments, the further treatment comprises bevacizumab or an iMiD, such as pomalidomide, thalidomide, lenalidomide, and / or apremilast. Additionally or alternatively, the further treatment comprises alpha irradiation and / or immune checkpoint inhibition and / or local or systemic chemotherapy treatment of a type known not to interfere with the immune response to alpha-emitter irradiation. The chemotherapy treatment may comprise one or more of cyclophosphamide (CP), doxorubicin, gemcitabine, oxaliplatin and / or cisplatin.

[0049] In some embodiments, the further treatment may alternatively or additionally include an anti-inflammatory drug such as an NSAID, for example a Cox2 inhibitor. Additionally, alternatively, or in addition, the further treatment includes administering one or more epigenetic drugs, such as DNMT inhibitors (e.g., decitabine, azacytidine, guadecitabine) and / or HDAC inhibitors (e.g., entinostat, vorinostat). In some embodiments, the further treatment is provided while the alpha emitter radiation is being applied (114). In other embodiments, the further treatment is provided after the alpha emitter radiation therapy is completed, e.g., after a majority of the radionuclides in the seeds have undergone nuclear reactions and / or after the seeds have been removed from the patient (114). In yet other embodiments, the further treatment is provided prior to the alpha emitter radiation therapy (114). In some embodiments, the supportive treatment is provided within less than 72 hours, less than 48 hours, or even less than 32 hours of one of the immune checkpoint modulation sessions and / or radiation therapy treatments. The timing of providing the further treatment may be selected according to the particular type of further treatment. Further treatments may be provided depending on the tumor type (114).

[0050] Treatment kit Figure 2 is a schematic diagram of a kit 200 for treatment of a patient according to the method of Figure 1. The kit 200 includes a sterile package 202 containing one or more alpha emitter radiotherapy seeds 204 for insertion into a tumor and one or more doses 216 of a drug / drugs for immune checkpoint inhibition. The seeds 204 may be provided in a vial or other casing 206 that prevents radiation from exiting the casing. In some embodiments, the casing is filled with a strong viscous liquid, such as glycerin, to prevent leakage of radiation from the casing 206, as described in PCT Application PCT / IB2019 / 051834, entitled "Radiotherapy Seeds and Applicators," the disclosure of which is incorporated herein by reference. In some embodiments, the kit 200 further includes a seed applicator 208 that is used to introduce the seeds 204 into a patient, as described in PCT Application PCT / IB2019 / 051834. The applicator 208 may be provided with one or more seeds 204 preloaded therein. Pursuant to this option, separate seeds 204 in the casing 206 are provided if a greater number of preloaded seeds are required. Alternatively, the seeds 204 in the casing 206 are not provided in the kit 200, and only the seeds in the applicator 208 are included in the kit 200.

[0051] As shown, doses 216 of immune checkpoint inhibitor are provided preloaded in one or more needles 210. In other embodiments, doses 216 are provided in one or more containers or vials and needles are provided separately within sterile packaging 202 or are not provided in kit 200 at all.

[0052] In some embodiments, the kit 200 further comprises one or more drugs 220 required for the supportive immunomodulatory treatment (114). In some embodiments, the kit 200 includes multiple separate compartments, separated by appropriate isolation, for materials requiring storage at different temperatures. For example, a first compartment may contain dry ice to keep the material in the first compartment at about -20°C, and a second compartment contains ice to maintain the material in the second compartment at about 4°C.

[0053] The radiotherapy seeds 204 may include a metallic or non-metallic support configured for insertion into the subject's body. The seeds 204 further include radionuclide atoms, such as, for example, radium-224, on an outer surface, as described, for example, in U.S. Patent 8,894,969, which is incorporated herein by reference. The radionuclide atoms are generally bound to the seeds such that the radionuclide atoms do not leave the support, but their daughter radionuclides may leave the seeds 204 upon radioactive decay due to recoil from the decay. The percentage of daughter radionuclides that leave the support upon decay is called the desorption probability. Binding of the radiotherapy atoms to the seeds is achieved, in some embodiments, by a heat treatment. Alternatively, or in addition, a coating covers the seeds and atoms to prevent release of the radionuclide atoms upon radioactive decay and / or to regulate the rate of release of the daughter radionuclides. The daughter radionuclide may pass through the coating and out of the seed 204 due to recoil, or recoil may carry the daughter radionuclide to the coating from where it may be released by diffusion.

[0054] Seed 204, in some embodiments, includes a seed for complete implantation within a patient's tumor and may have any suitable shape, such as a rod or plate. Instead of being completely implanted, seed 204 may be only partially implanted within the patient and may be part of a needle, wire, the tip of an endoscope, the tip of a laparoscope, or any other suitable probe. In some embodiments, the seeds 204 are cylindrical and have a length of at least 1 millimeter, at least 2 millimeters, or even at least 5 millimeters. The seeds 204 may have a length between 5 and 60 mm (millimeters). The seeds 204 may have a diameter of 0.7 to 1 mm, although in some cases larger or smaller diameter sources are used. For small spacing treatment designs in particular, the seeds 204 may have a diameter of less than 0.7 mm, less than 0.5 mm, less than 0.4 mm, or even 0.3 mm or less.

[0055] experiment FIG. 3 shows the results of an experiment conducted by the applicant to test the method of FIG. 1. In the experiment, Blb / c mice bearing SCC tumors were treated as follows: the aPD-1 group received an inactive source and mouse anti-PD-1 intraperitoneally at a dose of 10 mg / kg on days 2, 6, 9, and 13. The DaRT group received 6.5 mm DaRT seeds loaded with 75 kBq Ra-224 on day 0 and a control antibody. The inactive (control) group received an inactive source in addition to the control antibody. The DaRT+aPD-1 group received 6.5 mm DaRT seeds loaded with 75 kBq Ra-224 on day 0 and anti-PD1 intraperitoneally at a dose of 10 mg / kg on days 2, 6, 9, and 13. No effect on tumor growth was observed for aPD-1 treatment compared to the control. DaRT significantly reduced tumor growth compared to the control. The combined treatment inhibited tumor growth compared to the control and DaRT groups. This indicates that beyond the obvious killing effect of DaRT by diffusion of alpha-emitting atoms, another type of killing (likely T cell-mediated) affects tumor size in the presence of checkpoint blockade. This suggests that DaRT activates the immune system in response to checkpoint blockade. This experiment was repeated with similar results (aPD-1 doses given on days 2, 4, 8, and 12).

[0056] Figure 4 shows the results of an experiment conducted by applicant to test the method of Figure 1. In the experiment, C57BL / 6 mice bearing pancreatic ductal adenocarcinoma (PDAC) tumors were treated as follows: the aPD-1 group received an inactive source and mouse anti-PD-1 administered intraperitoneally at a dose of 10 mg / kg on days 1, 4, 7, 10, and 14. The DaRT group received 6.5 mm DaRT seeds loaded with 80 kBq Ra-224 on day 0 and a control antibody. The inactive (control) group received an inactive source and a control antibody. The DaRT+aPD-1 group received 6.5 mm DaRT seeds loaded with 80 kBq Ra-224 on day 0 and anti-PD1 intraperitoneally at a dose of 10 mg / kg on days 1, 4, 7, 10, and 14. The GEM group received gemcitabine (GEM) intraperitoneally at a dose of 60 mg / Kg on days 0, 3, 7, 10, 14, and 17.

[0057] On day 14, only the combination group showed a significant reduction in tumor volume compared to the control group. The combination treatment inhibited tumor growth in the same trend as observed for SCC tumors compared to DaRT (Figure 3). Notably, this experiment was terminated at a relatively early time point, day 20.

[0058] Figure 5 shows the results of an experiment conducted by the applicant to test the method of Figure 1. In the experiment, Blb / c mice bearing SCC tumors were treated as follows: the aPD-1 group received an inactive source and intraperitoneally mouse anti-PD-1 at a dose of 10 mg / kg on days 2, 5, 8, 11, and 14. The DaRT group received 6.5 mm DaRT seeds loaded with 75 kBq Ra-224 on day 0 and a control antibody. The inactive (control) group received an inactive source and a control antibody. The DaRT+aPD-1 group received 6.5 mm DaRT seeds loaded with 75 kBq Ra-224 on day 0 and intraperitoneally anti-PD1 at a dose of 10 mg / kg on days 2, 5, 8, 11, and 14. In this experiment, FACS analysis of the % of intratumoral activated dendritic cells (DCs) was used. On day 7, tumors were excised and enzymatically dissociated with collagenase (1.5 mg / ml), hyaluronidase (0.75 mg / ml) and DNase (0.1 mg / ml). The resulting single cell suspension was incubated for 30 min at 4°C with the following antibody mixture: CD11c-PE-cy7, CD86-BV650, CD11b-BB515 (FITC), Ly6G-BV421, Ly6C-PE-CF594 (PI), CD45-APC, MHC class II-PE. After two washes in FACS buffer (PBS + 2% fetal bovine serum + 5 mM EDTA), samples were read on a Stratedigm S1000EXi FACS instrument. Gating strategy: DCs were identified as CD45 + , CD11c and MHC-II double positive cells. CD86 was stained as an activation marker. Analysis revealed an increase in the percentage of activated dendritic cells (DCs) early in DaRT compared to the inactive group. These data suggest that alpha emitter-induced cell death may lead to enhanced identification of new tumor antigens by promoting peptide presentation and DAMP signals leading to DC activation and subsequent T cell recruitment and activation. This supports that DaRT activates the immune system in response to checkpoint blockade.

[0059] Figure 6 shows the results of an experiment conducted by the applicant to test the method of Figure 1. In the experiment, the same procedures as described with respect to Figure 5 were used. The experiment shows the effect of the combination of DaRT and anti-PD1 on T lymphocyte tumor infiltration and functionality, assessed by immunohistochemical staining of CD3, CD8, and Granzyme B molecules. The analysis was performed on tumors excised 16 days after DaRT insertion (two independent experiments) that were subjected to immunohistochemistry. Briefly, tumors were frozen in OCT and cryosectioned at a thickness of 5 μm. The tissue sections were then fixed in acetone for 20 minutes, air-dried, and stained with a Leica Bond III machine. Blocking was performed with 5% normal goat serum (NGS), 5% bovine serum albumin (BSA) in PBS for 1 hour. The primary antibodies used were rabbit anti-mouse CD3 (abcam AB-ab16669) diluted 1:400, rabbit anti-mouse CD8 alpha (abcam ab217344) 1:500, rabbit anti-mouse granzyme B (abcam ab255598) 1:200 diluted in blocking solution. The secondary antibody was rabbit-HRP conjugate provided as part of the Leica Bond III kit with DAB substrate and hematoxylin. The results clearly show the synergistic effect of anti-PD1 immune checkpoint inhibitors together with DaRT in increasing CD3+, CD8+, and granzyme B (Figures 6A, 6B, and 6C, respectively) density in the tumors. Representative pictures of aPD-1 alone and DaRT+aPD-1 tumors (Figure 6D) show a clear increase in CD3 T cell content in the dual combination therapy. Collectively, these results indicate a stronger antitumor immune response supporting a better outcome in combination therapy compared with monotherapy.

[0060] Figure 7 shows the results of an experiment conducted by the applicant to test the method of Figure 1. In the experiment, the same procedures and the same FACS staining protocol were used as described in Figure 5. Gating strategy: PMN-MDSC were identified by CD45 + , CD11b + , Ly6G + Ly6Cl owDaRT-injection was identified as a cell population. FACS analysis of spleens 16 days after DaRT insertion from two independent experiments revealed a decrease in the % of polymorphonuclear myeloid-derived suppressor cells in the DaRT, aPD-1 alone and combination (DaRT + anti-PD1) groups when compared to inactive. Interestingly, the combination therapy shows a significant decrease in peripheral MDSCs when compared to aPD-1 alone. As peripheral MDSCs correlate with prognosis, this result supports a possible therapeutic advantage in using both therapies together.

[0061] conclusion It will be appreciated that the methods and apparatus described above are to be construed to include apparatus for performing the methods and methods for using the apparatus. It should be understood that features and / or steps described with respect to one embodiment may be used with other embodiments, and that not all embodiments of the invention will have all of the features and / or steps shown in a particular figure or described with respect to one of the particular embodiments. Tasks do not necessarily have to be performed in the exact order described.

[0062] It should be noted that some of the above-described embodiments may include details of structures, acts, or structures and acts that may not be essential to the present invention and are described as examples. The structures and acts described herein may be replaced with equivalents that perform the same function, even if the structures or acts are different, as is known in the art. The above-described embodiments are cited as examples, and the present invention is not limited to those specifically shown and described above. Rather, the scope of the present invention includes both combinations and subcombinations of the various features described above, as well as variations and modifications thereof that may occur to a person skilled in the art upon reading the above description and that are not disclosed in the prior art. Thus, the scope of the present invention is limited only by the elements and limitations as used in the claims, and the terms "comprise", "include", "have" and variations thereof, when used in the claims, shall mean "including, but not necessarily limited to".

Claims

**Claim 1**: A medicament for treating a patient's tumor, comprising a substance that modulates immune checkpoints, wherein the administration pattern of the medicament comprises administering a therapeutically effective amount of the substance to the tumor in one or more sessions, and implanting into the tumor seeds carrying radium-224 for alpha-radiation therapy of the tumor within less than two weeks after administering the substance. **Claim 2** The medicament according to claim 1, wherein the administration pattern of the medicament comprises starting to administer the substance within less than five days after implanting the seeds. **Claim 3** The medicament according to claim 1, wherein the administration pattern of the medicament comprises starting to administer the substance at least 12 hours after implanting the seeds. **Claim 4** The medicament according to claim 1, wherein the administration pattern of the medicament comprises starting to administer the substance at least 72 hours after implanting the seeds. **Claim 5** The medicament according to claim 1, wherein the administration pattern of the medicament comprises starting to administer the substance at least 12 hours before implanting the seeds. **Claim 6** The medicament according to claim 1, wherein the administration pattern of the medicament comprises starting to administer the substance at least 72 hours before implanting the seeds. **Claim 7**: The medicament according to claim 1, wherein the substance comprises an anti-angiogenic agent. **Claim 8**: The medicament according to claim 1, wherein the substance comprises an immune checkpoint inhibitor. **Claim 9**: The medicament according to claim 1, wherein the substance comprises a small molecule inhibitor. **Claim 10**: The medicament according to claim 1, wherein the substance comprises nivolumab, pembrolizumab, semiprimab, tripalimab, or sintilimab. **Claim 11**: The medicament according to claim 1, wherein the substance comprises atezolizumab, avelumab, or durvalumab. **Claim 12**: The medicament according to claim 1, wherein the substance comprises a CTLA4 checkpoint inhibitor. **Claim 13**: The medicament according to claim 1, wherein the substance comprises relatlimab. **Claim 14**: The medicament according to claim 1, wherein the substance comprises LY3321367. **Claim 15**: The medicament according to claim 1, wherein the substance comprises TIM-3, TIGIT, or tiragolumab. **Claim 16**: The medicament according to claim 1, wherein the substance comprises epacadostat. **Claim 17**: The medicament according to claim 1, wherein the substance comprises enoblituzumab. **Claim 18**: The medicament according to claim 1, wherein the substance comprises HLX23 or ORIC-533. **Claim 19**: The medicament according to claim 1, wherein the substance comprises monalizumab.

20. The medicament according to claim 1, wherein the substance comprises pexidartinib or lankumizumab.

21. The medicament according to claim 1, wherein the substance comprises pepinemab.

22. The medicament according to claim 1, wherein the substance comprises enapotamab.

23. The medicament according to claim 1, wherein the substance comprises tabalumab or cudarolimab.

24. The medicament according to claim 1, wherein the substance comprises boptelimumab, sotigalimab or elotuzumab.

25. The medicament according to claim 1, wherein the substance comprises an immune checkpoint bispecific antibody.

26. The medicament according to claim 1, wherein the substance comprises an LAG3 checkpoint inhibitor.

27. The medicament according to claim 1, wherein the substance comprises a PD-1 checkpoint inhibitor.

28. The medicament according to claim 1, wherein the substance comprises a PDL-1 checkpoint inhibitor.

29. The seed is: A support having a length of at least 1 millimeter; and When the seed is transplanted into a tumor, no more than 20% of the radium-224 atoms will exit the support to the tumor within 24 hours without decay, but at least 5% of the daughter radionuclides of the radium-224 atoms will exit the support during decay, and the radium-224 atoms are bound to the support. The medicament according to any one of claims 1 to 28, comprising

30. The medicament according to any one of claims 1 to 28, wherein the administration pattern of the medicament comprises administering the substance at least 24 hours after the transplantation of the seed.

31. A kit for treating a patient, comprising At least one source for at least partially introducing into the body of the subject, on which an alpha-emitting atom is mounted; At least one immune checkpoint regulator; and A package containing at least one source and at least one immune checkpoint regulator The kit comprising

32. An alpha-emitting device designed for use in alpha-emitting radiotherapy treatment of a patient's tumor, wherein the alpha-emitting radiotherapy treatment pattern comprises treating the tumor with an alpha-emitting device, and then, within less than 6 weeks after the start of the alpha-emitting radiotherapy, administering a therapeutically effective amount of an immune checkpoint regulator in one or more sessions.

33. The alpha emitter radiotherapy treatment pattern includes treating a tumor with an alpha emitter device and then, within less than two weeks after the start of alpha emitter radiotherapy, administering a therapeutically effective amount of an immune checkpoint modulator in one or more sessions, the alpha emitter device according to claim 32.

34. An alpha emitter device designed for use in alpha emitter radiotherapy treatment of a population having tumors treated with a therapeutically effective amount of an immune checkpoint modulator in one or more sessions within less than six weeks after the start of alpha emitter radiotherapy.

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