Intratumoral alpha-emitter radiation and activation of cytoplasmatic sensors for intracellular pathogen

Abstract

To provide a combination of tumor therapy, specifically intratumoral alpha-emitter radiation and activation of cytoplasmatic sensors for intracellular pathogen.SOLUTION: The present invention provides a method of treating a patient with a tumor, and kits (200) for such treatment. The method includes the steps for: administering, to the patient, a substance (204) which activates cytoplasmatic sensors for intracellular pathogen in the tumor; and treating the tumor with intra-tumoral alpha-emitter radiotherapy within two weeks of administering the substance which activates cytoplasmatic sensors for intracellular pathogen in the tumor.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates generally to tumor therapy, and in particular to the combination of intratumoral alpha emitter emission and activation of a cytoplasmic sensor for intracellular pathogens (hereinafter "ACSIP"). [Background technology]

[0002] Cancer is the leading cause of death in many countries around the world, and as such, vast resources are dedicated to treating tumors such as cancer, implying a wide variety of such treatments.

[0003] One class of tumor therapy is tumor resection to kill tumor cells in vivo. In addition to killing cells in vivo, in some cases, resection induces an antitumor immune response to eliminate remaining distant tumor cells. This occurs due to the dispersion of tumor antigens and danger signals released by dead and / or dying tumor cells. Tumor antigens are captured by antigen-presenting cells, which then present them to T cells via the cross-presentation pathway, as described, for example, in Masafumi Nakayama, "Antigen presentation by MHC-clad cells," Frontiers in immunology 5 (2015):672 (Non-Patent Document 1).

[0004] Several ablation methods have been proposed, including thermal, microwave, laser, electrical, photodynamic, chemical (e.g., using reactive oxygen species (ROS)), and radioablation. Radioablation can be applied externally (e.g., external beam radiotherapy) or internally (e.g., brachytherapy) and can include various types of radiation, such as alpha, beta, and gamma radiation. A discussion of these methods can be found, for example, in Keisari, Yona, "Tumor abolition and antitumor immune stimulation by physicochemical tumor ablation," Front Biosci 22 (2017): 310-347 (Non-Patent Document 2). The ablation method used for a particular patient is generally selected according to the type of tumor, its location, its stage, and / or other parameters of the tumor.

[0005] Another class of tumor therapy, called immunotherapy, involves enhancing the patient's immune response against tumor cells. Many immunotherapy methods have been proposed, including checkpoint inhibitors, Toll-like receptor (TLR) agonists (e.g., CpG), local gene therapy, cytokine therapy, antibodies against specific protein targets, CAR-T cell therapy, dendritic cell vaccines, adoptive transfer of tumor-infiltrating lymphocytes and oncolytic virus therapy. These methods are discussed, for example, in Papaioannou, Nikos E. et al., "Harnessing the immune system to improve cancer treatment," Annals of Translational Medicine 4.14 (2016) (Non-Patent Document 3). Immunotherapeutic strategies may include local therapies, as described in Aznar, M. Angela et al., "Intratumoral delivery of immunotherapy acts locally, thinks globally," Journal of Immunology 198.1 (2017):31-39 (Non-Patent Document 4). In general, the particular method used for each patient is selected depending on the type of tumor or its stage. Multiple combinations of the above treatment types have been tested in preclinical and clinical trials, as described, for example, in Table 1 of Aznar's reference above.

[0006] Salazar, U.S. Patent Application Publication No. 2009 / 0088401, describes a method for autovaccination that involves the induction of immunogenic cell death at one or more target tumor sites, followed by injection of dsRNA at the same tumor sites. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2009 / 0088401 [Non-patent literature]

[0008] [Non-Patent Document 1] Masafumi Nakayama, "Antigen presentation by cells bearing MHC" Frontiers in immunology 5 (2015): 672 [Non-Patent Document 2] Keisari, Yona "Tumor abolition and antitumor immune stimulation by physicochemical tumor ablation" Front Biosci 22(2017):310-347 [Non-Patent Document 3] Papaioannou, Nikos E. et al., "Harnessing the immune system to improve cancer treatment," Journal of Translational Medicine 4.14 (2016) [Non-Patent Document 4] Aznar, M. Angela et al. "Intratumoral delivery of immunotherapy: act locally, think globally" Journal of Immunology 198.1(2017):31-39 Summary of the Invention

[0009] One aspect of some embodiments of the present invention relates to tumor treatment based on the synergy between activation of cytoplasmic sensors for intracellular pathogens (ACSIP) and intratumoral alpha emitter radiotherapy.

[0010] According to an embodiment of the invention, there is provided a method of treating a patient having a tumor, comprising administering to the patient a substance that activates a cytoplasmic sensor for an intracellular pathogen in the tumor; and treating the tumor with intratumoral alpha emitter radiotherapy within two weeks of administering the substance that activates a cytoplasmic sensor for an intracellular pathogen in the tumor.

[0011] Optionally, administering the substance comprises administering an agent that induces intracellular production of a molecule that stimulates a cytoplasmic sensor for intracellular pathogens. Optionally, administering the agent comprises administering a DNA methyltransferase (DNMT) inhibitor, such as decitabine, azacytidine, and / or guadecitabine. Alternatively or additionally, administering the agent comprises administering a histone deacetylase (HDAC) inhibitor, such as entinostat and / or vorinostat. Optionally, administering the substance comprises administering the pathogen mimic together with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic. Optionally, administering the pathogen mimic together with a delivery agent comprises administering a single dose of the intracellular pathogen mimic in a single session or in two sessions. In some embodiments, administering the pathogen mimic together with a delivery agent comprises administering at least three doses of the intracellular pathogen mimic in each separate session. Optionally, administering the substance comprises administering the substance together with a delivery agent suitable for targeted delivery. Optionally, the delivery agent comprises a lipid-based delivery agent, a polymer-based delivery agent, a cationic polymer complex or cationic lipid, a liposome or lipoplex, a polymer micelle and / or a dendrimer delivery agent. Alternatively or additionally, the delivery agent comprises a solid nanoparticle, for example a solid lipid nanoparticle or a solid polymer nanoparticle. Still alternatively or additionally, the delivery agent comprises a metal-based nanoparticle system. In some embodiments, the delivery agent comprises polyethyleneimine (PEI).

[0012] In some embodiments, administering a pathogen mimic comprises administering a bacterial mimic and / or administering a viral mimic. Optionally administering a viral mimic comprises administering double-stranded RNA (dsRNA). Optionally administering a dsRNA comprises administering polyIC. In some embodiments, polyIC is delivered with polyethylenimine (PEI), for example by administering BO-112. Alternatively or additionally, administering a dsRNA comprises administering 5'ppp-dsRNA, 3p-hpRNA, poly(dA:dT), poly-ICLC, poly(A:U) and / or CpG-rich RNA. Optionally administering a pathogen mimic comprises administering a pathogen mimic that is recognized as non-self by an intracellular receptor. Optionally administering a substance that activates a cytoplasmic sensor for an intracellular pathogen comprises administering a plurality of different substances that activate a cytoplasmic sensor for an intracellular pathogen.

[0013] Optionally, administering a plurality of different agents that activate a cytoplasmic sensor for an intracellular pathogen includes administering a first agent that induces intracellular production of a molecule that stimulates a cytoplasmic sensor for an intracellular pathogen, and a second agent that has a pathogen mimic with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic. Optionally, the method further includes administering to the patient a supportive treatment of cyclophosphamide. Optionally, treating the tumor with alpha emitter radiotherapy includes inserting a brachytherapy source carrying an alpha emitting radionuclide into the tumor. Optionally, the brachytherapy source, when inserted into the tumor, releases the alpha emitting radionuclide at a rate less than 25% of the alpha emitting radionuclide on the source per day. Optionally, the brachytherapy source includes a radium source. Optionally, treating the tumor with alpha emitter radiotherapy is performed only after completing administration of the agents. Optionally, administering the agent includes administering the agent with nanoghosts that target the agent to the tumor. Optionally, administering the substance comprises administering the one or more substances in at least two separate sessions separated by at least 12 hours. Optionally, administering the one or more substances comprises administering in at least three separate sessions separated by at least 20 hours. Optionally, the method comprises identifying a primary tumor in the patient and administering the substance comprises administering the substance to the identified primary tumor. Optionally, treating the tumor with alpha emitter radiation therapy comprises treating with alpha emitter radiation therapy within 80 hours of administering the last dose of the substance. Optionally, treating the tumor with alpha emitter radiation therapy comprises treating with alpha emitter radiation therapy within 60 hours of administering the last dose of the substance. Optionally, treating the tumor with alpha emitter radiation therapy comprises treating with alpha emitter radiation therapy at least 12 hours after administering the last dose of the substance.

[0014] Optionally, the method further comprises administering to the patient a supportive therapy that counteracts the accelerated tissue repair induced by alpha-emitter radiotherapy, for example, administering to the patient a supportive therapy that downregulates inflammation and / or administering to the patient a supportive therapy that downregulates checkpoint expression. In some embodiments, the supportive therapy comprises cyclophosphamide, pomalidomide, and / or beruzosertib. Alternatively or additionally, the supportive therapy comprises checkpoint blockade. Optionally, the supportive therapy comprises anti-PD-1, anti-PD-L1, and / or anti-CTLA4. In some embodiments, the supportive therapy comprises one or more TLR agonists, one or more MSDC inhibitors, one or more Treg inhibitors, one or more DNA repair inhibitors, and / or one or more anti-angiogenic factors. In some embodiments, the method further comprises performing surgery to remove the tumor at least one week after starting treatment of the tumor with alpha-emitter radiotherapy. Optionally, treating the tumor with intratumoral alpha-emitter radiation therapy includes treating the tumor with intratumoral alpha-emitter radiation therapy within one week of administering a substance that activates a cytoplasmic sensor for an intracellular pathogen in the tumor.

[0015] According to one embodiment of the present invention, a kit is provided for treatment of a patient, comprising: at least one radiation source for at least partial introduction into a body of a subject and having alpha-emitting atoms attached thereto; at least one substance that activates a cytoplasmic sensor for an intracellular pathogen; and a sterile package containing the at least one radiation source and the at least one substance.

[0016] Optionally, the alpha-emitting atoms are attached to the source in a manner that allows for controllable release of the alpha-emitting atoms or their daughter radionuclides from the source. Optionally, the alpha-emitting atoms include radium atoms. Optionally, the at least one substance includes a DNA methyltransferase (DNMT) inhibitor. Optionally, the DNA methyltransferase (DNMT) inhibitor includes decitabine. Optionally, the at least one substance further includes a pathogen mimic with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic. Optionally, the at least one substance includes a pathogen mimic with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic. Optionally, the sterile package further includes a supportive therapeutic agent that combats accelerated tissue repair induced by alpha-emitter radiotherapy. Optionally, the supportive therapeutic agent includes an agent that downregulates inflammation.

[0017] According to further embodiments of the invention, polyIC is further provided in the manufacture of a medicament for administration to a tumor in a patient. PEI The use of the above, wherein the administration pattern of the drug is a therapeutically effective amount of polyIC PEI into the tumor in one or more sessions, followed by polyIC PEI polyIC, which involves treating the tumor with intratumoral alpha-emitter radiotherapy within one week of administration PEI The use of

[0018] According to an embodiment of the present invention there is further provided the use of a DNA methyltransferase (DNMT) inhibitor in the manufacture of a medicament for administration to a tumour in a patient, wherein the administration pattern of the medicament comprises administering a therapeutically effective amount of the DNMT inhibitor.

[0019] The various options and alternatives described above can be used in the alternative or together in any suitable combination, unless the options are specifically contradictory. [Brief description of the drawings]

[0020] [Figure 1]1 is a flow chart of a treatment method according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram of a combined alpha emission and ACSIP kit according to one embodiment of the present invention. [Diagram 3] 1 is a graph showing the results of an experiment testing the effect of combined treatment of alpha radiation and ACSIP on breast cancer tumorigenesis, according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] One aspect of some embodiments of the present invention relates to a combination tumor treatment that includes activating a cytoplasmic sensor for intracellular pathogens (ACSIP) in a patient's tumor, and applying alpha-emitter radiation to the tumor 12-90 hours after the treatment that induces the ACSIP. Applicants have found that the combination of a specific immunotherapy that induces ACSIP followed by a specific tumor resection that applies alpha-emitter radiation has a substantially greater therapeutic effect than either treatment separately.

[0022] The applicants of the present application believe that ACSIP, which increases the expression of MHC1 molecules on tumor cells, also increases the likelihood of presentation of not yet presented tumor antigens by tumor cells prior to cell death induced by intratumoral alpha emitter radiation. Induction of tumor cell death by alpha emitter radiation following presentation of a broad range of tumor antigens promotes recruitment and activation of antigen-presenting cells by releasing damage-associated molecular pattern (DAMP) signals. Because antigen-presenting cells activate specific T cell responses, more antigen-specific T cells are activated to recognize a broader range of tumor antigens, resulting in stronger and longer-lasting systemic and specific adaptive immune responses against a broader range of tumor antigens. In addition, the death of tumor cells containing factors that induce ACSIP also leads to the release of pathogen-associated molecular patterns (PAMPs), and cytokines that promote recruitment and activation of immune responses. Together, alpha radiation and ACSIP induction may generate a snowball effect that increases tumor cell killing, which increases the immune response, which further increases tumor cell killing, and so on. The positive results of combining alpha radiation therapy with ACSIP may be due to the slow release of alpha-emitting atoms. This distributes the effects of radiation therapy over time, resulting in a synergistic effect with ACSIP. ACSIP delivered prior to radiation therapy may decondense the DNA of tumor cells, making them more sensitive to radiation therapy. Thus, combining alpha-emitter radiation with induction of ACSIP results in significant tumor kill, both locally and systemically, in a minimal period of time, with minimal adverse effects and increased efficiency.

[0023] In some embodiments, the induction of ACSIP is carried out by administering a pathogen mimic to the cytoplasm of tumor cells using a delivery agent suitable for cytoplasmic delivery.Instead of the intracytoplasmic administration of a pathogen mimic, the induction of ACSIP is achieved by stimulating endogenous intracellular pathogens and / or activating the promoter of intracytoplasmic pathogen sensor of tumor cells, for example, by administering a DNA methyltransferase (DNMT) inhibitor such as decitabine.

[0024] (Treatment method) 1 is a flow chart of a treatment method 100, according to an embodiment of the invention. In addition to identifying a patient's tumor (102), the treatment method 100 begins with inducing ACSIP in the patient in one or more sessions (104). A limited period of time (106) following ACSIP induction (104), alpha emitter radiotherapy (also referred to herein as alpha emitter radiotherapy) of the tumor is initiated (108).

[0025] In some embodiments, one or more supportive immunomodulatory therapies are provided prior to and / or during induction of ACSIP while alpha-emitter radiation therapy is provided and / or after alpha-emitter radiation therapy is completed (114). In some embodiments, after alpha-emitter radiation therapy is completed, the effectiveness of the treatment is evaluated (110). In some embodiments, the evaluation is followed by surgery (112) 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 radiation therapy. Although surgery to remove the cancerous tumor is generally performed as soon as possible, the applicant has discovered that after applying the combined ACSIP and radiation therapy treatment, it is better to wait until the treatment has taken effect before removing the tumor. Alternatively, surgery is performed at another appropriate time, or not at all if deemed unnecessary or infeasible. Further alternatively or additionally, no evaluation is performed.

[0026] (Tumor type) The treatment method 100 can be used to treat any tumor type, including cancerous tumors, benign neoplasms, in vivo neoplasms (pre-malignant), malignant neoplasms (cancer), and neoplasms of uncertain or unknown behavior. In some embodiments, the method of FIG. 1 is used to treat relatively solid tumors, such as breast cancer, kidney cancer, pancreatic cancer, skin cancer, head and neck cancer, colon cancer, ovarian cancer, bladder cancer, and prostate cancer. In other embodiments, the method of FIG. 1 is used to treat non-solid tumors. The method of FIG. 1 can be used for both primary and secondary growths.

[0027] Exemplary tumors that can be treated with the method of FIG. 1 include tumors of the digestive tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small intestine cancer and / or large intestine cancer, esophageal cancer, tyrosinephrine with esophageal cancer, gastric carcinoma, pancreatic carcinoma, pancreatic endocrine tumor), endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, biliary tract tumor, prostate cancer, prostate adenocarcinoma, renal 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, trophoblast tumor, testicular germ cell tumor, immature teratoma of the ovary, uterine, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental site trophoblast tumor, epithelial adult tumor, ovarian cancer, serous ovarian cancer, ovarian sex cord tumor, cervical cancer, small cell and non-small cell lung cancer, nasopharyngeal, breast carcinoma (e.g., ductal carcinoma, invasive intraductal carcinoma, breast cancer type 3, breast cancer type 4, breast cancer type 5, breast cancer type 6, breast cancer type 7, breast cancer type 8, breast cancer type 9, breast cancer type 10, breast cancer type 11, breast cancer type 12, breast cancer type 13, breast cancer type 14, breast cancer type 15, breast cancer type 16, breast cancer type 17, breast cancer type 18, breast cancer type 19, breast cancer type 20, breast cancer type 21, breast cancer type 22, breast cancer type 23, breast cancer type 24, breast cancer type 25, breast cancer type 26, breast cancer type 27, breast cancer type 28, breast cancer type 29, breast cancer type 30, breast cancer type 31, breast cancer type 32, breast cancer type 33, breast cancer type 34, breast cancer type 35, breast cancer type 36, breast cancer type 37, breast cancer type 38, breast cancer type 49, breast cancer type 41, breast cancer type 42, breast cancer type 43, breast cancer type 44, breast cancer type 45, breast cancer type 56, breast cancer type 57, breast cancer typebreast cancer), squamous cell carcinoma (e.g., head and neck), neurogenic tumors, astrocytoma, ganglioneuroblastoma, neuroblastoma, glioma, adenocarcinoma, adrenal tumor, hereditary adrenal cortical carcinoma, brain malignant tumors (tumors), and various other cancers (e.g., bronchogenic large cell, ductal, Ehrlich-Retard astrocytoma, epidermal, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinal cell, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma) , epidermoblastoma, epithelial erythroleukemia (e.g., Friend, lymphoblastic), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., pleomorphic, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma, insulinoma, pancreatic islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, lymphosarcoma, melanoma, breast tumor, astrocytoma, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, Multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, neural tissue glial tumor, neural tissue neural tumor, neuroma, neuroblastoma, oligodendroglioma, osteochondroma, myeloma, osteosarcoma (e.g., Ewing), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, histiocytic, Jensen, osteosarcoma, reticular cell), schwannoma, subcutaneous tumor, teratoma (e.g., multipotent), teratomas follicular tumors, testicular tumors, thymoma and trichoepithelioma, gastric carcinoma, fibrosarcoma, glioblastoma multiforme; multiple glioblastomas, Li-Fraumeni syndrome, liposarcoma, Lynch carcinoma family syndrome II, male germ cell tumors, medullary thyroid carcinoma, multiple meningiomas, endocrine tumor myxosarcoma, paraganglioma, familial nonpigmented, trichofolliculoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, rhabdoid tumor, soft tissue sarcoma, and Turcot's syndrome with glioblastoma.;

[0028] In some embodiments, the method of Figure 1 is applied to tumors that are known to be substantially affected by alpha radiation themselves. In other embodiments, the method of Figure 1 is applied to tumors that are not substantially affected by alpha radiation themselves, e.g., do not shrink in size at all, or do not shrink in size by more than 5% or 10%. Experiments performed by applicants have shown that tumors that are not substantially affected by alpha radiation themselves can be dramatically reduced in size when targeted with a combination of ACSIP and alpha radiation according to the method of Figure 1.

[0029] (Pathogen Mimicry) In some embodiments, induction of ACSIP (104) is performed by delivering a pathogen mimic to the tumor cells. The delivered pathogen mimic, in some embodiments, comprises a viral mimic. In other embodiments, an intracellular bacterial mimic is delivered to the tumor cells instead of or in addition to the viral mimic. In some embodiments, the pathogen mimic comprises an intracellular pathogen mimic that is recognized as non-self by an intracellular receptor.

[0030] In the embodiment where the pathogen mimic comprises a virus mimic, the virus mimic can comprise a DNA virus mimic or an RNA virus mimic, for example, a ssRNA virus mimic or a dsRNA virus mimic.The dsRNA can optionally comprise a RIG-I-like receptor (RLR) agonist, such as a polyinosinic acid:polycytidylic acid (polyIC) molecule, as described, for example, in PCT Publication WO2017 / 085228, entitled "New pharmaceutical composition comprising particles comprising complexes of double-stranded polyribonucleotide and polyalkyleneimine".

[0031] In some embodiments, polyIC has a high molecular weight (HMW), for example, a molecular weight of more than 1.2 kb, more than 1.5 kb, more than 2.5 kb, or more than 4 kb. However, it should be noted that polyIC virus mimics can also include low molecular weight (LMW) polyIC with a molecular weight of less than 1 kb or less than 0.8 kb. Alternatively, the molecular weight of polyIC is selected according to the type of tumor.

[0032] Alternatively, any of the other viral mimetics described in PCT Publication WO 2017 / 085228, which is incorporated herein by reference, can be used. In other embodiments, the viral mimic delivered comprises 5' triphosphate double stranded RNA (5'ppp-dsRNA), 5' triphosphate hairpin RNA (3p-hpRNA) and / or poly(3p-hpRNA) and / or other suitable viral mimetics such as one or more of Poly(dA:dT), poly-ICLC, poly(A:U), and / or CpG rich RNA.

[0033] In other embodiments, DNA virus mimics containing CDS or STING ligands are used, such as any of those listed at https: / / www.invivogen.com / dsdna and / or https: / / www.invivogen.com / cyclic-dinucleotide, which are incorporated herein by reference. In one embodiment, CpG ODN virus mimics are used.

[0034] The use of RNA mimics has the advantage that ACSIP induction (104) works through a different pathway than alpha-emitter radiotherapy, which generally targets cellular DNA.

[0035] Alternatively, or in addition to using pathogen mimics to induce ACSIP, oncolytic viruses are used to induce ACSIP.

[0036] (Route of Administration) In some embodiments, tumor identification (102) includes identification of one or more primary tumors, e.g., a primary cancerous tumor and / or one or more secondary and / or metastases, and delivery (104) is performed in vivo by direct intratumoral injection into the identified tumor or tumors.

[0037] In other embodiments, delivery to tumors and / or metastases is by systemic administration using any suitable method of targeted delivery, for example, delivery by nanoghosts as described in US Patent Application Publication No. 2017 / 0224618, entitled: "Liposome Compositions and Uses Thereof," which is incorporated herein by reference. Optionally, the pathogen mimic is delivered with a specific tumor marker that targets the tumor, so that the tumor marker, along with the mimic, attaches to the tumor cells. Targeting is performed as described, for example, in US Patent Application Publication No. 2018 / 0346596, entitled: "Chimeric Proteins for Targeting DSRNA," or Alexander Levitzki, "Targeting the Immune System for Cancer Combat Using Polyinosine / Cytosine (PolyIC)-Bearing Chemoreceptor Homing Vectors," Frontiers in Oncology, 2012, the disclosures of which are incorporated herein by reference.

[0038] Optionally, prior to administering the pathogen mimic to the patient, the size of the tumor and / or metastases is estimated and the amount of pathogen mimic to be administered is selected accordingly.

[0039] In some embodiments, the pathogen mimic is administered to the patient along with a delivery agent (e.g., a reagent) that directs the pathogen mimic to cells of the tumor. Optionally, the delivery agent is one that can bypass membrane and / or endosomal receptors, such as the endosome / TL3 receptor. Optionally, the pathogen mimic and the delivery agent are administered together as a single dose. Optionally, the delivery agent encapsulates and / or complexes the pathogen mimic. For example, in one particular embodiment, high molecular weight polyIC pathogen mimic is incubated with polyethyleneimine (PEI) at a nucleic acid / polymer ratio of between 6 and 8 to form the complex (hereinafter, polyIC PEI In another embodiment, BO-112, described in PCT Publication WO 2017 / 085228, is used. This complex is injected intratumorally at a dose of 20-70 mg of polyIC (depending on tumor volume).

[0040] Alternatively, the delivery agent comprises polyethyleneimine (PEI). Alternatively, the delivery agent is based on lipid-based, polymer-based, or other suitable nanoparticles, for example, as described in Lee, Hung-Yen, Kamal A. Mohammed, and Najmunnisa Nasreen, "Nanoparticle-Based Targeted Gene Therapy for Lung Cancer," American Journal of Cancer Research 6.5 (2016): 1118, the disclosure of which is incorporated herein by reference. In some embodiments, the delivery agent comprises a cationic polymer complex or a cationic lipid. Alternatively or additionally, the delivery agent comprises a liposome, a lipoplex, a dendrimer, or a polymer micelle. Still alternatively or additionally, the delivery agent comprises a solid nanoparticle, such as a solid lipid nanoparticle or a solid polymer nanoparticle. In some embodiments, the delivery agent comprises a metal-based nanoparticle system.

[0041] Delivery may be carried out using any suitable method known in the art, such as any of the methods described in Langut, Yael, et al., "PSMA-homing dsRNA chimeric protein vectors kill prostate cancer cells and activate anti-tumor bystander responses," Oncotarget 8.15 (2017):24046 and / or Duewell P et al., "RIG-I-like helicase induces immunogenic cell death of pancreatic cancer cells and sensitizes tumors to killing by CD8+ T cells," Cell Death and Differentiation 21.12 (2014):1825, the disclosures of which are incorporated herein by reference.

[0042] (Stimulation of endogenous pathogens) In some embodiments, ACSIP is induced by stimulating epigenetic changes in tumor cells, for example by stimulating endogenous pathogens such as endogenous retroviruses and / or stimulating activation of cytoplasmic pathogen sensors that activate the promoters of these sensors through epigenetic changes (104). The epigenetic changes are caused by epigenetic agents. In some embodiments, the epigenetic agents include DNA methyltransferase (DNMT) inhibitors such as decitabine, azacytidine and / or guadecitabine. Stimulation is performed using any suitable method known in the art, such as the method described in Chiappinelli et al., "Inhibiting DNA methylation in cancer via dsRNA containing endogenous retroviruses induces an interferon response," Cell, Volume 162, Issue 5, August 27, 2015, available at https: / / www.cell.com / cell / fulltext / S0092-8674(15)00848-X. The disclosure of which is incorporated herein by reference in its entirety. In other embodiments, the epigenetic agent comprises a histone deacetylase (HDAC) inhibitor, such as entinostat or vorinostat.

[0043] Alternatively, the epigenetic agent is administered systemically. In other embodiments, the epigenetic agent is administered using targeted delivery, for example, using nanoghosts that direct the epigenetic agent to tumors and possibly metastases. Alternatively still, the epigenetic agent is delivered locally by direct injection into the target tumor.

[0044] (Multiple Therapy Sessions) In some embodiments, ACSIP induction (104) is performed once before alpha emitter radiotherapy in a single session. Alternatively, ACSIP induction (104) is performed in multiple sessions, perhaps at least two, at least three, or at least four sessions, at least 24 hours apart. The limited period (106) after ACSIP induction (104) is counted from the last ACSIP induction session according to this alternative. The multiple sessions use the same ACSIP induction method in the alternative. Or, different sessions have different ACSIP induction methods. In yet other embodiments, two or more ACSIP inductions are performed simultaneously in the same session or within less than one hour of each other.

[0045] One embodiment found to provide particularly promising results includes an initial ACSIP induction session to stimulate epigenetic changes in tumor cells, a second session 24 hours later to both stimulate epigenetic changes in tumor cells and deliver a pathogen mimic, a third session 24 hours after the second session involving stimulation of epigenetic changes in tumor cells, and a fourth session 24 hours after the third session to both stimulate epigenetic changes in tumor cells and deliver a pathogen mimic.

[0046] In one particular implementation of this embodiment, ACSIP induction (104) involves a combination of four decitabine-based sessions every 24 hours prior to alpha radiation, and administration of polyIC with one of the delivery agents described above, twice, 72 hours and 24 hours prior to alpha radiation, with or without PEI, with alpha radiation optionally being activated one day after the last session.

[0047] (Alpha radiation) Alpha emitter radiotherapy optionally includes brachytherapy by inserting seeds carrying alpha emitting atoms, such as radium-224 or radium-223, into the tumor. Optionally, the alpha emitting atoms are attached to the seed such that the atoms do not leave the seed, but as the radionuclide decays, daughter radionuclides leave the seed. Brachytherapy seeds optionally release daughter radionuclide atoms at a rate of at least 0.1%, 0.5%, or at least 1% of the number of radionuclide atoms bound to the seed per 24 hours. In some embodiments, the daughter radionuclide atoms are slowly released from the seed at a rate of less than 25%, less than 10%, less than 5%, or less than 3% of the radionuclide atoms bound to the seed per 24 hours.

[0048] Instead of attaching alpha-emitting atoms to the seeds such that the atoms do not leave the seeds without radionuclide decay, the alpha-emitting atoms are attached to the seeds in a manner such that the atoms controllably leave the seeds in a manner other than by radionuclide decay at a rate of at least 0.1% per 24 hours, as described, for example, in PCT Publication WO 2020 / 193464, entitled "Controlled Release of Radionuclides," which is incorporated herein by reference.

[0049] In some embodiments, the alpha emitter radiation comprises diffuse alpha emitter radiation therapy (DaRT). Alpha emitter radiation therapy is optionally performed using any of the methods and / or devices described in U.S. Patent No. 8,834,837, U.S. Patent Application Publication 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.

[0050] Alpha emitter radiotherapy is optionally initiated by inserting one or more brachytherapy seeds into the tumor, the brachytherapy seeds including alpha-emitting atoms on the outer surface of the seed (108). Alternatively, alpha emitter radiotherapy is initiated by activating previously inserted seeds carrying alpha-emitting atoms. Optionally, according to this alternative, the seeds are inserted into the patient with a bioabsorbable coating that prevents alpha radiation and / or daughter radionuclides from leaving the seed. The bioabsorbable coating optionally includes polylactide (PLA), polyglycolide (PGA), or a copolymer of PLA and PGA, adjusted to achieve a desired absorption rate of the coating. Alternatively or additionally, the coating includes copolylactic / glycolic acid (PLGA). The polymer of the coating optionally has a molecular weight in the range of 5,000 to 100,000. The material of the coating dissolves in the patient by any of the methods known in the art, such as one or more of ultrasound energy, reaction with body temperature, and / or reaction with bodily fluids. Additional discussion of bioabsorbable polymers that may be used in accordance with embodiments of the present invention after adjusting the desired absorption rate is described in U.S. Patent No. 8,821,364 and U.S. Patent Application Publication No. 2002 / 555666, which are incorporated herein by reference. In some embodiments, initiation (108) involves applying a stimulus that dissolves the coating, thus allowing alpha radiation and / or daughter radionuclides to leave the seed. In other embodiments, initiation (108) is accomplished by dissolution of the coating upon contact with tumor tissue, without further physician initiation.

[0051] Alpha emitter radiation therapy is optionally applied to the patient for at least 24 hours, at least 5 days, or even at least 10 days. In some embodiments, the brachytherapy seeds are removed from the patient after the designated treatment period. For example, the seeds are optionally removed during surgery for tumor removal. Alternatively, the seeds are not removed. In some embodiments, the seeds comprise a biodegradable material.

[0052] Alternatively, alpha emitter radiation therapy is initiated only after induction of ACSIP is complete. Alternatively, no substance that induces ACSIP is provided to the patient during the alpha emitter radiation therapy procedure. In some embodiments, no substance that induces ACSIP is provided to the patient for at least 24 hours or at least one week after completion of alpha source radiation therapy, e.g., after removal of the brachytherapy source or source used. In other embodiments, one or more ACSIP inducers are provided while alpha emitter radiation therapy is being administered.

[0053] The limited period of time (106) between ACSIP induction (104) and the initiation of alpha emitter radiotherapy (108) is selected to enable ACSIP induction. For example, if ACSIP is induced by an intracellular viral mimic, the limited period of time (106) is selected to act as a suitable buffer period to allow for upregulation of MHC1 expression on the membrane of tumor cells due to the pathogen mimic. Alternatively or additionally, the length of the period (106) is selected to be sufficient to allow time for tumor cells to respond to the delivered mimic.

[0054] The period (106) is optionally at least 6 hours, at least 9 hours, at least 12 hours, at least 24 hours, or at least 36 hours, so that activation of intracellular pathogen recognition begins to take effect before alpha-emitter radiotherapy is applied. Optionally, the limited period (106) is shorter than 2 weeks, shorter than 10 days, shorter than 1 week, shorter than 120 hours, shorter than 96 hours, shorter than 72 hours, or even shorter than 48 hours, so that the effect of activation of intracellular pathogen recognition is effective even when alpha-emitter radiotherapy is applied. In some embodiments, the limited period (106) is shorter than 30 hours, or even shorter than 20 hours, for example, in tumors with many blood vessels or otherwise more rapidly responsive to ACSIP. If ACSIP is provided in multiple separate sessions, the period (106) is optionally selected to allow maximum effect of ACSIP treatment on the patient during radiotherapy. Optionally, the duration of ACSIP treatment is less than 10 days, less than 1 week, or even 5 days or less, such that the time period from the first ACSIP treatment session is not longer than the maximum desired wait time (e.g., 10 days between the first ACSIP treatment session and radiation therapy) and the time period between the last ACSIP treatment session and radiation therapy is not shorter than the minimum desired wait time (e.g., 12 hours).

[0055] Although the above description relates to the application of alpha emitter radiotherapy after ACSIP treatment, in some embodiments of the invention, alpha emitter radiotherapy is provided substantially simultaneously with or even prior to ACSIP treatment. Alpha emitter radiotherapy is provided to the patient within a given time (e.g., 2 weeks) before or after ACSIP treatment, the given time being selected according to the time frame of the expected effect of the treatment.

[0056] In some embodiments, one or more parameters of the tumor are monitored to determine the most suitable time to apply alpha emitter radiation therapy after activation of intracellular pathogen recognition. Monitoring optionally includes imaging the tumor using an appropriate modality (X-ray, ultrasound, PET-CT, MRI, CT, etc.) to identify when the tumor begins to change due to activation of intracellular pathogen recognition. Alternatively, monitoring includes performing blood tests to identify the level of the attribute. However, it should be noted that in some embodiments, DaRT radiation is applied before the effects of ACSIP are detectable.

[0057] (Additional treatment) Providing supportive treatment (114) includes, in some embodiments, providing one or more treatments to counteract undesirable side effects of radiation therapy and / or ACSIP induction (104). Optionally, the supportive treatment includes one or more treatments to counteract accelerated tissue repair induced by alpha emitter radiation therapy, as such accelerated tissue repair supports residual tumor cells and promotes tumor recurrence. Alternatively or additionally, the supportive treatment includes one or more anti-inflammatory treatments to downregulate inflammation following tissue damage caused by radiation therapy and / or ACSIP. Further alternatively or additionally, the supportive treatment includes one or more treatments to downregulate checkpoint expression (e.g., PD-L1 expression) caused by radiation therapy and / or ACSIP. In some embodiments, the supportive treatment includes one or more treatments to prevent DNA repair, so as to thwart 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 viral attack of ACSIP.

[0058] In some embodiments, supportive care includes treatments that have two or even three of the above effects.For example, providing supportive care of pomalidomide administration has the effects of opposing tissue repair, downregulating inflammation, and downregulating PD-L1 expression.Beruzosertib is another example of a drug that can be used to both prevent DNA repair and downregulate PD-L1 expression.

[0059] In other embodiments, supportive care includes multiple different treatments, for example, checkpoint blockade (e.g., anti-PD-1 and / or CTLA4) along with a TLR3 agonist that stimulates ACSIP from outside the tumor cell.

[0060] In some embodiments, the supportive treatment includes supportive immunomodulation, such as myeloid-derived suppressor cell (MDSC) 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.

[0061] Alternatively or additionally, providing the additional treatment (114) includes providing an immunotherapy treatment. The immunotherapy treatment optionally includes administration of one or more checkpoint blockade agents, such as anti-PD-1, anti-PD-L1, anti-CTLA4, anti-TIGIT, anti-LAG-3, anti-TIM-3, anti-CD134 and / or anti-CD137. Alternatively or additionally, the immunotherapy treatment includes administration of 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).

[0062] Alternatively or additionally, the additional treatment comprises administering a type of DNA repair inhibitor that has been found to increase, not affect, or only minimally interfere with the immune response induced by alpha-radioactive radiotherapy.In some embodiments, the DNA repair inhibitor administered comprises an ATR inhibitor, such as beruzosertib, AZD6738, and / or NU6027.Alternatively or additionally, the DNA repair inhibitor comprises 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).

[0063] Alternatively or additionally, the additional treatment comprises an antiangiogenic factor of a type that has been found to have no effect or only minimally interfere with the immune response induced by alpha radioactive radiotherapy and / or ACSIP. In some embodiments, the additional treatment comprises an iMiD, such as pomalidomide, thalidomide, lenalidomide and / or apremilast.

[0064] Further alternatively or additionally, the additional treatment comprises a local or systemic chemotherapy treatment of a type that has been found not to interfere with the immune response to alpha radiation and / or ACSIP and / or DaRT radiation. Optionally, the chemotherapy treatment comprises one or more of cyclophosphamide (CP), doxorubicin, mitoxantrone, gemcitabine, oxaliplatin, and / or cisplatin.

[0065] In some embodiments, the additional treatment includes checkpoint inhibition with one or more agents or drugs that downregulate expression of checkpoint molecules, such as, for example, pomalidomide (IMiD) and / or beruzosertib (ATR inhibitor).

[0066] Alternatively or additionally, in some embodiments, the additional therapy comprises an anti-inflammatory drug, such as an NSAID, e.g., a Cox2 inhibitor.

[0067] Further alternatively or additionally, the additional treatment includes administration of one or more epigenetic drugs, such as DNMT inhibitors (e.g., decitabine, azacitidine, guadecitabine) and / or HDAC inhibitors (e.g., entinostat, vorinostat).

[0068] In some embodiments, the additional treatment is provided while the alpha radiation is being provided (114). In other embodiments, the additional treatment is provided after the alpha-source radiation therapy is completed, e.g., after a majority of the radionuclides in the brachytherapy seeds have undergone nuclear reactions and / or after the brachytherapy seeds have been removed from the patient (114). In yet other embodiments, the additional treatment is provided prior to the alpha-emitter radiation therapy (114).

[0069] In some embodiments, supportive care is provided less than 72 hours, less than 48 hours, or even less than 32 hours from one of the ACSIP sessions and / or radiation therapy treatments.

[0070] The timing of providing the additional treatment is optionally selected depending on the specific type of additional treatment. For example, if a checkpoint inhibitor is provided, it is administered during or after alpha-emitter radiotherapy, perhaps 3 days or even 1 week after the start of alpha-emitter radiotherapy, and / or after the completion of alpha-emitter radiotherapy.

[0071] Optionally, additional treatments are offered depending on the tumor type ( 114 ).

[0072] (Treatment kit) Figure 2 is a schematic diagram of a kit 200 for treating 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 (204) of an agent for ACSIP induction.

[0073] As an option, the seeds 204 are provided within a vial or other casing 206 that prevents radiation from exiting the casing. In some embodiments, the casing is filled with a viscous liquid, such as glycerin, that prevents radiation from escaping the casing 206, as described in PCT Application PCT / IB2019 / 051834, entitled "Radiotherapy Seeds and Applicators," 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 the patient, as described in PCT Application PCT / IB2019 / 051834. As an option, the applicator 208 is provided with one or more seeds 204 preloaded therein. In accordance with this option, separate seeds 204 within the casing 206 are provided in case more than the 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 .

[0074] As shown, doses 216 of ACSIP are provided preloaded into 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 the sterile package 202, or are not provided in the kit 200 at all.

[0075] In some embodiments, the kit 200 further comprises one or more drugs 220 required for supportive immunomodulatory therapy (114).

[0076] In some embodiments, the kit 200 includes multiple separate compartments, separated by suitable insulating material, for materials requiring storage at different temperatures. For example, a first compartment may contain dry ice that keeps the materials in the first compartment at approximately -20°C, while a second compartment may contain ice that keeps the materials in the second compartment at approximately 4°C.

[0077] (experiment) FIG. 3 shows the results of an experiment conducted by applicant to test the method of FIG. 1. In the experiment, a high molecular weight (HMW) PolyIC dsRNA viral mimic (20 μg / 40 μl) was found to induce circulating dsRNA in approximately 40 mm 3 PolyIC was delivered in vivo into the cytoplasm of primary tumor cells using the delivery agent PEI in mice bearing 4T1 triple-negative breast cancer tumors with a volume of 100 μg / mL. PolyIC was complexed with PEI at a ratio of nucleic acid / polymer = 6. The viral mimetic delivery was performed twice, the second delivery 48 h after the first delivery. The waiting time (106) between the delivery of the second mimetic and the start of alpha-emitter radiotherapy by brachytherapy was 24 h. Alpha-emitter radiotherapy was performed by 70 kBq (kilobecquerel) seeds of radium-224 with a length of 8 mm. A control group of mice received an inert source without radium-224.

[0078] Figure 3 shows the average tumor volume in mice 29 days after the start of treatment on the Y-axis as a function of days after treatment on the X-axis. As can be seen in Figure 3, delivery of the intracellular viral mimic reduces tumor size by up to about 20%. Alpha radiation therapy on its own reduces tumor size by up to about 34%. The combination of alpha emitter treatment with naked viral mimic where no delivery reagent is provided reduces tumor size by up to about 64%. The combination of alpha emitter radiation therapy and intracellular viral mimic delivery reduces tumor size by over 82%, which is significantly more than would be expected with each treatment individually.

[0079] The following table shows the effect of the method of FIG. 1 using two imaging techniques on the percentage of mice with lung metastases. [Table 1]

[0080] This table summarizes the results for mice participating in the experiment described in Figure 3, measured by CT or M-Cherry fluorescent imaging 29 days after the first viral mimetic delivery. The numbers in the table indicate the percentage of mice that were positive for lung metastases. Significantly more mice were negative for lung metastases in the group that received the combination of alpha radiation and intracellular viral mimetic delivery treatment (77% metastasis free) compared to the groups that received the separate treatments, or PolyIC without PEI combined with DaRT.

[0081] Another experiment was conducted to test the effect of the method of Figure 1 on long-term immune responses. 3 ) were treated with a combination of alpha emitter radiotherapy and intracellular viral mimetic delivery therapy (same treatment protocol as described in Figure 3, seed data: 7 mm, 80 kBq; 20 μg / 40 μl polyIC PEI ). 24 days after tumor cell inoculation, the residual tumors were excised and the animals were observed for long-term survival (metastasis-associated death) and examined for long-term immune memory. 25% of the mice (4 out of 16) survived for 9 months after inoculation, indicating clearance of metastases. On the other hand, mice inoculated with 100% of the same number of tumor cells as treated with inert seeds and intratumoral PBS had their tumors excised before this time point (day 17) and had already developed lung metastases. Spleens of surviving (cured) mice were harvested and splenocytes were mixed with tumor cells in a ratio of 100:1 (spleen cells:tumor cells). The mixed suspension was inoculated into naïve mice. Splenocytes from naïve mice served as controls. DaRT and polyIC PEI Splenocytes from mice treated with IFN-γ significantly delayed tumor development compared to splenocytes from naive mice, with tumor delays of up to 48% lasting up to 19 days after co-inoculation of splenocytes and tumor cells, indicating that this treatment induced efficient long-term immune memory even after 9 months.

[0082] In further experiments, the applicants administered a low dose of cyclophosphamide (100 mg / kg, 1 day before the start of treatment, i.e., the first polyIC PEI We tested the method of Figure 1 in combination with an additional immunomodulatory treatment to inhibit T regulatory cells using a 6.5 mm and 65 kBq DaRT seed and 30 μg / 60 μl polyIC PEI 4T1 tumors (25 mm 3 ) in mice with polyIC. Addition of T regulatory cell inhibition PEI The treatment further delayed tumor onset by up to 43% compared to DaRT. Moreover, after tumor resection (24 days after tumor inoculation), additional treatment with CP did not reduce long-term survival, which was 71% in both groups at the end of the experiment (180 days after DaRT). However, it extended survival by 30%, e.g., during the period from 85 to 145 days after DaRT, compared to DaRT, polyIC PEI , and CP-treated groups had 100% survival, whereas DaRT and polyIC PEI In the group treated with DaRT + polyIC, 71% of the mice survived. PEI Treatment with DaRT+polyIC PEI Both treatments, DaRT plus cyclophosphamide, induced long-term immune memory and delayed the onset of tumor rechallenge in treated (cured) mice compared to naive mice. Furthermore, Applicants conducted additional experiments to test the method of Figure 1 in combination with cyclophosphamide in a neoadjuvant setting, allowing long-term survival rates following combined treatment to be compared to those of its individual components: DaRT alone and immune modulation by CP plus polyIC. PEI Alone. 4T1 tumor (23 mm 3 ) were treated with CP followed by 30 μg / 60 μl of polyIC as described above. PEITwo doses of DaRT were administered and 6.5 mm, 85 Kbq DaRT seeds were inserted. Residual tumors were resected 14 days after DaRT (24 days after tumor inoculation). Inactive seeds and vehicle injections served as controls. The treatment in Figure 1, when used as a neoadjuvant treatment, in combination with cyclophosphamide, prevented metastasis formation and cured mice in 75% of mice, whereas only 33% were cured using DaRT alone and polyIC PEI 25% were cured using +CP + inert seeds. 12% were cured using inert seeds and vehicle injection. Cured mice were defined as surviving 155 days or more from inoculation.

[0083] Additionally, applicants will test the method of Figure 1 by induction of ACSIP through stimulation of endogenous pathogens using low-dose decitabine and compare the effect of this treatment on tumor development using viral mimic delivery of polyIC PEI SQ2 (squamous cell carcinoma)-bearing mice were seeded with 85 Kbq DaRT and 25 μg / 50 μl polyIC 72 and 24 h before daily administration of 1 mg / kg ip DaRT or decitabine for 4 days. PEI Alpha-emitter radiotherapy in combination with decitabine was PEI DaRT combined with alpha-emitter radiation therapy delayed tumor onset as well as DaRT alone. Both treatments delayed tumor onset by approximately 66% compared to DaRT alone. Furthermore, the treatments maintained the ability to induce long-term immune memory, as shown by an improved delay in rechallenge tumor onset of approximately 82% in treated and tumor-resected mice compared to naive mice inoculated with the same number of tumor cells.

[0084] To test the method of Figure 1 by inducing ACSIP by stimulation of endogenous pathogens using low-dose decitabine in a 4T1 breast cancer mouse model (large tumors), Applicants conducted two additional experiments. Mice were treated with 75 kBq DaRT seed and 30 μg / 60 μl polyIC 72 h prior to DaRT. PEI, 50 μg / 100 μl polyIC 24 hours before DaRT or decitabine PEI Alpha-emitter radiotherapy combined with decitabine also significantly improved the proliferation of polyIC cells in the 4T1 breast mouse model compared to DaRT alone. PEI In addition, DaRT was combined with decitabine and polyIC PEI Combining DaRT with decitabine and DaRT doubled the effect compared to the same treatment with non-radioactive seeds and quadrupled it compared to DaRT alone. Furthermore, in this cancer model, activating the TLR3 pathway with the TLR3 agonist "naked" polyIC (without PEI) in addition to decitabine and DaRT activated the same RIG-1 pathway as decitabine and polyIC. PEI It has been shown to improve results by 41% compared to activation in combination with DaRT.

[0085] Finally, experiments have been performed by the applicant to test the method of Figure 1 in the murine pancreatic tumor model Panc02. Furthermore, treatment was tested in this model in combination with the iMiD drug pomalidomide (POM).

[0086] 11 days after inoculation, 22 mm 3 Tumors with a volume of 100 μg were injected with polyIC at a dose of 30 μg / 60 μl. PEI The first intratumoral injection of polyIC was performed, followed 48 hours later by a second injection. PEI 24 hours after injection, DaRT seeds (6.5 mm 75 kBq) were inserted into the tumor. Four days later, 25 mg / kg POM was injected intraperitoneally in a volume of 300 μl every other day for 5 days, starting on day 4 after DaRT.

[0087] polyIC PEI Alpha-emitter radiotherapy combined with DaRT eliminated pancreatic tumors in 30% of mice. Adding the iMiD drug pomalidomide increased the rejection rate to 50%, whereas DaRT alone or DaRT + pomalidomide or inert seed + pomalidomide + polyIC PEINo tumor rejection was observed in the group treated with

[0088] (Conclusion) It will be understood that the above methods and apparatus should be construed as including methods of using the apparatus and apparatus for performing the methods. It will 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 one of the specific embodiments, in a particular figure, or described with respect to below. Tasks will not necessarily be performed in the exact order described.

[0089] It should be noted that some of the above embodiments may not be essential to the present invention and may include details of structures, acts, or structures and acts 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 known in the art. The above 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 are not disclosed in the prior art and that will occur to those skilled in the art upon reading the foregoing description. Thus, the scope of the present invention is limited only by the elements and limitations used in the claims, and the terms "comprises", "includes", "having" and their conjugates shall mean "comprises" when used in the claims. However, they are not necessarily limited thereto.

Claims

1. administering to the patient an agent that activates a cytoplasmic sensor for an intracellular pathogen in the tumor; treating the tumor with intratumoral alpha emitter radiation therapy within two weeks of administering an agent that activates the cytoplasmic sensor for an intracellular pathogen in the tumor; A method of treating a patient having a tumor comprising:

2. 2. The method of claim 1, wherein administering the substance comprises administering an agent that induces intracellular production of a molecule that stimulates the cytoplasmic sensor for the intracellular pathogen.

3. 3. The method of claim 2, wherein administering the agent comprises administering a DNA methyltransferase (DNMT) inhibitor.

4. 4. The method of claim 3, wherein administering the DNA methyltransferase (DNMT) inhibitor comprises administering decitabine.

5. 4. The method of claim 3, wherein administering the DNA methyltransferase (DNMT) inhibitor comprises administering azacytidine.

6. 4. The method of claim 3, wherein administering the DNA methyltransferase (DNMT) inhibitor comprises administering guadecitabine.

7. 3. The method of claim 2, wherein administering the agent comprises administering a histone deacetylase (HDAC) inhibitor.

8. 8. The method of claim 7, wherein administering the histone deacetylase (HDAC) inhibitor comprises administering entinostat.

9. 8. The method of claim 7, wherein administering the histone deacetylase (HDAC) inhibitor comprises administering vorinostat.

10. 10. The method of claim 1, wherein administering the agent comprises administering the pathogen mimic with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic.

11. 11. The method of claim 10, wherein administering the pathogen mimic with the delivery agent comprises administering a single dose of an intracellular pathogen mimic in a single session.

12. 11. The method of claim 10, wherein administering the pathogen mimic with a delivery agent comprises administering at least three doses of the intracellular pathogen mimic in each separate session.

13. The method of any of claims 1 to 12, wherein administering the substance comprises administering the substance together with a delivery agent suitable for targeted delivery.

14. The method of claim 13 , wherein the delivery agent comprises a lipid-based delivery agent.

15. The method of claim 13 , wherein the delivery agent comprises a polymer-based delivery agent.

16. The method of claim 13 , wherein the delivery agent comprises a cationic polymer complex or a cationic lipid.

17. The method of claim 13 , wherein the delivery agent comprises a liposome or a lipoplex.

18. The method of claim 13 , wherein the delivery agent comprises a polymeric micelle.

19. The method of claim 13 , wherein the delivery agent comprises a dendrimer.

20. The method of claim 13 , wherein the delivery agent comprises a solid nanoparticle.

21. 21. The method of claim 20, wherein the delivery agent comprises a solid lipid nanoparticle or a solid polymer nanoparticle.

22. The method of claim 13 , wherein the delivery agent comprises a metal-based nanoparticle system.

23. The method of claim 13 , wherein the delivery agent comprises polyethyleneimine (PEI).

24. 14. The method of claim 13, wherein administering the pathogen mimic comprises administering a bacterial mimic.

25. 14. The method of claim 13, wherein administering the pathogen mimic comprises administering a virus mimic.

26. 26. The method of claim 25, wherein administering the viral mimic comprises administering double-stranded RNA (dsRNA).

27. 27. The method of claim 26, wherein administering the dsRNA comprises administering polyIC.

28. 28. The method of claim 27, wherein the delivery agent comprises polyethyleneimine (PEI).

29. 30. The method of claim 28, wherein administering the viral mimic comprises administering BO-112.

30. 27. The method of claim 26, wherein the step of administering a dsRNA comprises administering a 5'ppp-dsRNA.

31. 27. The method of claim 26, wherein the step of administering a dsRNA comprises administering a 3p-hpRNA.

32. 27. The method of claim 26, wherein administering the dsRNA comprises administering poly(dA:dT).

33. 27. The method of claim 26, wherein administering the dsRNA comprises administering polyICLC.

34. 27. The method of claim 26, wherein administering the dsRNA comprises administering poly(A:U).

35. 27. The method of claim 26, wherein administering the dsRNA comprises administering a CpG-rich RNA.

36. 14. The method of claim 13, wherein administering the pathogen mimic comprises administering a pathogen mimic that is recognized as non-self by an intracellular receptor.

37. A method according to any one of claims 1 to 12, wherein the step of administering a substance that activates a cytoplasmic sensor for an intracellular pathogen comprises administering a plurality of different substances that activate a cytoplasmic sensor for an intracellular pathogen.

38. 38. The method of claim 37, wherein administering a plurality of different agents that activate a cytoplasmic sensor for the intracellular pathogen comprises administering a first agent that induces intracellular production of a molecule that stimulates a cytoplasmic sensor for the intracellular pathogen, and a second agent having a pathogen mimic with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic.

39. 40. The method of claim 38, further comprising the step of additionally administering to the patient supportive therapy with cyclophosphamide.

40. A method according to any preceding claim, comprising the step of inserting a brachytherapy source carrying an alpha emitting radionuclide into the tumour.

41. 41. The method of claim 40, wherein the brachytherapy source, when inserted into a tumor, releases alpha-emitting radionuclides at a rate less than 25% of the alpha-emitting radionuclide on the source per day.

42. 41. The method of claim 40, wherein the brachytherapy source comprises a radium source.

43. The method according to any of claims 1 to 12, wherein treatment of the tumor with alpha emitter radiotherapy is carried out only after administration of the substance is completed.

44. The method of any of claims 1 to 12, wherein administering the substance comprises administering the substance with nano-ghosts that target the substance to a tumor.

45. The method of any of claims 1 to 12, wherein administering the substances comprises administering one or more substances in at least two separate sessions separated from each other by at least 12 hours.

46. 46. ​​The method of claim 45, wherein administering the one or more substances comprises administering in at least three separate sessions separated from one another by at least 20 hours.

47. 13. The method of any preceding claim, comprising identifying a primary tumour in the patient, and wherein administering the substance comprises administering the substance to the identified primary tumour.

48. 13. The method of any of claims 1 to 12, wherein treating the tumor with alpha emitter radiotherapy comprises treating with alpha emitter radiotherapy within 80 hours of administering the last dose of the agent.

49. 13. The method of any of claims 1 to 12, wherein treating the tumor with alpha emitter radiotherapy comprises treating with alpha emitter radiotherapy within 60 hours of administering the last dose of the agent.

50. 13. The method of any of claims 1 to 12, wherein the step of treating the tumor with alpha emitter radiotherapy comprises treating with alpha emitter radiotherapy at least 12 hours after administration of the last dose of the substance.

51. 13. The method of any of claims 1 to 12, wherein the step of treating the tumor with alpha emitter radiotherapy comprises treating with alpha emitter radiotherapy at least 24 hours after administration of the last dose of the substance.

52. The method of any of claims 1 to 12, further comprising the step of additionally administering to the patient a supportive therapeutic agent against the accelerated tissue repair induced by the alpha emitter radiotherapy.

53. The method of any of claims 1 to 12, further comprising the step of additionally administering to the patient a supportive therapeutic agent that downregulates inflammation.

54. The method of any one of claims 1 to 12, further comprising the step of additionally administering to the patient a supportive therapeutic agent that downregulates checkpoint expression.

55. The method of any one of claims 1 to 12, further comprising the step of administering to the patient additional supportive therapy of cyclophosphamide.

56. 13. The method of any of claims 1-12, further comprising the step of further administering to the patient supportive care of pomalidomide.

57. The method of any one of claims 1 to 12, further comprising the step of administering to the patient additional supportive therapy for beruzosertib.

58. The method of any one of claims 1 to 12, further comprising the step of additionally administering to the patient a supportive checkpoint blockade therapy.

59. 59. The method of claim 58, further comprising the step of additionally administering to the patient an anti-PD-1 supportive therapy.

60. 59. The method of claim 58, further comprising the step of additionally administering to the patient an anti-PD-L1 supportive therapy.

61. 59. The method of claim 58, further comprising the step of additionally administering an anti-CTLA4 supportive therapy to the patient.

62. 60. The method of claim 58, further comprising the step of additionally administering to the patient one or more supportive TLR agonist therapy.

63. 60. The method of claim 58, further comprising the step of additionally administering to the patient one or more supportive treatments of MSDC inhibitors.

64. 60. The method of claim 58, further comprising the step of additionally administering to the patient one or more supportive therapies of Treg inhibitors.

65. 60. The method of claim 58, further comprising the step of additionally administering to the patient a supportive therapy of one or more DNA repair inhibitors.

66. 60. The method of claim 58, further comprising the step of additionally administering to the patient a supportive therapy of one or more anti-angiogenic factors.

67. The method of any of claims 1 to 12, further comprising the step of performing a surgical procedure to remove the tumor at least one week after starting treatment of the tumor with alpha radiation therapy.

68. 13. The method of any of claims 1-12, wherein treating the tumor with intratumoral alpha emitter radiotherapy comprises administering a substance that activates a cytoplasmic sensor for an intracellular pathogen in the tumor and treating the tumor with intratumoral alpha emitter radiotherapy within one week.

69. at least one radiation source at least partially introduced into the subject's body and having alpha-emitting atoms attached thereto; at least one substance that activates a cytoplasmic sensor for an intracellular pathogen; a sterile package containing the at least one radiation source and the at least one substance; A kit for treating a patient comprising:

70. 70. The kit of claim 69, wherein the alpha-emitting atoms are attached to the source in a manner that allows for controllable release of the alpha-emitting atoms or their daughter radionuclides from the source.

71. 70. The kit of claim 69, wherein the alpha-emitting atoms comprise radium atoms.

72. 72. The kit of any of claims 69-71, wherein the at least one substance comprises a DNA methyltransferase (DNMT) inhibitor.

73. 73. The kit of claim 72, wherein the DNA methyltransferase (DNMT) inhibitor comprises decitabine.

74. 73. The kit of claim 72, wherein the at least one substance further comprises a pathogen mimic associated with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic.

75. 72. The kit of any of claims 69-71, wherein the at least one substance comprises a pathogen mimic associated with a delivery agent suitable for cytoplasmic delivery of the pathogen mimic.

76. 72. The kit of any of claims 69-71, further comprising in said sterile package a supportive therapeutic agent against the accelerated tissue repair induced by alpha emitter radiotherapy.

77. 77. The kit of claim 76, wherein the supportive therapy comprises an agent that downregulates inflammation.

78. PolyIC in the manufacture of a drug for administration to a tumor in a patient PEI The use of the method according to claim 1, wherein the administration pattern of the drug comprises administering a therapeutically effective amount of polyIC PEI was administered to the tumor in one or more sessions, followed by polyIC PEI and administering intratumoral alpha-emitter radiation therapy to treat the tumor within one week of administration. PEI Use of.

79. 1. Use of a DNA methyltransferase (DNMT) inhibitor in the manufacture of a medicament for administration to a tumor in a patient, wherein a pattern of administration of the medicament comprises administering a therapeutically effective amount of the DNMT inhibitor.

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