Combination therapy of oncolytic vaccinia virus and checkpoint inhibitors

Abstract

A drug combination is provided that includes (i) a replication-competent oncolytic vaccinia virus and (ii) an immune checkpoint protein inhibitor, as well as kits that include the drug combination and methods of treating and / or inhibiting cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 488,623, filed April 21, 2017, and U.S. Provisional Patent Application No. 62 / 550,486, filed August 25, 2017, which applications are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to virology and medicine. In certain embodiments, the present invention relates to a therapeutic combination comprising a replication-competent oncolytic vaccinia virus and an immunomodulatory agent. [Background technology]

[0003] Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. An imbalance in either cell proliferation or cell death can lead to a cancerous state. For example, cancer of the cervix, kidney, lung, pancreas, colon, and brain are just a few of the many cancers that can result. In fact, the incidence of cancer is so high that cancer kills more than 500,000 people each year in the United States alone.

[0004] Replication-selective oncolytic viruses hold promise as cancer therapeutics. These viruses can cause tumor cell death through direct replication-dependent oncolysis and / or viral gene expression-dependent oncolysis. However, tumor-induced immunosuppression and premature viral clearance often result in only weak tumor-specific immune responses, limiting the potential of these viruses as cancer therapeutic agents.

[0005] Similarly, immune checkpoint inhibitors have shown some efficacy in treating certain cancers, but only a limited percentage of patients achieve objective clinical responses, and there remains a need for improved cancer treatments. Summary of the Invention [Means for solving the problem]

[0006] The present inventors have found that the concurrent administration of an immune checkpoint inhibitor and a replication-competent oncolytic vaccinia virus administered intratumorally results in synergistic anti-tumor effects in clinically relevant cancer models. In this concurrent administration, the agents are administered simultaneously, first in an initial administration and preferably in multiple consecutive administrations. Thus, in some embodiments, the present application provides a combination therapy for use in the treatment and / or prevention of cancer and / or the establishment of metastasis in a mammal, the combination therapy comprising the concurrent administration of (i) a replication-competent oncolytic vaccinia virus and (ii) an immune checkpoint inhibitor to the mammal, wherein the oncolytic vaccinia virus is administered intratumorally to the mammal. In certain aspects, the concurrent administration of the drug combination partners to the mammal provides enhanced, even synergistic, anti-tumor immunity compared to either treatment alone.

[0007] In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. In some embodiments, the tumor does not express an immune checkpoint protein or expresses an immune checkpoint protein at a relatively low level prior to administration of the replication-competent oncolytic vaccinia virus. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to an immune checkpoint protein, and is preferably a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.

[0008] In some embodiments, the immune checkpoint inhibitors of the combination are selected from the group consisting of cytotoxic T-lymphocyte antigen-4 (CTLA4 or CTLA-4), programmed cell death protein 1 (PD-1), B7-H3, B7-H4, T-cell membrane protein 3 (TEM3), galectin 9 (GAL9), lymphocyte activation gene 3 (LAG3), V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), B and T lymphocyte attenuator (BTLA), and IL-1. The immune checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1, B7-H3, T7-H4, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT, or a combination thereof. In a preferred embodiment, the immune checkpoint protein inhibitor is an antibody (e.g., a monoclonal antibody, a chimeric antibody, a human antibody, or a humanized antibody), an antibody fragment, or a fusion protein that specifically binds to an immune checkpoint protein or its ligand.

[0009] In some preferred embodiments, the immune checkpoint inhibitor of the combination is an antibody or antigen-binding fragment thereof that specifically binds to (and inhibits) PD-1, PD-L1, PD-L2, TIGIT, TIM3, LAG3, or CTLA4. By way of non-limiting example, a method of treating and / or preventing cancer in a mammal is provided, the method comprising concurrently administering to a subject effective amounts of (i) a replication-competent oncolytic vaccinia virus by intratumoral injection, and (ii) a CTLA4 inhibitor and / or a PD-1 inhibitor.

[0010] In some preferred embodiments, the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1 or PD-L1, and is preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. In some preferred embodiments, the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to CTLA4, and is preferably selected from the group consisting of ipilimumab and tremelimumab. In some embodiments, multiple checkpoint inhibitors are administered to a subject in parallel with an oncolytic vaccinia virus. In some embodiments, the subject is concurrently administered (a) a CTLA4 inhibitor, a PD-1 inhibitor, and a replication-competent oncolytic vaccinia virus; (b) a CTLA4 inhibitor, an IDO inhibitor, and a replication-competent oncolytic vaccinia virus; (c) a PD-1 inhibitor, an IDO inhibitor, and a replication-competent oncolytic vaccinia virus; or (d) a PD-1 inhibitor, a CTLA4 inhibitor, an IDO inhibitor, and a replication-competent oncolytic vaccinia virus; (e) a LAG3 inhibitor, a PD-1 inhibitor, and a replication-competent oncolytic vaccinia virus; or (f) a TIGIT inhibitor, a PD-1 inhibitor, and a replication-competent oncolytic vaccinia virus. In some embodiments, the replication-competent oncolytic vaccinia virus is a Wyeth strain, a Western Reserve strain, a Lister strain, or a Copenhagen strain. In some embodiments, the vaccinia virus comprises one or more genetic modifications that enhance the selectivity of the virus for cancer cells, and preferably the virus is engineered to lack functional thymidine kinase and / or lack functional vaccinia growth factor.In some embodiments, the vaccinia virus comprises a functional 14L gene and / or F4L gene.

[0011] In some embodiments, the vaccinia virus is a Wyeth, Western Reserve, Lister, or Copenhagen strain that has one or more genetic modifications, such as inactivation of the thymidine kinase (TK) gene and / or the vaccinia virus growth factor (VGF) gene, that enhance selectivity of the vaccinia virus for cancer cells. In related embodiments, the vaccinia virus is engineered to express a cytokine, such as, but not limited to, GM-CSF, IL-2, IL-4, IL-5 IL-7, IL-12, IL-15, IL-18, IL-21, IL-24, IFN-γ, and / or TNF-α, preferably a cytokine selected from IFN-γ, TNF-α, IL-2, GM-CSF, and IL-12. In other related embodiments, the replication-competent oncolytic vaccinia virus is selected from the group consisting of, for example, but not limited to, BAGE, GAGE-1, GAGE-2, CEA, AIM2, CDK4, BMI1, COX-2, MUM-1, MUC-1, TRP-1 TRP-2, GP100, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, SOX2, human papillomavirus-E6, human papillomavirus-E7, ART1, ART4, SART1, SART2, SART3, B-cyclin, β-catenin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2 / Eck, Fra-1 / Fos1 1, ganglioside / GD2, GnT-V, β1,6-N, Her2 / neu, Ki67, Ku70 / 80, IL-13Ra2, MAGE-1, MAGE-3, NY-ESO-1, MART-1, PROX1, PSCA, SOX10, SOX11, survivin, caspase-8, UPAR, CA-125, PSA, pl85HER2, CD5, IL-2R, Fap-α, tenascin, melanoma-associated antigen p97, WT-1, regulator of G protein signaling 5 (RGS5)G-protein signaling 5, survivin (BIRC5 = baculoviral inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible lipid droplet associated protein (HIG2), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Ql-like: RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFIl), lysosomal protein transmembrane 4 alpha (LAPTM4A); RAB1B, RAS oncogene family (RABIB), CD24, homo sapiens thymosin beta 4, X-linked (TMSB4X), homo sapiens S100 calcium binding protein A6 (S100A6), homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin 2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor 1 (ATPIFl), stromal antigen 2 (STAG2)2), nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1), etc. In some embodiments, the tumor antigen is a renal cell carcinoma tumor antigen. In some embodiments, the renal cell cancer tumor antigen is regulator of G-protein signaling 5 (RGS5), survivin (BIRC5 = baculoviral inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible lipid droplet-associated protein (HIG2), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Ql-like: RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFIl), lysosomal protein transmembrane 4 alpha (LAPTM4A); RAB1B, RAS and selected from the group consisting of oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium-binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin 2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor 1 (ATPIFl), stromal antibody 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1).

[0012] In some embodiments, the vaccinia virus is about 10 7 ~about 10 11 Amount of pfu, preferably about 10 8 ~10 10pfu, more preferably about 10 9 ~10 10 pfu. In some embodiments, the checkpoint inhibitor is administered in an amount of about 2 mg / kg to 15 mg / kg.

[0013] In some embodiments, the drug combination is administered to a mammal to treat and / or prevent cancer in the mammal. In some embodiments, the cancer is a solid tumor type cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, head and neck cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, mesothelioma, gastrointestinal cancer, leukemia, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, mesothelioma, colorectal cancer, sarcoma, or thyroid cancer. In other preferred embodiments, the drug combination is administered to a mammal to treat metastasis. In some embodiments, the subject has renal cell carcinoma.

[0014] In preferred embodiments, the mammal treated with the drug combination is a human subject. In related aspects, the subject in need of treatment is a human having a cancer that is refractory (or resistant) to treatment with one or more chemotherapeutic agents and / or refractory to treatment with one or more antibodies. In certain embodiments, the human has a cancer (e.g., colon cancer) that is refractory (or resistant) to treatment including an immune checkpoint inhibitor, and optionally also refractory to treatment with one or more chemotherapeutic agents. In other embodiments, the human in need of treatment is a human who has been identified as a candidate for treatment with one or more immune checkpoint inhibitors.

[0015] In some embodiments, the subject has previously failed at least one chemotherapy or immunotherapy. In some embodiments, the subject has a cancer that is refractory to immune checkpoint inhibitor therapy, preferably the cancer is resistant to treatment with an anti-PD-1 antibody and / or an anti-CTLA-4 antibody. In some embodiments, the subject is identified as a candidate for immune checkpoint inhibitor therapy. In some embodiments, the method includes administering to the subject an additional therapy selected from chemotherapy (alkylating agents, nucleoside analogs, cytoskeleton-modifying agents, cytostatic agents) and radiation therapy. In some embodiments, the method includes administering to the subject an additional oncolytic virus therapy (e.g., rhabdovirus, Semliki Forest Virus). In some embodiments, the subject is a human. In some embodiments, a first dose of a replication-competent oncolytic vaccinia virus and a first dose of an immune checkpoint inhibitor are administered to the subject simultaneously, followed by at least one subsequent sequential co-administration of the virus and the checkpoint inhibitor. In some embodiments, the method comprises at least first, second, and third sequential co-administrations of a replication-competent oncolytic vaccinia virus and a checkpoint inhibitor to a subject. In some embodiments, the method comprises at least first, second, third, and fourth sequential co-administrations of a replication-competent oncolytic vaccinia virus and a checkpoint inhibitor to a subject. In some embodiments, the co-administration of a first dose of a replication-competent oncolytic vaccinia virus and a first dose of an immune checkpoint inhibitor to a subject, and at least one subsequent sequential co-administration of the virus and the checkpoint inhibitor, is followed by administration of at least one dose of the checkpoint inhibitor alone to the subject. In some embodiments, the method comprises an interval of 1 to 3 weeks between the sequential co-administrations of the agents, preferably an interval of about 1 week, about 2 weeks, or about 3 weeks.

[0016] This application demonstrates that intratumoral administration of a replication-competent oncolytic vaccinia virus (i) attracts host immune cells (e.g., tumor-infiltrating T cells) to the tumor and (ii) induces the expression of several checkpoint proteins, including PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT, in tumor cells, thereby sensitizing the tumor cells to concurrent therapy with inhibitors of the checkpoint proteins.

[0017] Thus, in some aspects, the expression level of one or more of these checkpoint proteins is used as a biomarker to select human cancer patients for treatment with the combination therapy described herein based on their expression level. In some embodiments, expression can be measured using any assay for measuring protein levels. In some embodiments, protein expression can be measured using an assay such as, for example, FACS or Nanotring assay.

[0018] In related embodiments, the human in need of treatment has a tumor that does not express a checkpoint protein (e.g., a subject refractory to a checkpoint inhibitor) or a tumor that expresses a checkpoint protein at a relatively low level, in which case the oncolytic vaccinia virus components of the combination therapy are administered in an amount effective to induce expression of a checkpoint protein (e.g., PD-L1), thereby sensitizing the tumor to the immune checkpoint inhibitor of the combination. In some embodiments, the human may have a tumor that does not express PD-1, PD-L1, CTLA-4, LAG3, TIM3, and / or TIGIT, or that expresses one or more of these checkpoint proteins at relatively low levels, and the oncolytic vaccinia virus is administered in an amount effective to sensitize the tumor to an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIM3, and / or TIGIT. In other related embodiments, the level of a checkpoint protein is measured in a tumor prior to administration of the combination therapy of an oncolytic vaccinia virus and a checkpoint inhibitor, and the combination therapy is administered to a subject if it is determined that the checkpoint protein is not expressed or is expressed at a relatively low level in the tumor. In other related embodiments, a method of sensitizing a tumor to a checkpoint inhibitor is provided, the method comprising administering to a human having a tumor an amount of an oncolytic vaccinia virus effective to induce expression of a checkpoint protein in the tumor, and concurrently administering a checkpoint inhibitor to the human. In some aspects, a tumor that does not express a checkpoint protein or expresses a checkpoint protein at a relatively low level means that less than 50%, less than 25%, less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5% of tumor cell lines are positive for the checkpoint protein as assessed by immunohistochemistry (IHC) staining of tumor samples. See, e.g., Ilie et al., Virchows Arch, 468(5):511-525 (2016).In some aspects, the human has non-small cell lung cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, or colon cancer.

[0019] In yet another related embodiment, the human in need of treatment has an immunologically "cold" tumor, meaning that the tumor is essentially or relatively free of immune cells in the tumor microenvironment. Treatment with oncolytic vaccinia virus attracts immune cells (e.g., T cells) to the tumor and acts synergistically with co-administered checkpoint inhibitors to treat the tumor. "Cold" tumors may be identified by methods known in the art, including, but not limited to, single-line or multiplex immunohistochemistry (IHC) for immune markers such as CD3 and CD8 at the tumor center and invasive margin, flow cytometry for phenotyping, genetic analysis of tumor tissue, RNA profiling of tumor tissue, and / or cytokine profiling in serum.

[0020] The oncolytic vaccinia virus and immune checkpoint inhibitor of the combination are administered concurrently (e.g., simultaneously), and may be administered as part of the same formulation or in different formulations. Simultaneous (or parallel) administration means that the first dose of each of the combination partners is administered simultaneously or near simultaneously (within 24 hours of each other, preferably within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours, or 1 hour of each other), and preferably, at least one subsequent dose of each of the combination partners is administered simultaneously or near simultaneously. Thus, in one embodiment, the combination therapy described herein includes a first dose of a replication-competent oncolytic vaccinia virus co-administered with a first dose of a checkpoint inhibitor (e.g., treatment of a subject with the combination therapy necessarily includes at least a first administration, where the oncolytic vaccinia virus and checkpoint inhibitor are administered to the subject simultaneously), and preferably further includes at least one, two, three, four, or more additional consecutive co-administrations of the oncolytic vaccinia virus and checkpoint inhibitor. Thus, a concurrent treatment regimen using a combination of agents may include at least two, at least three, at least four, at least five, at least six, at least seven, or more consecutive doses of the agents co-administered. In preferred embodiments, the interval between consecutively co-administered doses of oncolytic vaccinia virus and checkpoint inhibitor ranges from about one day to about three weeks, or any interval therebetween, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In some preferred embodiments, the interval between consecutively co-administered doses of oncolytic vaccinia virus and checkpoint inhibitor is about one week or about two weeks. Following at least one initial co-administration of a dose of oncolytic vaccinia virus and immune checkpoint inhibitor, one or more doses of the checkpoint inhibitor alone may be administered to the subject.

[0021] In yet another aspect, the present invention provides a commercial package containing as active agents a combination of an oncolytic vaccinia virus described herein and an immune checkpoint inhibitor, together with instructions for their simultaneous use in the treatment and / or prevention of cancer as described herein. In a preferred aspect, the commercial package includes as active agents a combination of a Western Reserve, Copenhagen, Wyeth, or Lister strain of vaccinia virus and an inhibitor of PD-1, PD-L1, TIGIT, or CTLA4.

[0022] In some embodiments, the invention provides methods of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising (a) a replication-competent oncolytic vaccinia virus and (b) an inhibitor of an immune checkpoint protein. In some embodiments, the replication-competent oncolytic virus is administered intratumorally. In some embodiments, the replication-competent oncolytic virus is administered via intravenous administration. In some embodiments, the replication-competent oncolytic virus is administered via intra-arterial administration. In some embodiments, the replication-competent oncolytic virus is administered via intraperitoneal administration. In some embodiments, the replication-competent oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments, the invention provides a method of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus in an amount effective to induce expression of an immune checkpoint protein in the tumor, and (b) an inhibitor of the immune checkpoint protein. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered IV. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT.In some embodiments of the methods of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the methods of treatment, the immune checkpoint protein is PD-L1. In some embodiments of the methods of treatment, the immune checkpoint protein is LAG3. In some embodiments of the methods of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the methods of treatment, the immune checkpoint protein is PD-1. In some embodiments of the methods of treatment, the immune checkpoint protein is TIM3. In some embodiments of the methods of treatment, the tumor is a solid tumor. In some embodiments of the methods of treatment, the tumor is colorectal cancer. In some embodiments of the methods of treatment, the tumor is renal cell carcinoma.

[0023] In some embodiments of the dual combination therapy method, the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1. In some embodiments, the monoclonal antibody that selectively binds to PD-1 or PD-L1 is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab.

[0024] In some embodiments of the dual combination therapy method, the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4, hi some embodiments, the monoclonal antibody that selectively binds to CTLA-4 is selected from the group consisting of ipilimumab and tremelimumab.

[0025] In some embodiments of the dual combination therapy method, the tumor does not express, or expresses at relatively low levels, immune checkpoint proteins prior to administration of the replication-competent oncolytic vaccinia virus.

[0026] In some embodiments of the dual combination therapy method, the method includes measuring the expression level of an immune checkpoint protein in the tumor prior to administration of the combination.

[0027] In some embodiments, the invention provides methods of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus; (b) an inhibitor of PD-1 and / or PD-L1; and (c) an inhibitor of an immune checkpoint protein. In some embodiments, the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of the immune checkpoint protein. In some embodiments, the replication-competent oncolytic virus is administered intratumorally. In some embodiments, the replication-competent oncolytic virus is administered via intravenous administration. In some embodiments, the replication-competent oncolytic virus is administered via intra-arterial administration. In some embodiments, the replication-competent oncolytic virus is administered via intraperitoneal administration. In some embodiments, the replication-competent oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. In some embodiments, the invention provides a method of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus in an amount effective to induce expression of an immune checkpoint protein in the tumor, (b) an inhibitor of PD-1 and / or PD-L1, and (c) an inhibitor of an immune checkpoint protein, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments of the method of treatment, the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3.In some embodiments of the methods of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the methods of treatment, the immune checkpoint protein is TIM3. In some embodiments of the methods of treatment, the tumor is a solid tumor. In some embodiments of the methods of treatment, the tumor is colorectal cancer. In some embodiments of the methods of treatment, the tumor is renal cell carcinoma.

[0028] In some embodiments of the triple combination therapy method, the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1. In some embodiments, the monoclonal antibody that selectively binds to PD-1 or PD-L1 is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab.

[0029] In some embodiments of the triple combination therapy method, the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4. In some embodiments, the monoclonal antibody that selectively binds to CTLA-4 is selected from the group consisting of ipilimumab and tremelimumab.

[0030] In some embodiments of the triple combination therapy method, the tumor does not express immune checkpoint proteins or expresses immune checkpoint proteins at relatively low levels prior to administration of the replication-competent oncolytic vaccinia virus.

[0031] In some embodiments of the triple combination therapy method, the method includes measuring the expression level of a checkpoint protein in the tumor prior to administration of the combination.

[0032] Other embodiments of the invention are contemplated throughout this application. Any embodiment discussed with respect to one aspect of the invention applies equally to other aspects of the invention, and vice versa. It is understood that the embodiments in the Examples section are embodiments of the invention applicable to all aspects of the invention.

[0033] The following drawings form part of this application and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0034] [Figure 1]Figure 1A: Chart illustrating the parallel combination treatment regimen using intratumoral (IT) injection of mJX-594 and an anti-PD-1 checkpoint inhibitor antibody administered intraperitoneally. Eight-week-old BALB / c immunocompetent mice were injected with 5 × 10 Renca (renal carcinoma) cells. When tumors reached 50 mm3 or greater (day 0), mice were treated with PBS (control, days 0, 3, 6, and 9), anti-PD-1 antibody alone (days 0, 3, 6, and 9), mJX-594 alone (days 0, 2, and 4), or parallel delivery of anti-PD-1 and mJX-594 (coadministration of the agents on days 0, 2, and 4, followed by anti-PD-1 alone on day 6; mJX-594 administered intratumorally (IT) at 1 × 107 pfu and anti-PD-1 administered intraperitoneally (IP) at 10 mg / kg). FIG. 1B: Eight-week-old female BALB / c mice were injected with RENCA cells (2×10 6 cells) in 100 μl of PBS under the capsule of the left kidney. On day 10 post-implantation, mice bearing Renca tumors (50–100 mm as visualized using an IVIS® Spectrum in vivo imaging system) were treated intraperitoneally (ip) with the indicated regimens: (i) PBS (control), (ii) vaccinia virus (JX-929) monotherapy (6 × 10 PFU on days 10, 11, and 12 post-implantation for a total of three doses), (iii) anti-PD1 monotherapy (BioXcell, West Lebanon, NH, 100 μl) (on days 10, 11, and 12 post-implantation for a total of three doses), or (iv) JX929 + anti-PD1 concurrent treatment (administered on days 10, 11, and 12 post-implantation, respectively; JX-929 was administered in the morning and ICI was administered 9 hours apart in the afternoon of the same day). FIG. 1C: Balb / c mice bearing Renca tumors over 50 mm 3 were treated intratumorally with four doses of mJX594 (1×10 7 on each of days 0, 3, 6, and 9) or PBS control according to the treatment regimen indicated.

[0035] [Figure 2]Figure 2A: Graph showing the effect of the treatment regimen described in Figure 1A on tumor volume. Parallel combination treatment (PD1 + mJX594) significantly suppressed tumor growth (18 days after implantation) compared to all other treatment groups. Figure 2B: Photographs and graphs showing tumor weight in each treatment group described in Figure 1A. Parallel combination treatment (PD1 + mJX594) exerted a synergistic effect, significantly reducing tumor volume compared to either monotherapy.

[0036] [Figure 3] Parallel combination treatment with IT mJX-594 and anti-PD-1 significantly increased intratumoral T cell infiltration compared to the control and either monotherapy. Mice were treated according to the dosing regimen shown in Figure 1. Figure 3A: Images showing significantly increased CD8 T cell infiltration in both the peritumoral and intratumoral regions in the parallel combination treatment group compared to the control and either monotherapy group. Figure 3B: Graph showing significantly increased peritumoral and intratumoral CD8 T cell infiltration in the parallel combination treatment group compared to the control and either monotherapy group.

[0037] [Figure 4] Parallel combination treatment with IT mJX-594 and anti-PD-1 upregulates PD-L1 expression in tumors. Mice were treated according to the dosing regimen shown in Figure 1. Figure 4A: Images showing significantly increased PD-L1 expression levels in both the peritumoral and central tumor regions in the parallel combination treatment group compared with the control group and either monotherapy group (PD-L1 staining). Figure 4B: Images showing significantly increased apoptosis in tumors in the parallel combination treatment group compared with the control group and either monotherapy group.

[0038] [Figure 5A]CD8 T cells and CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) are increased in the parallel combination treatment group compared to the control group. Figure 5A: Flow cytometry graphs showing positivity for CD8 and Gr-1 in tumors from each treatment group. Compared to either monotherapy group, tumors from the parallel combination treatment group show a significant increase in CD8+ T cells. An increase in MDSCs is also shown compared to either monotherapy. Figure 5B: Bar graphs show flow cytometry results. [Figure 5B] Same as above.

[0039] [Figure 6] This chart illustrates a combination treatment regimen using IT injection of mJX-594 and intraperitoneally delivered anti-PD-1 (+ / - anti-CTLA4) checkpoint inhibitor antibodies. Eight-week-old BALB / c mice were injected subcutaneously with 5 × 10 Renca cells into the right flank. Treatment began when tumors reached 50–100 mm3 in size (day 0). On day 0, mice (bearing Renca tumors) were treated with PBS (control), sequentially delivered mJX-594 + anti-PD-1 antibody combination, parallel-delivered mJX-594 + anti-PD-1 combination, and parallel-delivered triple combination of mJX-594 + anti-PD-1 + anti-CTLA4. mJX-594 was administered IT at 1 × 10 pfu, anti-PD-1 was administered IP at 10 mg / kg, and anti-CTLA4 was administered IP at 4 mg / kg.

[0040] [Figure 7] Graph showing the effect of the treatment regimens described in Figure 6 on tumor volume. Parallel combination treatment with αPD1 + mJX594 and αPD1 + mJX594 + αCTLA4 significantly suppressed tumor growth from day 6 (post-treatment) compared to all other treatment groups. Both parallel combination treatment groups significantly delayed tumor growth compared to the control group and the sequential combination treatment group (mJX594 → αPD1), in which tumor regression was observed from day 12.

[0041] [Figure 8] This chart illustrates a combination treatment regimen using IT injection of mJX-594 and an anti-CTLA4 checkpoint inhibitor antibody delivered intraperitoneally. Eight-week-old BALB / c mice were injected subcutaneously with 5 × 10 Renca cells into the right flank. Treatment began when tumors reached 50–100 mm in size (day 0). On day 0, mice (bearing Renca tumors) were treated with PBS (control), anti-CTLA alone, mJX-594 alone, the combination of mJX-594 and CTLA4 delivered sequentially, and the combination of mJX-594 and CTLA4 delivered concurrently. mJX-594 was administered IT at 1 × 10 pfu, and anti-CTLA4 was administered at 4 mg / kg.

[0042] [Figure 9] Graph showing the effect of the treatment regimens described in Figure 8 on tumor volume. Parallel combination treatment with mJX594 + αCTLA4 significantly delayed tumor growth compared to sequential combination treatment with mJX594 + αCTLA4 and either monotherapy.

[0043] [Figure 10A] The parallel combination of IT mJX594 and anti-CTLA4 significantly increased tumor infiltration of CD8+ T cells and reduced MDSC levels compared to the sequential combination and either monotherapy. Figure 10A: Flow cytometry graphs showing positivity for CD8 and Gr-1 in tumors from each treatment group. Compared to the sequential treatment group and either monotherapy group, tumors from the parallel combination treatment group showed a significant increase in CD8+ T cells and a significant decrease in MDSCs. Figure 10B: Bar graphs show flow cytometry results. [Figure 10B] Same as above.

[0044] [Figure 11]This chart illustrates a combination treatment regimen using intravenous (IV) injection of mJX-594 and an anti-PD1 checkpoint inhibitor antibody delivered intraperitoneally. Eight-week-old BALB / c mice were injected subcutaneously with 5 × 10 Renca cells in the right flank. Treatment began when tumors reached 50–100 mm in size (day 0). On day 0, mice (bearing Renca tumors) were treated with PBS (control), anti-PD1 alone, mJX-594 alone, or co-delivered mJX-594 and anti-PD1. mJX-594 was administered IV at 2 × 10 pfu, and anti-PD1 was administered IP at 10 mg / kg.

[0045] [Figure 12] Graph showing the effect of the treatment regimens described in Figure 11 on tumor volume. Concurrent combination treatment with mJX594 IV + αPD1 was inferior to treatment with mJX594 alone and equivalent to treatment with αPD1 alone.

[0046] [Figure 13] Chart showing fold changes (relative to pre-treatment levels) of immune checkpoint proteins in Renca tumor-bearing mice treated intratumorally with four doses of 1 x 107 pfu of mJX594mJX594 (Wyeth vaccinia virus engineered to contain a disruption of the viral thymidine kinase gene and an insertion of murine GM-CSF) administered every 3 days.

[0047] [Figure 14] We present data on the number of mJX594 injections and tumor growth inhibition. To find the optimal immunotherapy with mJX594, various dose numbers were tested in Renca renal carcinoma. Tumor growth decreased with increasing dose number of mJX594.

[0048] [Figure 15] Images showing intratumoral recruitment of CD8+ T cells after mJX594 treatment.

[0049] [Figure 16] Image showing intratumoral recruitment of CD8+ T cells after mJX594 treatment. Aggregations of CD8+ lymphocytes resembling lymphoid follicles were observed in mJX594-treated tumors.

[0050] [Figure 17] These data demonstrate that mJX594 increases the number of intratumoral CD8+ T cells and enhances their effector function. The ratios of CD8+ T cells and regulatory T cells increased after mJX594 treatment. The expression of ICOS and granzyme B in CD8+ T cells increased after mJX594 treatment. The numbers of CD4+Foxp3+CD25+ regulatory T cells, as well as CD8+ and CD4+ T cells, were simultaneously expanded in the lymphocyte fraction, while the ratios of CD8+ effector T cells and regulatory T cells were further elevated compared with controls. Furthermore, the expression of ICOS and granzyme B (GzB), markers of costimulation and T cell activation, was also increased in CD8+ T cells.

[0051] [Figure 18] These data show that mJX594 treatment repolarized bone marrow cells (Ly6G-Ly6C+↑, Ly6G+Ly6Cint↓). mJX594 increased CD11b+Ly6G-Ly6C+ monocytic myeloid cells and decreased CD11b+Ly6G+Ly6Cint granulocytic myeloid cells. In a subset analysis of bone marrow cell fractions, we found an increase in CD11b+Ly6G-Ly6C+ monocytic myeloid cells and a decrease in CD11b+Ly6G+Ly6Cint granulocytic myeloid cells. This suggests that mJX594 treatment significantly increased the ratio of monocytic to granulocytic cells.

[0052] [Figure 19] Data outlining treatment with depletion antibody experiments. To elucidate which components of the immune system are responsible for the therapeutic benefit following mJX594 treatment, we examined the effects of depletion of CD8+ T cells, CD4+ T cells, and GM-CSF on tumor growth and anti-cancer immunity.

[0053] [Figure 20] These data demonstrate that depletion of T cells or GM-CSF significantly abolished the anti-cancer effects of mJX594. Both CD8+ and CD4+ T cells are essential mediators of the anti-cancer effects of mJX594 treatment, and GM-CSF may also confer immunotherapeutic benefits. While efficient tumor inhibition was detected with mJX594 monotherapy, depletion of either CD8+ or CD4+ T cells abolished the therapeutic effect.

[0054] [Figure 21] These data demonstrate that depletion of CD4+ T cells or GM-CSF reduces intratumoral CD8+ T cell infiltration after mJX594. Depletion of CD4+ T cells reduced intratumoral CD8+ T cells, suggesting that CD4+ T cells were involved in CD8+ T cell activation. GM-CSF depletion reduced both CD8+ and CD4+ T cells. Depletion of CD4+ T cells with mJX594 injection reduced intratumoral CD8+ T cells, suggesting that CD4+ T cells were involved in CD8+ T cell activation. In contrast, depletion of CD8+ T cells did not induce significant changes in CD4+ T cells, suggesting that CD8+ T cells did not affect CD4+ T cells. These data demonstrate that treatment with mJX594 induced the priming of CD8+ and CD4+ T cells, indicators of anti-cancer immunity. Infiltration of CD8+ T cells and CD4+ T cells is an indicator of anti-cancer effect.

[0055] [Figure 22] Summary of the experiment on triple combination therapy of mJX594, αPD-1, and αCTLA-4. mJX594

[0056] [Figure 23]These data demonstrate that the triple combination of mJX594, αPD-1, and αCTLA-4 significantly delayed tumor growth. Of note, the triple combination of mJX594, αPD-1, and αCTLA-4 resulted in complete regression of Renca tumors in some mice (37.5%). Compared to controls, the dual combination of αPD-1 and αCTLA-4 delayed tumor growth by 14.5%, mJX594 monotherapy inhibited tumor growth by 36.9%, and the triple combination demonstrated 76.5% tumor growth inhibition. Of note, the triple combination of mJX594, αPD-1, and αCTLA-4 resulted in complete tumor regression (complete response rate: 37.5%); no complete tumor regression was observed in tumors treated with either dual combination or mJX594 monotherapy.

[0057] [Figure 24] These data demonstrate that triple combination immunotherapy with mJX594, PD-1, and CTLA-4 prolongs overall survival. Mice treated with the triple combination therapy exhibited significant anti-cancer therapeutic effects. Furthermore, to determine whether these potent anti-cancer effects induced by the triple combination therapy could be translated into long-term survival benefits, survival analysis of tumor-bearing mice was performed. Mice treated with the triple combination therapy demonstrated a survival benefit compared with monotherapy or dual combination immunotherapy.

[0058] [Figure 25] Summary of experiments on triple combination treatment of mJX594, αPD-1, and αLAG3.

[0059] [Figure 26]Data showing that the triple combination of mJX594, αPD-1, and αLAG3 moderately delayed tumor growth. In this experiment, the triple combination did not show statistically significant differences compared to the dual combination of mJX594 and αPD-1. Compared to controls, the dual combination of mJX594 and αPD-1 delayed tumor growth by 41.9%, while αLAG3 monotherapy inhibited tumor growth by 5.7%. The triple combination demonstrated 30.1% tumor growth inhibition.

[0060] [Figure 27] Data showing that the triple combination of mJX594, αPD-1, and αLAG3 increased CD8+ and CD4+ T cells. Subset analysis of lymphocyte fractions revealed that the absolute numbers of intratumoral CD8+ and CD4+ T cells increased with dual and triple combination treatment.

[0061] [Figure 28] Summary of experiments on triple combination treatment of mJX594, αPD-1, and αTIGIT.

[0062] [Figure 29] Data showing that the triple combination of mJX594, αPD-1, and αTIGIT moderately delayed tumor growth. The triple combination showed no significant difference compared to the dual combination of mJX594 and αPD-1 in Renca tumors.

[0063] [Figure 30] Data showing that the triple combination of mJX594, αPD-1, and αTIGIT increased CD4+ and CD8+ T cells. Subset analysis of lymphocyte fractions revealed that the absolute numbers of intratumoral CD8+ and CD4+ T cells increased with dual and triple combination treatment.

[0064] [Figure 31]Data show that mJX594 exerted a synergistic effect with anti-PD-1 therapy to delay colon cancer growth. To overcome resistance to ICI monotherapy, we evaluated the combined effect of mJX594 and immune checkpoint blockade in a CT26 colon cancer model. In this model, αPD-1 monotherapy had little effect on tumor growth, and mJX594 monotherapy showed modest tumor growth inhibition. However, dual therapy with mJX594 and an αPD-1 antibody significantly delayed tumor growth.

[0065] [Figure 32] Data showing that the combination of mJX594 and anti-PD1 treatment increased intratumoral CD8+ T cells. Along with tumor growth inhibition, microscopic analysis showed significant recruitment of CD8+ T cells in both the peripheral and central regions of tumors treated with the combination therapy.

[0066] [Figure 33]mJX594 (JX) induces dynamic changes in immune-related genes in the immunosuppressive TME. Renca tumors were implanted sc into BALB / c mice and treated with a single it injection of 1 x 10 pfu of mJX-594 when tumors exceeded 50 mm3. (A) Representative images of JX-treated Renca tumors. Tumors stained for vaccinia virus (VV), CD31, CD8, CD11c, and PD-L1. (B) Quantification of the expression of VV, CD31, blood vessels, CD8, cytotoxic T cells, CD11c, dendritic cells, and PD-L1 cells. (C) Time course of VV, CD8, and PD-L1 in the tumor microenvironment after JX treatment. (D) Images showing upregulated PD-L1 expression (red) in various cell types (green) within the TME after JX treatment. PD-L1 expression was primarily observed on Pan-CK+ tumor cells (arrowheads). Note that some CD11b+ myeloid cells (arrowheads) also rarely expressed PD-L1, whereas CD3+ T cells did not. (E) Heatmap of NanoString immune-related gene expression. Red and green represent up- and down-regulated regions, respectively. (F) Volcano plot showing gene expression in JX-treated tumors. Genes associated with immune stimulation are shown. Red lines indicate p<0.05. (G) Comparison of gene expression associated with inhibitory immune checkpoints (ICs), agonistic ICs, Th1 responses, Th2 responses, TME, and myeloid cells. n=5 per group unless otherwise indicated. Values are mean ± SEM. *p<0.05 vs. control. Scale bar = 50 μm. Some data are also shown in Figure 13.

[0067] [Figure 34]JX inhibited tumor growth, increased T cell infiltration, and modulated myeloid cells. Mice bearing Renca tumors were treated itinerantly with PBS or 1 x 10 pfu of JX one to three times. (A-B) Comparison of tumor growth in JX-treated mice. Average (A) and individual (B) tumor growth curves over time. (C-D) Representative images (C) and comparison (D) of CD8+ T cells in the peritumoral or intratumoral regions of tumors treated one to three times with JX. (E) Representative flow cytometry plots showing CD8+ and CD4+ T cell fractions in tumors. (F) Absolute numbers of CD8+ and CD4+ T cells per gram of tumor calculated from flow cytometry. (G) Comparison of CD45+, CD8+, and CD4+ fractions in tumors treated with triple-dose JX. (H-J) Comparison of CD4+Foxp3+CD25+ (Treg), CD8 / Treg ratio, CD8+ICOS+, and CD8+GzB+ fractions in tumors. (K) Comparison of CD11b+Gr1+ myeloid cell fractions in tumors. (L) Representative flow cytometry plots showing CD11b+ myeloid cell fractions in tumors. (M) Comparison of Ly6G-Ly6C+ monocytic myeloid cell fractions, Ly6G+Ly6Cint granulocytic myeloid cells, and monocytic / granulocytic ratios relative to CD11b+ in tumors. Unless otherwise indicated, n = 5-6 per group. Values are mean ± SEM. *p < 0.05 vs. control. #p < 0.05 vs. JX x1; $p < 0.05 vs. JX x2. ns = not significant. Scale bar = 100 μm. Some data are also shown in Figures 19-21.

[0068] [Figure 35]Intratumoral injection of JX induces CD8+ lymphocyte infiltration in both local and distant tumors. Mice were injected sc with Renca tumors in the right flank and with Renca or CT26 tumors in the left flank. Arrows indicate itJX treatment. (A) Schematic of tumor implantation and treatment, and growth curves of JX-injected and uninjected Renca tumors. (B-C) Representative images of CD8+ T cells (green) in JX-injected and uninjected tumors (B) and comparison (C). (D) Schematic of implantation and treatment, and growth curves of JX-injected and uninjected Renca tumors (C) and comparison (F). (E-F) Representative images of CD8+ T cells (green) in JX-injected and distant tumors (E) and comparison (F). Unless otherwise indicated, n = 5 per group. Values are mean ± SEM. *p < 0.05 vs. control. ns: not significant. Scale bar: 50 μm.

[0069] [Figure 36] Antitumor immunity plays an important role in the overall therapeutic effect of JX. Mice were sc-inoculated with Renca and treated with itJX or with i.p. depleting antibodies against CD8+ T cells, CD4+ T cells, or mouse GM-CSF. (A) Treatment scheme. (B-C) Comparison of tumor growth in mice treated with JX or depleting antibodies. Mean (B) and individual (C) tumor growth curves over time. *p<0.05 vs. control; $p<0.05 vs. αGM-CSF. (D-E) Absolute numbers of CD8+ T cells (D) and CD4+ T cells (E) per gram of tumor treated with JX and immune cell-depleting antibodies. Unless otherwise specified, n=7-8 per group. Values are mean ± SEM. *p<0.05 vs. control; #p<0.05 vs. VX; $p<0.05 vs. αGM-CSF. ns is not significant.

[0070] [Figure 37]Combination therapy with JX and αPD-1 synergistically elicits CD8+ T cell-mediated tumor immunity. Renca tumor-bearing mice were treated with either PBS, JX, αPD-1 antibody, or JX + αPD-1 antibody. (A-B) Comparison of tumor growth in mice treated with JX and / or αPD-1 antibody. Average (A) and individual (B) tumor growth curves over time. (C-D) Representative images (C) and comparison (D) of CD8+ T cells, CD31+ blood vessels, activated caspase 3 (Casp3)+ apoptotic cells, and PD-L1+ cells in tumors treated with JX and / or αPD-1 antibody. (E) Schematic diagram depicting the overcoming of immunosuppressive TME by combination therapy with JX and αPD-1 blockade. Unless otherwise indicated, n = 7 per group. Values are mean ± SEM. *p < 0.05 vs. control. #p<0.05 for JX; $p<0.05 for αPD-1. ns = not significant. Scale bar = 100 μm. Some data are also shown in Figures 19–21.

[0071] [Figure 38] The efficacy of combination immunotherapy using intratumoral JX and systemic ICI was largely unaffected by the treatment schedule. Mice were implanted sc with Renca tumors and treated with JX plus ICI using various schedules. (A) Schematic diagram showing the various treatment schedules. Arrows indicate treatment with either intratumoral delivery of JX (red arrow) or systemic delivery of immune checkpoint blockade (blue arrow). (B-C) Comparison of tumor growth in mice treated with JX and αPD-1 antibody using different time schedules. Average (B) and individual (C) tumor growth curves over time. (D) Representative flow cytometry plots showing tumor-infiltrating CD8+ and CD4+ T cell fractions. (E-F) Comparison of absolute numbers of CD8+, CD4+, CD8+ICOS+, and CD8+GzB+ cells per gram of tumor. Unless otherwise indicated, n = 7 per group. Values are mean ± SEM. *p < 0.05 vs. control. ns is not significant.

[0072] [Figure 39]The triple combination of JX, αPD-1, and αCTLA-4 antibodies results in complete regression and improved overall survival. Mice were implanted sc with Renca tumors and treated with JX in the presence or absence of immune checkpoint blockade against PD-1 and CTLA-4. (A-B) Comparison of tumor growth in mice treated with JX and / or immune checkpoint blockade. Mean (A) and individual (B) tumor growth curves over time. (C) Waterfall plot showing maximum percent change from baseline in tumor size. (D) Kaplan-Meier plot for overall survival. (E) Comparison of tumor size after rechallenge with Renca tumor cells in mice with complete tumor regression. n = 8 per group unless otherwise specified. *p<0.05 vs. control. #p<0.05 vs. JX; $p<0.05 vs. αPD-1 + αCTLA-4. ns = not significant. Some of the data are also shown in Figures 23-24.

[0073] [Figure 40]Triple combination therapy delays tumor growth and metastasis in a spontaneous breast cancer model. Tumor growth was analyzed weekly in spontaneous mammary tumors in MMTV-PyMT mice beginning at 9 weeks of age. Samples were collected at 13 weeks of age. (A) Schematic diagram showing the treatment schedule. Arrows indicate treatment with either it-delivered JX or systemic delivery of αPD-1 and αCTLA-4 antibodies. (B) Representative images showing the macroscopic appearance of tumors. Dotted circles demarcate the boundaries of palpable mammary tumor nodules. (C) Comparison of total tumor burden. Tumor burden was calculated by summing the volume of each tumor nodule per mouse. (D) Comparison of the number of palpable tumor nodules. (E) Comparison of the volume of each tumor nodule. Each tumor nodule in MMTV-PyMT mice was plotted as an individual dot. (F) Kaplan-Meier curve for overall survival. (G) H&E tumor section showing intratumoral area. The acinar structures in the JX and JX+P+C groups were early, less invasive lesions (Ea) with clear borders and surrounding mammary adipose tissue (Adi). In contrast, the invasive ductal carcinomatous areas (Ca) in the control and P+C groups significantly invaded the surrounding tissue, forming solid sheets of tumor cells, with no acinar structures remaining. Scale bar, 200 μm. (H-J) Representative images and comparison of CD8+ T cells (H and I) and CD31+ tumor vessels (H and J) in the tumor. (K) Representative lung sections stained with H&E. Arrows indicate metastatic lesions. Scale bar, 200 μm. (L) Comparison of the number of metastatic colonies per lung section. Unless otherwise specified, n = 6–7 per group. Values are mean ± SEM. *p < 0.05 vs. control. #p<0.05 vs. JX; $p<0.05 vs. αPD-1 + αCTLA-4. ns = not significant. Scale bar = 100 μm.

[0074] [Figure 41] Vaccinia virus is not detected in distant tumors. Robust vaccinia virus (VV) replication (green) is observable in the injected tumor on the right, while vaccinia virus is not detected in the uninjected tumor on the left. Scale bar, 100 μm.

[0075] [Figure 42] Combination therapy with JX and ΛCTLA-4 synergistically elicits CD8+ T cell-mediated tumor immunity. Renca tumor-bearing mice were treated with either PBS, JX, αCTLA-4 antibody, or JX + αCTLA-4 antibody. (A-B) Comparison of tumor growth in mice treated with JX and / or αCTLA-4 antibody. Average (A) and individual (B) tumor growth curves over time. (C-E) Comparison with images of CD8+ T cells (C and D) and CD31+ blood vessels (C and E) in the tumor. (F-H) Absolute numbers of CD45+ immune cells (F), CD8+ T cells (G), and CD4+ cells (H) per gram of tumor treated with JX and / or αCTLA-4 antibody. Values are mean ± SEM. *p<0.05 vs. control. #p<0.05 vs. JX; $p<0.05 vs. αCTLA-4. ns indicates not significant. Scale bar, 100 μm.

[0076] [Figure 43] JX enhances the anti-cancer effects of ΛCTLA-4 immune responses, regardless of treatment schedule. (A-B) Comparison of tumor growth in mice treated with JX and αCTLA-4 antibody using different timing schemes. Average (A) and individual (B) tumor growth curves over time. (C) Representative flow cytometry plots showing tumor-infiltrating CD8+ and CD4+ T cell fractions in tumors. (D-E) Absolute numbers of CD8+, CD4+, CD8+ICOS+, and CD8+GzB+ cells per gram of tumor treated with JX and αCTLA-4 antibody. Values are mean ± SEM. *p<0.05 vs. control. ns indicates not significant. Some data are also shown in Figure 38. DETAILED DESCRIPTION OF THE INVENTION

[0077] I. Selected Definitions The terms "inhibiting," "reducing," or "preventing," or any variations of these terms, as used in the claims and / or specification, include any measurable decrease or complete inhibition that achieves the desired result.

[0078] As used herein, the term "combination" refers to the combined administration of an anticancer agent, i.e., an oncolytic vaccinia virus, and an immune checkpoint inhibitor, which can be administered individually or in different fixed combinations using distinct amounts of the combination partners. The term "combination" also defines a "kit" containing simultaneously administered combination partners. Preferably, the time interval between successive co-administrations of the combination partners is selected so that the combination of agents exhibits a synergistic effect. As used herein, the term "synergistic" or "synergistic effect" means that the effect achieved by the combination of anticancer agents encompassed by the present invention is greater than the sum of the effects produced when the anticancer agents, i.e., an oncolytic vaccinia virus, and an immune checkpoint inhibitor are used as monotherapy. Such a synergistic effect is beneficial in that it provides a higher effect at the same dose and / or prevents or delays the establishment of multidrug resistance.

[0079] As used herein, the term "refractory cancer" refers to a cancer that has failed to respond favorably to anti-neoplastic treatment, or a cancer that has recurred or returned after responding favorably to anti-neoplastic treatment. Thus, as used herein, "cancer refractory to treatment" refers to a cancer that has failed to respond favorably to treatment, or a cancer that is resistant to treatment, or a cancer that has recurred or returned after responding favorably to treatment. For example, such previous treatment may be a chemotherapy regimen, or may be an immunotherapy regimen including the administration of a monoclonal antibody that specifically binds to PD-1, PD-L1, or CTLA4.

[0080] The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0081] It is contemplated that any embodiment discussed herein can be implemented with respect to any method and composition of the invention, and vice versa. Furthermore, the compositions and kits of the invention can be used to practice the methods of the invention.

[0082] Throughout this specification, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0083] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or unless the alternatives are mutually exclusive, but the present disclosure supports the definition referring to alternatives only and referring to "and / or."

[0084] As used in this specification and the claims, the words "comprising" (and any form of including, e.g., "comprise," "comprises"), "having" (and any form of having, e.g., "have," "has"), "inluding" (and any form of including, e.g., "includes," "include") or "containing" (and any form of including, e.g., "contains," "contain") are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0085] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0086] Combination therapy of an oncolytic vaccinia virus and a checkpoint inhibitor has been found to provide unexpected improvements in cancer treatment. When the agents are administered concurrently and an oncolytic vaccinia virus is administered, the agents interact synergistically, even synergistically, to provide significantly improved antitumor effects compared to administration of either agent alone. Surprisingly, these effects are not significantly observed when the agents are administered sequentially. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intratumorally. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intravenously. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intraperitoneally. In some embodiments, the replication-competent oncolytic virus is then delivered exclusively via intratumoral administration. In some embodiments, the replication-competent oncolytic virus is then delivered exclusively via intra-arterial administration. In some embodiments, the checkpoint inhibitor is administered systemically. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the combination therapy results in a synergistic effect when the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically.

[0087] It has further been found that oncolytic vaccinia viruses upregulate the expression of checkpoint proteins, such as PD-1, PD-L1, CTLA-4, TIM3, LAG3, and TIGIT, in human tumors, thereby sensitizing the tumors to treatment with checkpoint inhibitors, and supporting that the combination therapy is useful not only for patients whose tumors express the checkpoint inhibitors of the combination, but also for patients whose tumors do not express the checkpoint inhibitors of the combination, or who express the checkpoint inhibitors at relatively low levels.

[0088] In some embodiments, a combination therapy for use in the treatment and / or prevention of cancer and / or the establishment of metastases in a mammal (preferably a human) is provided, the combination therapy comprising concurrently administering to the mammal (i) a replication-competent oncolytic vaccinia virus and (ii) one or more immune checkpoint inhibitors. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intravenously. In some embodiments, the replication-competent oncolytic vaccinia virus is administered only intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered only intra-arterially. In some embodiments, the combination therapy produces a synergistic effect when the replication-competent oncolytic virus is administered via intraperitoneal administration. In some embodiments, the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically, hi some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. II. Oncolytic Vaccinia Virus

[0089] Vaccinia virus is a large, complex, enveloped virus with a linear, double-stranded DNA genome of approximately 190 kbp and encoding approximately 250 genes. Vaccinia is best known for its role as the vaccine that eradicated smallpox. Since the eradication of smallpox, scientists have explored its use as a tool for gene delivery into biological tissues (gene therapy and genetic engineering). Vaccinia virus is unique among DNA viruses because it replicates exclusively in the cytoplasm of host cells. Therefore, its large genome must encode various enzymes and proteins required for viral DNA replication. During replication, vaccinia produces several infectious forms that differ in their envelope: intracellular mature virions (IMV), intracellular enveloped virions (IEV), cell-associated enveloped virions (CEV), and extracellular enveloped virions (EEV). IMV is the most common infectious form and is thought to be responsible for the spread of infection between hosts, whereas CEV is thought to play a role in cell-to-cell spread, and EEV is thought to be important for long-term, widespread dissemination within the host organism.

[0090] Any known oncolytic strain of vaccinia virus can be co-administered with a checkpoint inhibitor according to the methods described herein. In a preferred embodiment, the replication-competent oncolytic vaccinia virus is the Copenhagen strain, the Western Reserve strain, the Lister strain, or the Wyeth strain, and most preferably the Western Reserve strain or the Wyeth strain. The genome of the Western Reserve vaccinia strain has been sequenced (accession number AY243312). In some embodiments, the replication-competent oncolytic vaccinia virus is the Copenhagen strain. In some embodiments, the replication-competent oncolytic vaccinia virus is the Western Reserve strain. In some embodiments, the replication-competent oncolytic vaccinia virus is the Lister strain. In some embodiments, the replication-competent oncolytic vaccinia virus is the Wyeth strain.

[0091] Replication-competent oncolytic vaccinia viruses can be engineered to lack one or more functional genes to enhance the virus's cancer selectivity. In some preferred embodiments, oncolytic vaccinia viruses are engineered to lack thymidine kinase (TK) activity. TK-deficient vaccinia viruses require thymidine triphosphate for DNA synthesis, resulting in preferential replication in dividing cells, particularly cancer cells. In another aspect, oncolytic vaccinia viruses can be engineered to lack vaccinia virus growth factor (VGF). This secreted protein is produced early in the infection process and acts as a mitogen to prime surrounding cells for infection. In another aspect, oncolytic vaccinia viruses can be engineered to lack both VGF and TK activity. In other aspects, oncolytic vaccinia viruses can be engineered to lack one or more genes involved in evading host interferon (IFN) responses (e.g., E3L, K3L, B18R, or B8R). In some preferred embodiments, the replication-competent oncolytic vaccinia virus is a Western Reserve strain, a Copenhagen strain, a Lister strain, or a Wyeth strain, and lacks a functional TK gene. In other embodiments, the oncolytic vaccinia virus is a Western Reserve strain, a Copenhagen strain, a Lister strain, or a Wyeth strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Western Reserve strain, a Copenhagen strain, a Lister strain, or a Wyeth strain, and lacks a functional TK gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Western Reserve strain and lacks a functional TK gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Copenhagen strain and lacks a functional TK gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Lister strain and lacks a functional TK gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Wyeth strain and lacks a functional TK gene.In some embodiments, the oncolytic vaccinia virus is a Western Reserve strain, a Copenhagen strain, a Lister strain, or a Wyeth strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the oncolytic vaccinia virus is a Copenhagen strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the oncolytic vaccinia virus is a Lister strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the oncolytic vaccinia virus is a Wyeth strain that lacks a functional B18R gene and / or a B8R gene. In some embodiments, the replication-competent oncolytic vaccinia virus is a Western Reserve strain, a Copenhagen strain, a Lister strain, or a Wyeth strain, and lacks a functional TK gene and a functional B18R gene and / or a B8R gene. In some embodiments, the replication-competent oncolytic vaccinia virus contains a functional 14L gene and / or a F4L gene. In some embodiments, the replication-competent oncolytic vaccinia virus does not express a chemokine (e.g., the vaccinia virus does not express CXCL-11).

[0092] In some embodiments, the replication-competent oncolytic vaccinia virus of the combination comprises a functional 14L gene and / or an F4L gene.

[0093] In other embodiments, the replication-competent oncolytic vaccinia virus of the combination does not express a chemokine (eg, the vaccinia virus does not express CXCL-11).

[0094] Heterologous sequences (e.g., encoding cytokines and / or tumor antigens) can be placed under the control of a vaccinia virus promoter and integrated into the vaccinia virus genome. Alternatively, expression of heterologous sequences can be achieved by transfecting shuttle vectors or plasmids, such as those found in Table 1 of Current Techniques in Molecular Biology, (Ed. Ausubel, et al.) Unit 16.17.4 (1998). These contain vaccinia promoter control sequences and introduce heterologous sequences into cells infected with vaccinia virus by homologous recombination. When high-level expression is desired, the strong late vaccinia virus promoter is preferred. Early and intermediate promoters can also be used. In some embodiments, the heterologous sequence is placed under the control of a vaccinia virus promoter containing early and late promoter elements. Suitable early promoters include, but are not limited to, promoters of vaccinia virus genes encoding 42K, 19K, or 25K polypeptides. Suitable early-late promoters include, but are not limited to, promoters of vaccinia virus genes encoding 7.5K polypeptides. Suitable late promoters include, but are not limited to, the promoters of the vaccinia virus genes encoding polypeptides of 11K or 28K. In a related embodiment, heterologous sequences are inserted within the TK and / or VGF sequences to inactivate the TK and / or VGF sequences.

[0095] In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors. In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered intratumorally in combination with one or more checkpoint inhibitors. In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered intravenously (IV, or intravascularly) in combination with one or more checkpoint inhibitors. In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered intraperitoneally (IP) in combination with one or more checkpoint inhibitors. In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered intra-arterially in combination with one or more checkpoint inhibitors. In some embodiments, the replication-competent oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally, and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially, and the checkpoint inhibitor is administered systemically. Intratumoral administration generally involves injection into the tumor mass or into the tumor-associated vasculature. In some aspects, the tumor is imaged before or during viral administration. Intravascular administration generally involves injection into the vascular system and is a form of systemic administration. Intraperitoneal administration generally involves injection into the peritoneal cavity (body cavity). In some embodiments, the replication-competent oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, and both are administered systemically, for example, by IV administration.In some embodiments, a replication-competent oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally and the one or more checkpoint inhibitors are administered systemically, e.g., by IV administration. In some embodiments, a replication-competent oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replication-competent oncolytic vaccinia virus is administered intraperitoneally and the one or more checkpoint inhibitors are administered systemically, e.g., by IV administration. In some embodiments, a replication-competent oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replication-competent oncolytic vaccinia virus is administered intra-arterially and the one or more checkpoint inhibitors are administered systemically, e.g., by IV administration.

[0096] The oncolytic vaccinia viruses described herein can be administered in a single dose or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more). The virus can be administered in doses of 1×10 5 Plaque-forming units (PFU), 5 × 10 5 PFU, 1 × 10 6 PFU, at least 1 × 10 6 PFU, 5 × 10 6 or about 5 x 10 6 PFU, 1 × 10 7 , at least 1 x 10 7 PFU, 1 × 10 8 or about 1 x 10 8 PFU, at least 1 × 10 8 PFU, approximately or at least 5 × 10 8 PFU, 1 × 10 9 or at least 1×10 9 PFU, 5 × 10 9 or at least 5×10 9 PFU, 1 × 10 10 PFU or at least 1 × 10 10 PFU, 5 × 10 10 or at least 5×1010 PFU, 1 × 10 11 or at least 1×10 11 , 1×10 12 or at least 1×10 12 , 1×10 13 or at least 1×10 13 For example, the virus may be administered at a dose of about 10 6 ~10 13 pfu, about 10 7 ~10 13 pfu, about 10 8 ~10 13 pfu, about 10 9 ~10 12 pfu, about 10 8 ~10 12 pfu, about 10 7 ~10 12 pfu, about 10 6 ~10 12 pfu, about 10 6 ~10 9 pfu, about 10 6 ~10 8 pfu, about 10 7 ~10 10 pfu, about 10 7 ~10 9 pfu, about 10 8 ~10 10 pfu, or approximately 10 8 ~10 9 Preferably, the virus is administered in a dose of at least 10 pfu. 7 pfu, 10 7 ~10 10 pfu, 10 7 ~10 9 pfu, 10 7 ~10 8 pfu, 10 8 ~10 10 pfu, 10 8 ~10 9 pfu or 10 9 ~10 10 pfu doses.

[0097] A single dose of virus is intended to refer to an amount administered to a subject or tumor over a period of 0.1, 0.5, 1, 2, 5, 10, 15, 20, or 24 hours (including all values therebetween). The doses may be dispersed over time or by separate injections. Typically, multiple doses are administered to the same target area, such as near a tumor. In some embodiments, the virus dose is delivered by an injection device equipped with a syringe or a single-port needle or a multi-port with a single needle or a multi-prong syringe, or a combination thereof. A single dose of vaccinia virus may be administered, or multiple doses may be administered over a treatment period that may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. For example, the vaccinia virus may be administered every other day, once a week, every other week, or every three weeks for a period of 1, 2, 3, 4, 5, 6 months or more.

[0098] Vaccinia virus may be propagated using the methods described by Earl and Moss in Ausubel et al, 1994, or the methods described in International Patent Application Publication No. WO 2013 / 022764, both of which are incorporated herein by reference. III. Immune Checkpoint Inhibitors (ICIs)

[0099] Immune checkpoint proteins interact with specific ligands that send signals within T cells and inhibit T cell function. Cancer cells exploit this by expressing high levels of checkpoint proteins on their surface, thereby suppressing anti-cancer immune responses.

[0100] Immune checkpoint inhibitors (also referred to as ICIs) for use in the drug combinations described herein are any compounds capable of inhibiting the function of immune checkpoint proteins. Inhibition includes reduction of function as well as complete blockage. In particular, the immune checkpoint proteins are human checkpoint proteins. Therefore, it is preferred that the immune checkpoint inhibitor is an inhibitor of a human immune checkpoint.

[0101] Checkpoint proteins include, but are not limited to, CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT, and / or IDO. The pathway including LAG3, BTLA, B7-H3, B7-H4, TIM3, and KIR is recognized in the art as constituting an immune checkpoint pathway similar to the CTLA-4 and PD-1 dependent pathway (see, e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT, and / or IDO. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and / or TIM3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3.

[0102] In some embodiments, the immune checkpoint inhibitor of the combination is an antibody. As used herein, the term "antibody" encompasses natural and engineered antibodies, as well as full-length antibodies or functional fragments or analogs thereof (e.g., retaining an antigen-binding portion) capable of binding, for example, to a target immune checkpoint or epitope. Antibodies for use in accordance with the methods described herein may be from any origin, including, but not limited to, human, humanized, animal, or chimeric, and may be of any isotype, preferably IgG1 or IgG4 isotype, and may be glycosylated or aglycosylated. The term antibody also includes bispecific or multispecific antibodies, so long as the antibody exhibits the binding specificities described herein.

[0103] Humanized antibodies refer to non-human (e.g., mouse, rat, etc.) antibodies whose protein sequences have been altered to increase their similarity to human antibodies. Chimeric antibodies refer to antibodies that comprise one or more elements of one species with one or more elements of another species, such as a non-human antibody with at least a portion of a human immunoglobulin constant region (Fc).

[0104] Many antibody types can be engineered for use in the combinations of the present invention, representative examples of which include Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CHI domains), F(ab')2 fragments (bivalent fragments comprising two Fab fragments linked by at least one disulfide bridge at the hinge region), Fd fragments (composed of the VH and CHI domains), Fv fragments (composed of the VL and VH domains of one arm of an antibody), dAb fragments (composed of a single variable domain fragment (VH or VL domain)), and single-chain Fv (scFv) (composed of Fv fragments of two domains, VL and VH, fused together and finally containing a linker to form a single protein chain).

[0105] In some embodiments, the immune checkpoint protein inhibitor (also referred to as ICI) of the combination therapy is an antibody or fragment thereof that specifically binds to an immune checkpoint protein selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, and TIGIT. In particularly preferred embodiments, the immune checkpoint inhibitor is a monoclonal antibody, fully human antibody, chimeric antibody, humanized antibody, or fragment thereof that can at least partially antagonize CTLA4, PD-1, PD-L1, PD-L2, TIM3, LAG3, or TIGIT. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to CTLA4. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to PD-1. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to PD-L1. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to PD-L2. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to B7-H3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to B7-H4. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to TIM3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to GAL9. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to LAG3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to VISTA. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to KIR.In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to BTLA. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds to TIGIT.

[0106] In some embodiments, the drug combination comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. The fully human CTLA-4 nucleic acid sequence can be found in GenBank under accession number LI 5006. Monoclonal antibodies that specifically bind to CTLA4 include, but are not limited to, ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca / MedImmune), as well as the antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 086014, the contents of each of which are incorporated herein by reference. Examples include antibodies disclosed in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281 and 8,491,895, which are incorporated herein by reference, or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

[0107] In some embodiments, the drug combination comprises the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1. The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank under accession numbers U64863 and NP_005009.2. Monoclonal antibodies against PD-1 include, but are not limited to, lambrolizumab (e.g., disclosed as hPD109A in U.S. Pat. No. 8,354,509, incorporated herein by reference, and its humanized derivatives h409A11, h409A16, and h409A17), nivolumab (Opdivo®; Bristol-Myers Squibb; code name BMS-936558), disclosed in U.S. Pat. No. 8,008,449, incorporated herein by reference, pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)), or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described in, for example, International Patent Application Publication Nos. 2004 / 004771, 2004 / 056875, 2006 / 121168, 2008 / 156712, 2009 / 014708, 2009 / 114335, 2013 / 043569, and 2014 / 047350. In related embodiments, the checkpoint inhibitor of the drug combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

[0108] In some embodiments, the drug combination comprises the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1. Monoclonal antibodies against PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in International Patent Application Publication No. WO 2009 / 114335), BMS-936559 (MDX-1105), atezolizumab (Genentech / Roche; MPDL33280A), disclosed in U.S. Pat. No. 8,217,149, the contents of which are incorporated herein by reference, durvalumab (AstraZeneca / MedImmune; MEDI4736), disclosed in U.S. Pat. No. 8,779,108, which is incorporated herein by reference, MIH1 (Affymetrix, available from eBioscience (16.5983.82)), and avelumab (MSB0010718C; Merck KGaA), or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as the PD-L2-Fc fusion protein known as AMP-224 (disclosed in Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)).

[0109] In some embodiments, the drug combination comprises the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a PD-L2 inhibitor, such as MIH18 (disclosed in Pfistershammer et al., Eur J Immunol. 36:1104-1113 (2006)).

[0110] In some embodiments, the drug combination is a combination of a Western Reserve vaccinia virus strain, a Wyeth vaccinia virus strain, a Lister vaccinia virus strain, or a Copenhagen vaccinia virus strain with, for example, a soluble LAG3 (see U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009), IMP321 or LAG3-Ig), as disclosed in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Patent No. 5,773,578, incorporated herein by reference, or LAG3 inhibitors such as BMS-986016 or other fully human antibodies that block LAG3, as disclosed in U.S. Patent Application Publication No. 2011-0150892, incorporated herein by reference.

[0111] In some embodiments, the drug combination includes a Western Reserve vaccinia virus strain, a Wyeth vaccinia virus strain, a Lister vaccinia virus strain, or a Copenhagen vaccinia virus strain and a BLTA inhibitor, such as antibody 4C7, disclosed in U.S. Pat. No. 8,563,694, incorporated herein by reference.

[0112] In some embodiments, the drug combination includes the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a B7H4 checkpoint inhibitor, such as an antibody disclosed in U.S. Patent Application Publication No. 2014 / 0294861, which is incorporated herein by reference, or a soluble recombinant form of B7H4, such as disclosed in U.S. Patent Application Publication No. 20120177645, which is incorporated herein by reference.

[0113] In some embodiments, the drug combination comprises the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a B7-H3 checkpoint inhibitor, such as the antibody MGA271, disclosed as BRCA84D, or derivatives disclosed, for example, in U.S. Patent Application Publication No. 20120294796, which is incorporated herein by reference.

[0114] In some embodiments, the drug combination includes the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a TIM3 checkpoint inhibitor, such as the antibodies disclosed in U.S. Pat. No. 8,841,418, incorporated herein by reference, or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008).

[0115] In some embodiments, the drug combination includes the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a KIR checkpoint inhibitor, such as lirilumab (disclosed in Romagne et al., Blood, 114(13):2667-2677 (2009)).

[0116] In some embodiments, the drug combination comprises a Western Reserve vaccinia virus strain, a Wyeth vaccinia virus strain, a Lister vaccinia virus strain, or a Copenhagen vaccinia virus strain and a TIGIT inhibitor. The TIGIT checkpoint inhibitor preferably inhibits the interaction of TIGIT with poliovirus receptor (CD155), including, but not limited to, antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference), and U.S. Patent Application Publication Nos. 20160355589 and 20160176963 (incorporated herein by reference), and poliovirus receptor variants, such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

[0117] In some embodiments, the drug combination comprises a Western Reserve vaccinia virus strain, a Wyeth vaccinia virus strain, a Lister vaccinia virus strain, or a Copenhagen vaccinia virus strain and an IDO inhibitor. IDO is recognized as an immune checkpoint protein, and its expression in tumor cells contributes to immune tolerance by shutting down effector T cells. IDO is thought to contribute to resistance to anti-CTLA-4 therapy. IDO inhibitors for use in accordance with the methods described herein include, but are not limited to, tryptophan mimetics such as D-1MT (the D isoform of 1-methyl-DL-tryptophan (MT)), L-1MT (the L isoform of MT), MTH-Trp (methylthiohydantoin-dl-tryptophan; a transcriptional repressor of IDO), and β-carbolines, e.g., naphthoquinone-based agents, indole mimetics such as S-allyl-brassinin, S-benzyl-brassinin, 5-bromo-brassinin, and phenylimidazole-based agents, 4-phenylimidazole, exiguamine A, epacadostat, rosmarinic acid, norharmane, and NSC 401366. Preferred IDO inhibitors include INCB 024360 (epacadostat; 1,2,5-oxadiazole-3-carboximidamide, 4-((2-((aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-, (C(Z))-; Incyte), indoximod (NLG2101; D-1MT; NewLink Genetics), IDO peptide vaccine (University of Copenhagen), and NLG919 (NewLink Genetics).

[0118] In some embodiments, the drug combination comprises the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strains, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1, and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank under accession numbers U64863 and NP_005009.2. Monoclonal antibodies against PD-1 include, but are not limited to, lambrolizumab (e.g., disclosed as hPD109A in U.S. Pat. No. 8,354,509, incorporated herein by reference, and its humanized derivatives h409A11, h409A16, and h409A17), nivolumab (Opdivo®; Bristol-Myers Squibb; code name BMS-936558), disclosed in U.S. Pat. No. 8,008,449, incorporated herein by reference, pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)), or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described, for example, in International Patent Application Publication Nos. 2004 / 004771, 2004 / 056875, 2006 / 121168, 2008 / 156712, 2009 / 014708, 2009 / 114335, 2013 / 043569, and 2014 / 047350. In related embodiments, the checkpoint inhibitor of the drug combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1). The complete human CTLA-4 nucleic acid sequence can be found in GenBank under accession number LI 5006.Monoclonal antibodies that specifically bind to CTLA4 include, but are not limited to, ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca / MedImmune), as well as the antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 086014, the contents of each of which are incorporated herein by reference. Examples include antibodies disclosed in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281 and 8,491,895, which are incorporated herein by reference, or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

[0119] In some embodiments, the combination of agents comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. Monoclonal antibodies against PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in International Patent Application Publication No. WO 2009 / 114335), BMS-936559 (MDX-1105), atezolizumab (Genentech / Roche; PDL33280A), disclosed in U.S. Pat. No. 8,217,149, the contents of which are incorporated herein by reference, durvalumab (AstraZeneca / MedImmune; MEDI4736), disclosed in U.S. Pat. No. 8,779,108, which is incorporated herein by reference, MIH1 (Affymetrix, available from eBioscience (16.5983.82)), and avelumab (MSB0010718C; Merck KGaA), or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as the PD-L2-Fc fusion protein known as AMP-224 (disclosed in Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)). The complete human CTLA-4 nucleic acid sequence can be found in GenBank under accession number LI 5006.Monoclonal antibodies that specifically bind to CTLA4 include, but are not limited to, ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca / MedImmune), as well as the antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 086014, the contents of each of which are incorporated herein by reference. Examples include antibodies disclosed in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281 and 8,491,895, which are incorporated herein by reference, or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

[0120] In some embodiments, the drug combination comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1, and a soluble LAG3 (see U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009), IMP321 or LAG3-Ig, as disclosed in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Patent No. 5,773,578, incorporated herein by reference, or LAG3 inhibitors such as BMS-986016 or other fully human antibodies that block LAG3, as disclosed in U.S. Patent Application Publication No. 2011-0150892, incorporated herein by reference. The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank under accession numbers U64863 and NP_005009.2. Monoclonal antibodies against PD-1 include, but are not limited to, lambrolizumab (e.g., disclosed as hPD109A in U.S. Pat. No. 8,354,509, incorporated herein by reference, and its humanized derivatives h409A11, h409A16, and h409A17), nivolumab (Opdivo®; Bristol-Myers Squibb; code name BMS-936558), disclosed in U.S. Pat. No. 8,008,449, incorporated herein by reference, pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)), or antibodies comprising the heavy and light chain regions of these antibodies.Other anti-PD-1 antibodies are described in, for example, International Patent Application Publication Nos. 2004 / 004771, 2004 / 056875, 2006 / 121168, 2008 / 156712, 2009 / 014708, 2009 / 114335, 2013 / 043569, and 2014 / 047350. In related embodiments, the checkpoint inhibitor of the drug combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

[0121] In some embodiments, the drug combination comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a soluble LAG3 (see U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009), IMP321 or LAG3-Ig), as disclosed in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Patent No. 5,773,578, incorporated herein by reference, or LAG3 inhibitors such as BMS-986016 or other fully human antibodies that block LAG3, as disclosed in U.S. Patent Application Publication No. 2011-0150892, incorporated herein by reference. Monoclonal antibodies against PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in International Patent Application Publication No. WO 2009 / 114335), BMS-936559 (MDX-1105), atezolizumab (Genentech / Roche; MPDL33280A), disclosed in U.S. Pat. No. 8,217,149, the contents of which are incorporated herein by reference, durvalumab (AstraZeneca / MedImmune; MEDI4736), disclosed in U.S. Pat. No. 8,779,108, which is incorporated herein by reference, MIH1 (Affymetrix, available from eBioscience (16.5983.82)), and avelumab (MSB0010718C; Merck KGaA), or antibodies comprising the heavy and light chain variable regions of any of these antibodies.In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as the PD-L2-Fc fusion protein known as AMP-224 (disclosed in Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)).

[0122] In some embodiments, the drug combination includes the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strains, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1, and a TIM3 checkpoint inhibitor, such as those disclosed in U.S. Patent No. 8,841,418, incorporated herein by reference, or the anti-human TIM3-blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008). The complete nucleotide and amino acid sequences of human PD-1 can be found in GenBank under accession numbers U64863 and NP_005009.2. Monoclonal antibodies against PD-1 include, but are not limited to, lambrolizumab (e.g., disclosed as hPD109A in U.S. Pat. No. 8,354,509, incorporated herein by reference, and its humanized derivatives h409A11, h409A16, and h409A17), nivolumab (Opdivo®; Bristol-Myers Squibb; code name BMS-936558), disclosed in U.S. Pat. No. 8,008,449, incorporated herein by reference, pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)), or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described in, for example, International Patent Application Publication Nos. 2004 / 004771, 2004 / 056875, 2006 / 121168, 2008 / 156712, 2009 / 014708, 2009 / 114335, 2013 / 043569, and 2014 / 047350. In related embodiments, the checkpoint inhibitor of the drug combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

[0123] In some embodiments, the combination of agents includes the Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a TIM3 checkpoint inhibitor, such as the antibodies disclosed in U.S. Pat. No. 8,841,418, incorporated herein by reference, or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008). Monoclonal antibodies against PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in International Patent Application Publication No. WO 2009 / 114335), BMS-936559 (MDX-1105), atezolizumab (Genentech / Roche; MPDL33280A), disclosed in U.S. Pat. No. 8,217,149, the contents of which are incorporated herein by reference, durvalumab (AstraZeneca / MedImmune; MEDI4736), disclosed in U.S. Pat. No. 8,779,108, which is incorporated herein by reference, MIH1 (Affymetrix, available from eBioscience (16.5983.82)), and avelumab (MSB0010718C; Merck KGaA), or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as the PD-L2-Fc fusion protein known as AMP-224 (disclosed in Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)).

[0124] In some embodiments, the drug combination comprises a Western Reserve vaccinia virus strain, a Wyeth vaccinia virus strain, a Lister vaccinia virus strain, or a Copenhagen vaccinia virus strain and a TIGIT inhibitor. The TIGIT checkpoint inhibitor preferably inhibits the interaction between poliovirus receptor (CD155) and TIGIT, and examples of TIGIT checkpoint inhibitors include, but are not limited to, antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference), and U.S. Patent Application Publication Nos. 20160355589 and 20160176963 (incorporated herein by reference), and poliovirus receptor variants, such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

[0125] In some embodiments, the drug combination comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and an IDO inhibitor (indoleamine-pyrrole 2,3-dioxygenase). IDO inhibitors include metabolic inhibitors, preferably those that inhibit metabolic pathways, including, but not limited to, norharman (see Chiarugi A, et al., "Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages", Journal of Leukocyte Biology. 68(2):260-6. (2000)), rosmarinic acid (see Lee HJ, et al., "Rosmarinic acid inhibits indoleamine 2,3-dioxygenase expression in murine dendritic cells", Biochemical Pharmacology. 73(9):1412-21 (2007)), COX-2 inhibitors (see Cesario A, et al., "The interplay between indoleamine 2,3-dioxygenase 1 (IDO1) and cyclooxygenase (COX)-2 in chronic inflammation and cancer", Current Medicinal Chemistry. 18(15):2263-71(2011)), 1-methyltryptophan (Hou DY, et al., "Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses". Cancer Research. 67(2):792-801(2007) and Chauhan N, et al., (April 2009). "Reassessment of the reaction mechanism in the heme dioxygenases". Journal of the American Chemical Society. 131(12):4186-7(2009)), such as the specific racemer 1-methyl-D-tryptophan (known as indoximod) (clinical trial candidate), epacadostat (INCB24360), navoximod (GDC-0919) (see Jochems C, et al., "The IDOl selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells", Oncotarget. 7(25):37762-37772. (2016)), and / or BMS-986205. In some embodiments, the IDO inhibitor is selected from the group consisting of norharman, rosmarinic acid, a COX-2 inhibitor, 1-methyltryptophan, indoximod, epacadostat (INCB24360), navoximod (GDC-0919), and / or BMS-986205.

[0126] As known to those skilled in the art, alternative and / or equivalent names may be used for the specific antibodies described above, and such alternative and / or equivalent names are interchangeable in the context of the present invention.

[0127] In some embodiments, the drug combinations described herein include (i) two or more immune checkpoint inhibitors and (ii) a replication-competent oncolytic vaccinia virus. In a preferred embodiment, a PD-1 inhibitor and a CTLA-4 inhibitor are administered in parallel with the vaccinia virus. Other examples include, but are not limited to, the parallel administration of a LAG3 inhibitor and a PD-1 inhibitor with the vaccinia virus, or the parallel administration of a LAG3 inhibitor and a PD-L1 inhibitor. Other examples include the parallel administration of an IDO inhibitor and a CTLA-4 inhibitor and / or a PD-1 inhibitor. In some embodiments, the IDO inhibitor is selected from the group consisting of norharman, rosmarinic acid, a COX-2 inhibitor, 1-methyltryptophan, indoximod, epacadostat (INCB24360), navoximod (GDC-0919), and / or BMS-986205. IV. Cytokines

[0128] In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination comprises a heterologous sequence encoding a cytokine, which cytokine is expressed by the virus.

[0129] In some embodiments, a replication-competent oncolytic vaccinia virus is provided that is engineered to express a cytokine selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-24 (IL-24), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). In particularly preferred embodiments, the replication-competent oncolytic vaccinia virus is the Wyeth strain, Western Reserve strain, Copenhagen strain, or Lister strain. In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express GM-CSF. In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-2 (IL-2). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-4 (IL-4). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-5 (IL-5). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-7 (IL-7). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-12 (IL-12). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-15 (IL-15). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-18 (IL-18). In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-21 (IL-21), hi some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express interleukin-24 (IL-24), interferon-γ (IFN-γ).In some embodiments, the replication-competent oncolytic vaccinia virus is engineered to express tumor necrosis factor-α (TNF-α). In some embodiments, the replication-competent oncolytic vaccinia virus is mJX594 and is engineered to express GM-CSF. V. Tumor Antigens

[0130] In some embodiments, the replication-competent oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and optionally a cytokine, wherein the tumor antigen and optionally a cytokine are expressed in cells infected with the virus, preferably in tumor cells. Tumor antigens include tumor-specific antigens and tumor-associated antigens. The replication-competent oncolytic vaccinia virus may express a full-length tumor antigen or an immunogenic peptide thereof. In some embodiments, the cytokine is expressed in cells infected with the replication-competent oncolytic vaccinia virus. In some embodiments, the cytokine is expressed in cells infected with the virus. In some embodiments, the cell is a tumor cell.

[0131] In some embodiments, tumor antigens include, but are not limited to, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase-V, p-15, gp100, MART-1 / Melan A, TRP-1 (gp75), TRP-2, tyrosinase, cyclin-dependent kinase 4, β-catenin, MUM-1, CDK4, HER-2 / neu, human papillomavirus-E6, human papillomavirus E7, CD20, carcinoembryonic antigen (CEA), epidermal growth factor receptor, MUC-1, caspase-8, CD5, mucin-1, Lewisx, CA-125, p185HER2, IL-2R, Fap-a, tenascin, metalloprotease-associated antigen, CAMPATH-1, RCC: regulator of G protein signaling 5 (RGS5), survivin (B IRC5 = baculovirus inhibitor of apoptosis repeat 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible lipid droplet-associated protein (HIG2), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Ql-like: RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFIl), lysosomal protein transmembrane 4 alpha (LAPTM4A); RAB1B, RAS Examples of antigens include the oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium-binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin 2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor 1 (ATPIF1), stromal antibody 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). In some embodiments, the antigen is an antigen listed in U.S. Patent No. 9,919,047, which is incorporated herein by reference in its entirety.In some embodiments, the tumor antigen is a renal cell cancer tumor antigen. In some embodiments, the renal cell cancer tumor antigen is a renal cell cancer tumor antigen, such as regulator of G protein signaling 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis protein repeat 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible lipid droplet-associated protein (HIG2), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Ql-like: RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFIl), lysosomal protein transmembrane 4 alpha (LAPTM4A); RAB1B, RAS and selected from the group consisting of oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium-binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin 2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor 1 (ATPIFl), stromal antibody 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1).In some embodiments, the tumor antigen may be, but is not limited to, KS 1 / 4 pan-cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphatase, prostate-specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, CEA, polymorphic epithelial mucin antigen, milk fat globule antigen, colon tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), Burkitt's lymphoma antigen-38.13, CD19, B lymphoma antigen-CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3), tumor-specific transplantation type of cell-surface antigen (TSTA), or other antigens. antigens) (e.g., virally-induced tumor antigens including T antigens of DNA tumor viruses, and envelope antigens of RNA tumor viruses), carcinoembryonic antigen-alpha-fetoprotein, e.g., colon CEA, bladder tumor carcinoembryonic antigen, differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigens, leukemia T-cell antigen-Gp37, neoglycoproteins, sphingolipids, breast cancer antigens (e.g., EGFR (epidermal growth factor receptor) HER2 antigen (pl85). HER2 ) and HER2 neu epitope), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigens (e.g., I antigen present in fetal erythrocytes and primary endoderm, I antigen present in adult erythrocytes and preimplantation embryos, I (Ma) present in gastric adenocarcinoma, Ml8 and M39 present in breast epithelium, SSEA-1 present in bone marrow cells, VEP8, VEP9, Myl, VIM-D5, and D present in colon cancer) 156 -22, TRA-1-85 (blood group H) present in colon adenocarcinoma, C14, F3 present in lung adenocarcinoma, AH6 present in gastric cancer, Y hapten present in embryonal carcinoma cells, Le y, TL5 (blood group A) present in A431 cells, EGF receptor, E1 series (blood group B) present in pancreatic cancer, FC10.2 present in embryonal carcinoma cells, gastric adenocarcinoma antigen present in adenocarcinoma, CO-514 (blood group Le a ), NS-10 present in adenocarcinoma, CO-43 present in the EGF receptor of A431 cells (blood group Le b ), G49, MH2 (blood group ALe) present in colon adenocarcinoma b / Le y ), 19.9 present in colon cancer, gastric cancer mucin, T5A7 present in bone marrow cells, R present in melanoma 24 , 4.2 present in embryonal carcinoma cells, G D3 , D1.1, OFA-1, G M2 , OFA-2, G D2 and M1:22:25:8, and SSEA-3 and SSEA-4 present in 4- to 8-cell stage embryos), T-cell receptor-derived peptides from cutaneous T-cell lymphoma, C-reactive protein (CRP), cancer antigen-50 (CA-50), breast cancer-associated cancer antigen 15-3 (CA15-3), cancer antigen-19 (CA-19) and gastrointestinal cancer-associated cancer antigen-242, cancer-associated antigen (CAA), chromogranin A, epithelial mucin antigen (MC5), human epithelial-specific antigen (E1A), These include Lewis(a) antigen, melanoma antigen, melanoma-associated antigens 100, 25 and 150, mucin-like cancer-associated antigen, multidrug resistance-associated protein (MRPm6), multidrug resistance-associated protein (MRP41), Neu oncogene protein (C-erbB-2), neuron-specific enolase (NSE), P-glycoprotein (mdrl gene product), multidrug resistance-associated antigen, p170, multidrug resistance-associated antigen, prostate-specific antigen (PSA), CD56 and NCAM.

[0132] In some embodiments, other tumor antigens include, but are not limited to, AIM2 (absent in melanoma 2), BMI1 (BMI1 polycomb ring finger oncogene), COX-2 (cyclooxygenase-2), EGFRvIII (epidermal growth factor receptor variant III), EZH2 (enhancer of the thymus homolog 2), LICAM (human LI cell adhesion molecule), Livin, Livin beta, MRP-3 (multidrug resistance protein 3), Nestin, OLIG2 (oligodendrocyte transcription factor), SOX2 (SRY-related HMG-box 2), ART1 (T cell recognition antigen 1), ART4 (T cell recognition antigen 4), SARTl (T cell recognition squamous cell carcinoma antigen 1), SART2, SART3, B-cyclin, Gli1 (glioma-associated oncogene homolog 1), Cav-1 (caveolin-1), cathepsin B, CD74 (cluster of differentiation antigen 74), E-cadherin (epithelial calcium-dependent adhesion), EphA2 / Eck (EPH receptor A2 / epithelial kinase), Fra-l / Fosl These include 1 (fos-related antigen 1), Ki67 (nuclear proliferation-associated antigen of the antibody Ki67), Ku70 / 80 (human Ku heterodimeric protein subunit), IL-13Ra2 (interleukin-13 receptor subunit alpha-2), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), PROX1 (prospero homeobox protein 1), PSCA (prostate stem cell antigen), SOX10 (SRY-related HMG box 10), SOX11, survivin, UPAR (urokinase-type plasminogen activator receptor), and WT-1 (Wilms tumor protein 1). VI. Treatment Regimens and Drug Formulations

[0133] The replication-competent oncolytic vaccinia virus and immune checkpoint inhibitor of the drug combination are administered simultaneously, and the oncolytic vaccinia virus is delivered by intratumoral injection. Simultaneous administration may be in the form of a single fixed combination containing these agents, or by simultaneously administering each agent in separate formulations. In some embodiments, the replication-competent oncolytic vaccinia virus and the PD-1 or PD-L1 immune checkpoint inhibitor of the drug combination are administered simultaneously. In some embodiments, the replication-competent oncolytic vaccinia virus and the CTLA-4 immune checkpoint inhibitor of the drug combination are administered simultaneously. In some embodiments, the replication-competent oncolytic vaccinia virus and the TIGIT immune checkpoint inhibitor of the drug combination are administered simultaneously. In some embodiments, the replication-competent oncolytic vaccinia virus, the PD-1 or PD-L1 immune checkpoint inhibitor, and the CTLA-4 immune checkpoint inhibitor of the drug combination are administered simultaneously. In some embodiments, the replication-competent oncolytic vaccinia virus, the PD-1 or PD-L1 immune checkpoint inhibitor, and the TIGIT immune checkpoint inhibitor of the drug combination are administered simultaneously. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, and / or intraperitoneally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intravenously. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intraperitoneally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intraarterially. In some embodiments, the replication-competent oncolytic vaccinia virus is administered exclusively intratumorally. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically.In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen, and optionally a cytokine, wherein the tumor antigen and optionally a cytokine are expressed in cells infected with the virus, preferably in tumor cells.

[0134] In some embodiments, the present invention provides methods of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus; and (b) an inhibitor of an immune checkpoint protein. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is PD-L1. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is PD-1. In some embodiments of the method of treatment, the immune checkpoint protein is TIM3. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally, IV, and / or intraperitoneally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered IV. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intraperitoneally. In some embodiments, the replication-competent oncolytic vaccinia virus is administered intra-arterially. In some embodiments, the replication-competent oncolytic vaccinia virus is administered exclusively intratumorally. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically.In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen, and optionally a cytokine, wherein the tumor antigen and optional cytokine are expressed in cells infected with the virus, preferably in tumor cells.

[0135] In some embodiments, the present invention provides methods of treating a tumor in a human, the method comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus; (b) an inhibitor of PD-1 and / or PD-L1; and (c) an inhibitor of an immune checkpoint protein. In some embodiments of the method of treatment, the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma. In some embodiments, the replication-competent oncolytic vaccinia virus is administered via intratumoral, IV, and / or intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered via intratumoral administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered via IV administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered via intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered exclusively via intratumoral administration. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically.In some embodiments, the replication-competent oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen, and optionally a cytokine, which are expressed in cells infected with the virus, preferably in tumor cells.

[0136] In some embodiments of the methods of treatment, the tumor does not express immune checkpoint proteins or expresses immune checkpoint proteins at relatively low levels prior to administration of the replication-competent oncolytic vaccinia virus. In some embodiments, a high level is indicated by a tumor proportional score of 50% or greater. In some embodiments, a high level is indicated by a tumor proportional score of greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, about 99%, or about 100%. In some embodiments, a high level relative to a previously treated tumor is indicated by a tumor proportional score of greater than 1%. In some embodiments, a high level is indicated by 50% or more PD-L1-expressing tumor cells (e.g., more than 50% of tumor cells express PD-L1). In some embodiments, a high level is indicated by greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, about 99%, or about 100% PD-L1-expressing tumor cells. In some embodiments, a high level for a previously treated tumor is indicated by greater than 1% of tumor cells expressing PD-L1 (e.g., greater than 1% of tumor cells express PD-L1). In some embodiments, any PD-L1 diagnostic test can be employed to measure PD-L1 expression. In some embodiments, when the PD-L1 checkpoint is measured, the PD-L1 DaKo Companion Diagnostic test is employed to measure the level of PD-L1.

[0137] In some embodiments of the methods of treatment, the methods include measuring the expression level of a checkpoint protein in the tumor prior to administering the combination.

[0138] The administration of the oncolytic vaccinia virus and immune checkpoint inhibitor will follow the general protocol for the administration of each specific treatment, taking into account the toxicity, if any, associated with the treatment. It is expected that treatment cycles will be repeated as necessary. It is also contemplated that various standard therapies and surgical interventions may be applied in addition to the combination therapy of the present invention.

[0139] Treatment regimens can vary and often depend on tumor type, tumor location, disease progression, and the patient's health and age. Certain tumor types require more aggressive treatment, but at the same time, certain patients cannot tolerate more arduous protocols.

[0140] In certain embodiments, the tumor being treated may not be resectable, at least initially. Treatment with the combination therapy of the present invention may increase the resectability of the tumor by shrinking it at the border or eliminating certain infiltrated areas. After treatment, resection may become possible. Additional treatment after resection may help remove microscopic residual disease at the tumor site.

[0141] Determination of synergistic interactions between one or more elements, the optimal range for efficacy, and the absolute dose range of each element for efficacy can be definitively measured by administering the elements at different w / w ratio ranges and doses to patients in need of treatment. For humans, the complexity and cost of conducting clinical trials on patients makes this type of testing impractical as a primary model for synergy. However, observing synergy in one species can predict efficacy in other species. As described herein, animal models exist for measuring synergy, and the results of these studies can also be used to predict effective doses, plasma concentration ratio ranges, and absolute doses and plasma concentrations required in other species by applying pharmacokinetic / pharmacodynamic methods. The established correlation between effects seen in tumor models and humans suggests that synergy in animals can be demonstrated, for example, in human xenograft tumor models.

[0142] In some embodiments, the combination is used to treat and / or prevent cancer in a mammal. In some embodiments, the cancer includes, but is not limited to, brain cancer, head and neck cancer, esophageal cancer, skin cancer, lung cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, kidney cancer, testicular cancer, rectal cancer, breast cancer, and pancreatic cancer. In some embodiments, the cancer is selected from the group consisting of brain cancer, head and neck cancer, esophageal cancer, skin cancer, lung cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, kidney cancer, testicular cancer, rectal cancer, breast cancer, and pancreatic cancer. In a preferred embodiment, the combination is used to treat and / or prevent metastasis. In other preferred embodiments, the combination is used to treat cancers including, but not limited to, hepatocellular carcinoma, colon cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, and liver cancer. In some embodiments, the combination is used to treat cancers selected from the group consisting of hepatocellular carcinoma, colon cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, and liver cancer. In some embodiments, the combination is used to treat colon cancer, particularly metastatic colon cancer. In some embodiments, the mammal being treated is a human. In another preferred embodiment, the combination is used to treat cancers resistant to one or more immune checkpoint inhibitors (e.g., the cancer is resistant to immunotherapy with a PD-1 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, and / or a TIGIT inhibitor). In some embodiments, the cancer is a solid cancer or solid tumor.

[0143] The method includes concurrently administering a therapeutically effective amount of a replication-competent oncolytic vaccinia virus and an immune checkpoint inhibitor. A therapeutically effective amount of the oncolytic virus is defined as an amount sufficient to induce oncolysis, i.e., the destruction or lysis of cancer cells. Preferably, the oncolytic vaccinia virus and the immune checkpoint inhibitor are administered in synergistic amounts. The term includes slowing, inhibiting, or reducing tumor growth or size, and in some instances, tumor elimination. In some embodiments, the effective amount of the oncolytic vaccinia virus results in systemic dissemination of the therapeutic virus upon infection of a tumor, e.g., an uninjected tumor. In some embodiments, the effective amount of the oncolytic vaccinia virus is an amount sufficient to induce oncolysis, i.e., the destruction or lysis of cancer cells.

[0144] In some embodiments, the treatment and / or prevention of cancer is one that demonstrates at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, at least a 99%, or about a 100% decrease and / or reduction in tumor size and / or presence after treatment. In some embodiments, the treatment and / or prevention of cancer is one that demonstrates complete tumor regression after treatment. In some embodiments, the treatment and / or prevention of cancer is one that demonstrates complete tumor remission after treatment. In some embodiments, the cancer is refractory or resistant to treatment with an immune checkpoint inhibitor. In some embodiments, the cancer is refractory or resistant to treatment with an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In some embodiments, the cancer is resistant to treatment with an anti-PD-1 antibody. In some embodiments, the cancer is resistant to treatment with an anti-CTLA-4 antibody. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus and an immune checkpoint inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and / or TIM3. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, an inhibitor of PD-1 or PD-L1, and an immune checkpoint inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, an inhibitor of PD-1 or PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor, a LAG3 inhibitor, a TIGIT inhibitor, or a TIM3 inhibitor.In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-L1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a LAG3 inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-L1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a LAG3 inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a TIGIT inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-L1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a TIGIT inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a TIM3 inhibitor. In some embodiments, the treatment comprises administering a replication-competent oncolytic vaccinia virus, a PD-L1 inhibitor, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a TIM3 inhibitor.

[0145] In some embodiments, the replication-competent oncolytic vaccinia virus is administered by intratumoral administration, IV administration, and / or intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered by intratumoral administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered by IV administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered by intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered by intra-arterial administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered exclusively by intratumoral administration. In some embodiments, the replication-competent oncolytic vaccinia virus is administered exclusively by intratumoral administration.

[0146] The checkpoint inhibitors disclosed herein can be administered by a variety of routes, including orally or parenterally, e.g., intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracapsularly, intratumorally, intravascularly, intradermally, or by passive or enhanced absorption through the skin, e.g., using a skin patch or transdermal iontophoresis, respectively. In some embodiments, the checkpoint inhibitor is administered systemically. The checkpoint inhibitor can also be administered to the site of the pathological condition, e.g., intravenously or intra-arterially, into a blood vessel supplying a tumor. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and / or TIM3.

[0147] In some embodiments, the replication-competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication-competent oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically.

[0148] The total amount of agent administered in practicing the method of the present invention can be administered to a subject as a single dose, either by bolus or infusion, over a relatively short period of time, or can be administered using a divided treatment protocol in which multiple doses are administered over an extended period of time. Those skilled in the art will know that the amount of a composition for treating a pathological condition in a subject depends on many factors, including the subject's age and general health, as well as the route of administration and the number of treatments given. Taking these factors into account, those skilled in the art will adjust the specific dose as necessary. Generally, the formulation, route of administration, and frequency of administration of the composition will be initially determined using Phase I and Phase II clinical trials.

[0149] In certain embodiments, the checkpoint inhibitor is administered at a dose of 0.01-0.05 mg / kg, 0.05-0.1 mg / kg, 0.1-0.2 mg / kg, 0.2-0.3 mg / kg, 0.3-0.5 mg / kg, 0.5-0.7 mg / kg, 0.7-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 4-5 mg / kg, 5-6 mg / kg, 6-7 mg / kg, 7-8 mg / kg, 8-9 mg / kg, 9-10 mg / kg, at least 10 mg / kg, or any combination thereof. Suitable dosages of checkpoint inhibitors range from about 0.5 mg / kg to 25 mg / kg, preferably from about 1 mg / kg to about 20 mg / kg, and more preferably from about 2 mg / kg to about 15 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or every several months. In certain embodiments, the checkpoint inhibitor is administered as one dose, two doses, three doses, four doses, five doses, or six or more doses. Preferably, the checkpoint inhibitor is administered intravenously (e.g., intravenous infusion or injection) or intratumorally. As a non-limiting example, ipilimumab is preferably administered by intravenous infusion at a dose of 3 mg / kg every three weeks for a total of four doses. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and / or TIM3. A. Additional Anticancer Treatment

[0150] One or more additional chemotherapeutic agents may be administered with the combination of the present invention, including, but not limited to, any combination of 5-fluorouracil (FU), folinic acid (FA) (or leucovorin), methotrexate, capecitabine (Xeloda; an oral prodrug of 5-FU), oxaliplatin (Eloxatin), bevacizumab (Avastin), cetuximab (Erbitux), and panitumumab (Vectibix). These agents may be administered according to known treatment protocols. Generally, the additional chemotherapeutic agents are administered intravenously, with the exception of capecitabine, which is an oral formulation.

[0151] In other embodiments, the methods of the present invention further comprise administering an additional cancer therapy, such as, for example, radiation therapy, hormone therapy, surgery, and combinations thereof.

[0152] Radiation therapy includes, but is not limited to, gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA-damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radioactivity emitted, and uptake by neoplastic cells.

[0153] Approximately 60% of people with cancer undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, and / or alternative therapies.

[0154] Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery). It is further contemplated that the present invention may be used in conjunction with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0155] The removal of part or all of the cancerous cells, tissue, or tumor may result in a cavity in the body. Treatment may be achieved by perfusion, direct injection, or local application of an additional anti-cancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Dosages for these treatments may also vary.

[0156] Another form of therapy for use in conjunction with the methods of the present invention is hyperthermia, which involves exposing a patient's tissues to elevated temperatures (up to 106°F (approximately 41°C)). External or internal heating devices are involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves applying heat to a small area, such as a tumor. Heat may be generated externally with radiofrequency waves from a device outside the body that targets the tumor. Internal heating involves the use of thin, heated wires or hollow tubes filled with warm water, implanted microwave antennas, or sterile probes containing radiofrequency electrodes.

[0157] A patient's organs or limbs are heated in a localized therapy. This can be achieved, for example, using a high-energy producing device such as a magnet. Alternatively, a portion of the patient's blood is withdrawn, heated, and then perfused into the area to be internally heated. Whole-body heating can also be performed in cases where the cancer has spread throughout the body. Warm water blankets, hot wax, induction coils, and warming chambers may be used for this purpose.

[0158] Hormonal therapy may also be used in conjunction with the present invention or in combination with any of the other cancer therapies described above. The use of hormones may be employed in the treatment of certain cancers, such as breast, prostate, ovarian, and cervical cancer, to lower the levels or block the action of certain hormones, such as testosterone or estrogen. B. Compositions and Formulations

[0159] The replication-competent oncolytic vaccinia virus of the drug combination is administered to treat cancer and / or administered directly to tumor cells. Accordingly, the pharmaceutical composition of the present disclosure is formulated for a desired route of administration (e.g., intratumoral injection, intravenous administration, intraarterial administration, and / or intraperitoneal administration). In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intratumoral administration, intravenous administration, intraarterial administration, and / or intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intratumoral administration. In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intravenous administration. In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intraarterial administration. In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intraperitoneal administration. In some embodiments, the replication-competent oncolytic vaccinia virus of the drug combination is formulated for administration by intratumoral administration only.

[0160] Intratumoral injection of oncolytic vaccinia virus may be performed using a syringe or any other method used for injecting solutions, so long as the expression construct can pass through a needle of the specific gauge required for injection. A new needle-free injection system has recently been reported (U.S. Pat. No. 5,846,233, incorporated herein by reference). This system has a nozzle defining an ampoule chamber to hold the solution, and an energy device for forcing the solution from the nozzle to the delivery site. A syringe system for use in gene therapy has also been described (U.S. Pat. No. 5,846,225, incorporated herein by reference), which allows for precise multiple injections of a predetermined amount of solution at any depth.

[0161] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils, can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Suitable dosage forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the dosage form must be sterile and fluid to the extent that syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained by using a coating such as lecithin, for example, to maintain the required particle size in the case of dispersions, and by using surfactants. Prevention of microbial influence can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include an isotonic agent, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using the composition with an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0162] For intratumoral injection of an aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this regard, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of fluid for subcutaneous infusion or injected at a proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0163] Sterile injectable solution is prepared by incorporating the required amount of active compound in suitable solvent with various other ingredients as listed above as necessary.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle containing basic dispersion medium and the other ingredients as listed above that are required.For the sterile powder used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which can obtain powder of active ingredient plus any additional desired ingredients from the solution previously sterile-filtered.

[0164] The compositions disclosed herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and from organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. Formulations can be prepared, for example, by administering a pharmaceutically acceptable carrier. They are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules, and the like.

[0165] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0166] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce allergic or similar unexpected reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is also well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid prior to injection can also be prepared. [Example]

[0167] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any manner. Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the aims and advantages set forth above, as well as those inherent therein. The examples, along with the methods described herein, are representative of presently preferred embodiments and are illustrative and not intended to limit the scope of the invention. Those skilled in the art will recognize modifications therein and other uses that are encompassed within the spirit of the invention as defined by the claims. Example 1

[0168] We evaluated the ability of combination therapy with oncolytic vaccinia virus and checkpoint inhibitors to induce tumor regression in a syngeneic mouse model of metastatic renal cell carcinoma (Renca). The tumor growth pattern closely mimics human adult renal cell carcinoma, particularly with regard to spontaneous metastasis to the lung and liver. The Renca model is hypervascular, resistant to anti-PD-1 antibody immunotherapy, and immunocompetent. Because the tissue origin is infection-independent, the Renca model is relevant to all cancers. Materials and Methods

[0169] Mice and Cell Lines—Specific pathogen-free male BALB / c mice were housed in filter-lined cages and provided with water and food on a 12-hour reverse day / night cycle. All mice were anesthetized by intramuscular injection of an anesthetic combination (80 mg / kg ketamine and 12 mg / kg xylazine) and subsequently sacrificed. The Renca renal carcinoma cell line and the CT26 colon carcinoma cell line were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. Viral amplification

[0170] mJX594 is a Western Reserve vaccinia virus engineered to contain a disruption of the viral thymidine kinase gene and an insertion of mouse GMCSF-GFP (mGMCSF-GFP) under the control of a synthetic early-late promoter. HeLaS3 cells at 100% confluency were infected with mJX594 at a multiplicity of infection (MOI) of 1–3 and placed in a 37°C CO2 incubator for 1.5 hours. DMEM containing 2.5% FBS was then added and incubated for 48–72 hours. Cells were harvested by centrifugation, and the supernatant was discarded. Cells were resuspended in 10 mM Tris-Cl, pH 9.0, homogenized in a Daunce homogenizer, and centrifuged. The cell pellet was resuspended in 10 mM Tris-Cl, pH 9.0, centrifuged, and the supernatant was combined with the first supernatant. The sonicated lysate was layered on top of 36% sucrose and centrifuged at 32,900 x g for 80 minutes at 4°C, after which the pellet was resuspended in 10 mM Tris-Cl, pH 9.0, and stored below -60°C. ICI inhibitors (also referred to herein as immune checkpoint inhibitors)

[0171] Antibodies against CTLA-4 and PD-1 were purchased from BioXcell. 9D9 monoclonal antibody reacts with mouse CTLA-4. Isotype: mouse IgG2b. J43 monoclonal antibody reacts with mouse PD-1. Isotype: Armenian hamster IgG. Tumor model and treatment schedule

[0172] To create a clinically relevant renal tumor model, a suspension of Renal tumor cells (renal carcinoma, syngeneic) (5 × 10 5 Cells / 100 μl) were injected subcutaneously into the right dorsal flank of 8-10 week old immunocompetent male Balb / c mice. The mean tumor volume was 50 mm 3 When the IgG4-associated leukemia was exceeded, mice were randomized to receive the treatment regimen described below.

[0173] Tumor size was measured every 3 days in all groups using digital calipers. Tumor volume was calculated using the formula 0.5 × A × B 2 where A is the largest diameter of the tumor and B is its perpendicular diameter. On the designated days thereafter, the mice were sacrificed by CO2 and tissues were harvested for further analysis. Histological analysis-

[0174] For immunofluorescence studies, samples were fixed in 1% PFA, dehydrated overnight in 20% sucrose solution, and embedded in tissue freezing medium (Leica). Frozen blocks were cut into 50 μm sections. Samples were blocked with 5% goat (or donkey) serum in PBST (0.03% Triton X-100 in PBS) and then incubated with the following primary antibodies for 3 hours at room temperature (RT): anti-GFP (rabbit, Millipore), anti-CD31 (hamster, clone 2H8, Millipore), anti-VEGFR2 (rabbit, Cell Signaling), anti-CD8a (rat, BD Pharmingen), anti-CD11b (rat, BD Pharmingen), anti-FoxP3 (rat, eBioscience), anti-caspase 3 (rabbit, R&D Systems), anti-vaccinia (rabbit, Abcam), and anti-PD-L1 (rat, eBioscience). After several washes, the samples were incubated with the following secondary antibodies for 2 hours at room temperature: FITC-, Cy3-, or Cy5-conjugated anti-hamster IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch), Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch), or Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen). Samples were then mounted using fluorescent mounting medium (DAKO), and immunofluorescence images were captured using a Zeiss LSM880 confocal microscope (Carl Zeiss). Morphological analysis-

[0175] Densitometry of blood vessels, CD8 T cells, or apoptotic areas was performed using ImageJ software (http: / / rsb.info.nih.gov / ij). Random 0.42 mm 2 CD31, CD8, or caspase-3 per area of + The area of tumor tissue was measured in the peritumoral and intratumoral regions. All measurements were performed in at least five different fields per mouse. Flow cytometry

[0176] The harvested tumor tissue was minced and incubated in FACS buffer (PBS, 1% FBS) containing collagenase D (Roche) and DNase I (Roche) for 1-2 hours at 37°C in a shaking water bath. The digested cells were filtered through a 40 μm nylon mesh to remove cell clumps. RBCs were removed by incubating the cell suspension in ACK lysis buffer for 5 minutes at room temperature. The resulting single cells were incubated for 30 minutes with the following antibodies in FACS buffer: PerCP-cy5.5-conjugated anti-mouse CD45 (rat, eBioscience), APC-conjugated anti-mouse CD3e (hamster, eBioscience), FITC-conjugated anti-mouse CD4 (rat, eBioscience), PE-conjugated anti-CD8a (rat, eBioscience), FITC-conjugated anti-mouse CD11b (rat, eBioscience), APC-conjugated anti-mouse Gr1 (rat, eBioscience), and APC-conjugated anti-mouse CD11c (hamster, eBioscience). Statistical analysis-

[0177] Values are expressed as mean ± standard deviation. Statistical differences between means were determined by unpaired Student's t-test or one-way analysis of variance followed by the Student-Newman-Keuls test. Statistical significance was set at p<0.05. result Antitumor effect of vaccinia virus + anti-PD-1 combination therapy

[0178] For combination therapy with anti-PD-1 and mJX-594, 5 × 10 5 Renca tumor cells were injected into the right dorsal flank of BALB / c mice until tumor size reached 50–100 mm. 3 Treatment was initiated when the tumor reached 1 × 10 (day 0). Mice were randomized into four treatment groups: (i) control group: intratumoral injection of PBS every 3 days; (ii) mJX-594 monotherapy group: 1 × 10 7 (iii) anti-PD-1 monotherapy group: 10 mg / kg of the antibody was injected intraperitoneally every 3 days on days 0, 2, and 4 for a total of 4 times; (iv) mJX-594 + anti-PD-1 combination group: mJX-594 and anti-PD-1 were administered in parallel. mJX-594 was injected intratumorally every 2 days on days 0, 2, and 4 for a total of 3 times, and anti-PD-1 was administered intraperitoneally on days 0, 2, 4, 6, and 9 for a total of 5 times (see Figure 1A).

[0179] Tumor growth inhibition was observed in the mJX-594 monotherapy group and the mJX-594 / anti-PD-1 combination group (combination group) compared with the control (see Figure 2A). Tumor growth inhibition was more pronounced in the combination group (see Figure 2A; compare "mJX594" and "PD1" with "PD1+mJX594"). Tumor growth inhibition was not observed in the anti-PD-1 monotherapy group (see Figure 2A; compare "control" with "PD1"). Tumor weight was shown to be reduced in the mJX-594 monotherapy group (see Figure 2B). A significant reduction in tumor weight was observed in the combination group, which was significantly greater than that observed in the mJX-594 monotherapy group (see Figure 2B). Therefore, concurrent administration of mJX-594 and anti-PD-1 resulted in significant reductions in tumor weight and volume compared with either monotherapy.

[0180] CD8 T cell infiltration was increased in both the peritumoral and intratumoral regions in the PD-1 monotherapy group, mJX-594 monotherapy group, and parallel combination group (see Figure 3A). While CD8 T cell infiltration was increased in the peripheral region compared to the central region in the anti-PD-1 monotherapy group, the mJX-594 group showed a higher number of CD8 T cells infiltration in both the central and peripheral regions (see Figure 3B). Concurrent administration of mJX-594 and anti-PD-1 antibody significantly increased intratumoral T cell infiltration, as measured by CD8+ staining in the peritumoral and intratumoral regions, compared to the control and either monotherapy alone (see Figure 3B). Decreased vascular density was also observed in the treatment groups compared to the control (see Figure 3B).

[0181] PD-L1 expression levels in both the peripheral and central tumor regions were increased in the anti-PD-1 monotherapy group, mJX-594 monotherapy group, and parallel combination group compared with the control group (see Figure 4A). While the anti-PD-1 monotherapy group showed increased PD-L1 expression levels in the peripheral region compared with the central region, the mJX-594 monotherapy group showed comparable increases in PD-L1 expression levels in both the peripheral and central regions (see Figure 4A). Concurrent administration of JX-595 and an anti-PD-1 antibody increased intratumoral PD-L1 expression compared with monotherapy with either agent alone (see Figure 4A). Renca tumors are resistant to anti-PD-1 immunotherapy. The increased tumor PD-L1 expression in the parallel combination treatment group reflects the sensitization of these tumors to anti-PD-1 therapy, accompanied by the infiltration of CD8+ T cells into the tumor, and is an indicator of therapeutic efficacy. These patterns suggest that, at baseline, T cells are immunosuppressed and unable to infiltrate the tumor microenvironment. mJX-594 induces inflammation and vasodilation, allowing T cells to exert their antitumor effects. Concurrent administration of mJX-594 and an anti-PD-1 antibody results in T cell activation and infiltration into the tumor core.

[0182] Increased intratumoral apoptosis was observed in the PD-1 monotherapy group, mJX-594 monotherapy group, and parallel combination group compared to controls, as measured by caspase 3 staining (see Figure 4B). Significant increases in intratumoral apoptosis were observed in the parallel combination group compared to either monotherapy group (see Figure 4B). The apoptosis and antivascular effects in the parallel combination group (shown in Figure 3B) combined suggest extensive tumor necrosis in the parallel combination group.

[0183] Changes in the immune microenvironment, as measured by CD4 and CD11b, were observed after concurrent treatment with mJX-594 and an anti-PD-1 antibody compared with control and monotherapy with either agent (see Figure 5A). Importantly, tumor infiltration of CD8 T cells was highest in the concurrent combination group. Depletion of CD8+ cells using an anti-CD8 antibody reduced tumor growth inhibition, confirming the role of these cells in the anti-tumor effect (data not shown). Myeloid-derived suppressor cells (MDSCs) were increased in the mJX-594 and concurrent combination groups compared with the control group. Traditionally, CD11b+Gr1+ cells have been considered simply immunosuppressive cells. However, recent results demonstrate that these cells are not so simply defined. The relative increase in MDSCs observed in the concurrent combination group with αPD1 contrasts with the decrease in MDSCs observed in the concurrent combination group with αCTLA4 (Figures 10A-10B).

[0184] The effects of sequential and parallel co-administration of mJX-594 with PD-1 ± CTLA4 on tumor growth were evaluated. 5 Renca (renal carcinoma, syngeneic) cells were injected subcutaneously into the right flank of 8-week-old immunocompetent BALB / c mice. Tumor size was 50 mm. 3 Once this was reached, treatment was initiated (day 0).

[0185] Mice were divided into four treatment groups: (i) control group: injected with PBS on days 0, 3, 6, 9, 12, and 15; (ii) mJX-594 + anti-PD-1 sequential combination group: mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-PD-1 was injected intraperitoneally on days 6, 9, 12, and 15; and (iii) mJX-594 and anti-PD-1 parallel combination group: mJX-594 and anti-PD-1 were administered in parallel. mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-PD-1 was administered intraperitoneally on days 0, 3, 6, 9, 12, and 15; (iv) mJX-594 + anti-PD-1 + anti-CTLA4 triple parallel combination group: mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-PD-1 and anti-CTLA4 were injected intraperitoneally on days 0, 3, 6, 9, 12, and 15 (see Figure 6).

[0186] Compared to controls, tumor growth was suppressed in the sequential and parallel administration groups of mJX-594 + anti-PD-1, as well as in the triple parallel administration group of mJX-594 + anti-PD-1 + anti-CTLA4 (see Figure 7). Surprisingly, parallel administration of mJX-594 and anti-PD-1 suppressed (delayed) tumor growth to a greater extent than sequential administration of these agents (see Figure 7). Further tumor growth delay was observed in the triple parallel administration group (combination (mJX-594 + αPD1 + αCTLA4)) (see Figure 7).

[0187] Comparison of tumor size in each mouse between treatment groups confirmed tumor regression in the combination group compared with the control. Tumor regression was observed from day 12 in the serial combination group, while tumors in the parallel and triple parallel combination groups showed a tendency to shrink from day 6.

[0188] Surprisingly, these results suggest that the dosing regimen and potentially the route of administration can significantly impact the antitumor efficacy of combination therapy. In particular, when vaccinia virus and checkpoint inhibitors (anti-PD-1, CTLA-4) are administered in parallel, the vaccinia virus synergizes with the checkpoint inhibitors to induce potent antitumor immune responses. In some cases, the vaccinia virus induced potent antitumor immune responses when administered intratumorally as part of the parallel administration. The synergistic antitumor effect observed with the parallel administration of vaccinia virus and checkpoint inhibitors was particularly surprising, because it is understood in the art that immune checkpoint inhibitors inhibit the replication of oncolytic viruses, such as vaccinia (Rojas et al., J. Immunol., 192(1 Supplement):142.3(2014)). Vaccinia virus + anti-CTLA4

[0189] 5×10 5 Renal carcinoma (renal carcinoma) cells were injected subcutaneously into the right flank of 8-week-old immunocompetent BALB / c mice. Tumors were 50–100 mm in size. 3 Once this was reached, treatment was initiated (day 0).

[0190] Mice were randomized into five treatment groups: (i) control group: PBS was injected intratumorally on days 0, 3, 6, 9, 12, and 15; (ii) mJX-594 monotherapy group: 1 × 10 7pfu of mJX-594 was injected intratumorally on days 0, 3, 6, and 9; (iii) anti-CTLA4 monotherapy group: 4 mg / kg of antibody was injected intraperitoneally on days 0, 3, 6, 9, 12, and 15; (iv) mJX-594 + anti-CTLA4 sequential combination group: mJX-594 and anti-CTLA4 were administered sequentially. mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-CTLA4 was injected intraperitoneally on days 6, 9, 12, and 15; (v) mJX-594 and anti-CTLA4 simultaneous combination group: mJX-594 and anti-CTLA4 were administered sequentially. mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-CTLA4 was injected intraperitoneally on days 0, 3, 6, 9, 12, and 15 (see Figure 8).

[0191] Tumor size was measured every 3 days in all groups. When observation was completed (day 16), mice were sacrificed by CO2, and tumors were removed and subjected to flow cytometry analysis (CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), Gr1+ / CD11b+ MDSCs).

[0192] Tumor growth was observed to be suppressed in all treatment groups compared to controls (see Figure 9). Significantly greater tumor growth suppression was observed in the combination treatment group. The greatest tumor growth suppression was observed in the parallel treatment group (see Figure 9).

[0193] Comparison of tumor size between treatment groups confirmed tumor regression in the combination group compared with the monotherapy and control groups. Increased CD8 T cell infiltration was observed in all treatment groups compared with controls (see Figure 10A). While MDSC levels were increased in the mJX-594 monotherapy group compared with controls, no significant change was observed in the sequential combination treatment group, and a decrease in MDSC levels was observed in the parallel combination treatment group (see Figure 10B). Concurrent administration of JX929 and immune checkpoint inhibitors

[0194] JX929 (6 × 10) administered IT in parallel with an intraperitoneally administered PD-1 checkpoint inhibitor 7 The antitumor effect of JX929 on the tumor-like growth factor receptor (TK) gene expression in the mouse Renca model was tested. - / GF - phenotype) and do not express GM-CSF. Cell line-

[0195] Mouse RENCA cells (ATCC) were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37°C in 5% CO . In vivo experiments

[0196] Eight-week-old female BALB / c mice were inoculated with RENCA cells (2 × 10 6 10 cells) were injected into the subcapsular space of the left kidney. Ten days after implantation, mice bearing Renca tumors (50 mm as visualized using the IVIS® Spectrum in vivo imaging system) were 3 ~100mm 3 ) according to the regimen shown in Figure 1B, (i) PBS (control), (ii) vaccinia virus (JX-929) monotherapy (6 × 10 7 Treatment was performed intraperitoneally (i.p.) with PFU (100 μl, BioXcell, West Lebanon, NH) (a total of three doses on days 10, 11, and 12 after transplantation), (iii) anti-PD1 monotherapy (BioXcell, West Lebanon, NH) (a total of three doses on days 10, 11, and 12 after transplantation), or (iv) JX929 + anti-PD1 concomitant treatment (administered on days 10, 11, and 12 after transplantation, respectively; JX-929 was administered in the morning and ICI was administered in the afternoon of the same day, with a 9-hour interval).

[0197] Mice were sacrificed 2 days after the last treatment for further histological and flow cytometric analysis. Flow cytometry

[0198] Peripheral blood samples were collected, and red blood cells were lysed with RBC lysis buffer. Cells were washed with PBS containing 1% FBS and then stained with monoclonal mouse anti-CD8, rabbit anti-CD4, and rabbit anti-CD3 antibodies (Santa Cruz Biotechnology, CA, USA). Cells were fixed with 4% paraformaldehyde and then incubated with FITC-conjugated goat anti-rabbit or goat APC-conjugated anti-mouse antibodies (Santa Cruz Biotechnology, CA, USA). 10,000 cells from each sample were analyzed using a FACS Calibur instrument (BD Biosciences, CA, USA). Histological analysis-

[0199] Mice were euthanized, and vital organs, including tumor-bearing kidneys and lungs, were harvested and fixed in 10% neutralized formalin (BBC Biochemical, Washington, USA). Tissues were embedded in paraffin, and sections (4 μm thick) were stained with hematoxylin and eosin for basic histological analysis. For immunofluorescence and immunohistochemistry, sections were stained by standard methods using a mouse monoclonal antibody specific for CD8 (Santa Cruz Biotechnology, California, USA). Sections were then incubated with either an FITC-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology) for immunofluorescence or the Vectastain® Elite ABC-Peroxidase kit (Vector Laboratories, California, USA) and visualized with Vector SG (Vector Laboratories) for immunohistochemistry. The weight and volume of tumors harvested from each treatment group were measured and compared. ELISpot Assay

[0200] IFNγ-secreting cells were assessed using the ELISpot Mouse IFNγ Kit (Mabtech, Cincinnati, OH) according to the manufacturer's protocol. Spleens were isolated and prepared as single-cell suspensions. Spleen cells were mixed with RENCA tumor cells or vaccinia virus-infected mouse splenocytes at a 5:1 ratio and incubated for 24 hours at 37°C. The intensity of specific spots was analyzed using ImageJ software (NIH). Statistical analysis-

[0201] All values were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Instat3 (GraphPad Software, Inc., California, USA). Multiple comparisons were analyzed using one-way analysis of variance (ANOVA) with a paired Bonferroni post-hoc comparison test. mJX594 treatment induces the expression of checkpoint proteins.

[0202] 50mm 3 Balb / c mice bearing Renca tumors exceeding 1 × 10 were injected with mJX594 (1 × 10 on each of days 0, 3, 6, and 9) according to the treatment regimen shown in Figure 1C. 7 ) was administered four times intratumoral doses or as a PBS control.

[0203] The levels of immune checkpoint proteins in tumors from control and mJX-594-treated animals were measured on day 0 (before treatment) and at sacrifice on day 12. Figure 13 illustrates the fold change in checkpoint proteins after treatment in mJX-594-treated mice compared to control mice. As shown in Figure 13, mJX594 treatment induces the expression of checkpoint proteins, including PD-1 (4-fold increase), PD-L1, PD-L2, CTLA-4 (more than 2-fold increase), LAG3, TIM3 (more than 3-fold increase), and TIGIT (more than 2-fold increase). Treatment with a replication-competent oncolytic vaccinia virus resulted in dynamic changes in the tumor immune microenvironment, including a significant increase in the checkpoint proteins PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT, thereby sensitizing tumors to blockade of each of these checkpoint proteins with the respective checkpoint inhibitors. Clinical trials have demonstrated that tumor-infiltrating cells and checkpoint protein (e.g., PD-L1) expression are indicators of amenability to treatment with checkpoint inhibitors (e.g., anti-PD-L1 therapy), supporting the efficacy of the combination therapies described herein not only in patients with tumors that express particular checkpoint proteins, but also in patients with tumors that express low levels (or no expression) of particular checkpoint proteins. Example 2

[0204] Remodeling the tumor microenvironment by intratumoral oncolytic vaccinia virus enhances the efficacy of immune checkpoint blockade summary

[0205] Cancer immunotherapy is a powerful and durable treatment method, but its clinical benefits have not yet been universal. Herein, we employed mJX-594, a targeted and GM-CSF-armed oncolytic vaccinia virus (VV), as a combination partner of immune checkpoint inhibitors (ICIs) in mice bearing transplanted renal, colon, and spontaneous breast cancers. Intratumoral injection of VV induced extensive remodeling of the tumor microenvironment, transforming non-T cell, non-inflammatory tumors into CD8+ / CD9+ / CD8 ... + Tumors transformed into T cell-mediated inflammatory tumors with increased T cell numbers and enhanced effector functions. Furthermore, VV and ICI combination therapy induced tumor regression, along with improved survival and anti-metastatic effects. Our findings indicate that VV, in combination with ICI, elicits robust anti-cancer immunity and overcomes immunotherapy resistance. Introduction

[0206] Cancer immunotherapy using immune checkpoint inhibitors (ICIs) targeting PD-1 or CTLA-4 has demonstrated potent and durable therapeutic efficacy and has emerged as a new weapon in the fight against cancer (Hegde et al., 2016; Topalian et al., 2015; Wolchok and Chan, 2014). However, the clinical efficacy of ICIs has been limited to tumors with a T cell-promoted tumor microenvironment (TME) (Gajewski, 2015; Topalian et al., 2016). In poorly immunogenic tumors with few tumor-infiltrating lymphocytes (TILs), the TME lacks the signature of type I interferons and chemokines for T cell recruitment (Gajewski et al., 2013). Furthermore, tumor vasculature and stromal components can create barriers to the intratumoral trafficking of T cells and their effector functions against tumor cells (De Palma and Jain, 2017; Rivera and Bergers, 2015; Sharma et al., 2017). Therefore, additional therapeutic interventions are needed for these non-T cell inflammatory tumors to properly remodel the TME and make these tumors more susceptible to ICI treatment.

[0207] Oncolytic viruses (OVs) have been proposed as a novel class of anticancer therapy, and OVs with different backbones and transgenes are currently being evaluated in clinical trials (Bell, 2014; Lichty et al., 2014). While the success of OVs was initially measured in the past decade through their rapid replication and enhanced tumor lytic potential, OVs are now increasingly recognized as immunotherapeutics. This is because the most robust and durable responses after oncolytic virotherapy are associated with successful induction of antitumor immunity, accompanied by an increase in tumor-specific effector and memory T cells (Bell, 2014; Chiocca and Rabkin, 2014; Thorne, 2014). However, the therapeutic efficacy of OVs is significantly hindered by the immunosuppressive TME, and therefore, releasing the brakes on the immune system is crucial to maximizing the immunotherapeutic efficacy of OVs (Bell and Ilkow, 2017; Hou et al., 2016; Liu et al., 2017). Therefore, the combination of OV and ICI is a rational and preferable strategy to overcome poor immunogenicity and immunosuppressive TME.

[0208] JX-594 (Pexa-thymodin devasilepvec, Pexa-vec) is an oncolytic vaccinia virus (VV) engineered to express the immune-activating transgene GM-CSF and with a disrupted viral thymidine kinase gene (Kirn and Thorne, 2009). JX-594 has demonstrated remarkable anticancer activity and low toxicity in preclinical and clinical trials, making it one of the most feasible and promising OV platforms for clinical development (Breitbach et al., 2011a; Cripe et al., 2015; Heo et al., 2013; Park et al., 2008). Apart from its oncolytic and vascular-disrupting activities, JX-594 has been proposed to exhibit in situ cancer vaccine effects. This is because JX-594 can elicit adaptive immune responses against tumor antigens due to selective tumor destruction and subsequent release of additional tumor antigens (Breitbach et al., 2011b; Breitbach et al., 2015a). JX-594 is currently undergoing a phase III randomized clinical trial in advanced hepatocellular carcinoma (HCC) (Abou-Alfa et al., 2016). However, most studies have not yet characterized its immunomodulatory function in the primary TME after JX-594 treatment as well as in distant lesion sites (Kim et al., 2018). Furthermore, the optimal combination of JX-594 with immunotherapies, such as ICIs, is still under investigation and validation.

[0209] Herein, we report the mouse variant of JX-594 (mJX-594, WR.TK - To comprehensively analyze the dynamic remodeling of the TME using mGM-CSF, explore its immunotherapeutic potential, and provide rational combination strategies with ICIs in poorly immunogenic tumor models. result mJX-594 transforms immunosuppressive non-inflammatory tumors into inflammatory tumors

[0210] To determine the immunomodulatory potential of the oncolytic virus mJX-594, we examined the time course of tumor microenvironment changes following a single mJX-594 injection in poorly immunogenic Renca tumors. mJX-594 levels were already high on day 1 post-injection, peaked on day 3, and were nearly undetectable by day 7 (Figures 33A and 33B). In contrast, tumor vasculature showed an opposite response to virus levels. Tumor vascular density was significantly reduced between days 1 and 3 post-injection but recovered from day 7 onward (Figures 33A and 33B). This suggests that mJX-594 induced potent, but transient, destruction of tumor blood vessels. Of note, CD8+ cells in the intratumoral region, which play a crucial role in anti-cancer immunity, were also involved. + Cytotoxic T cell populations began to increase significantly at 5 days post-injection, peaked at 7 days, and remained at high density for 2 weeks (Figures 33A and 33B), clearly demonstrating a pronounced and long-lasting conversion of non-inflammatory tumors to T cell-inflamed tumors by mJX-594. In comparison, CD11c + Dendritic cells (DCs) appeared transiently on day 3 but subsequently declined (Figures 33A and 33B). PD-L1 expression was lowest on day 0 and was upregulated after mJX-594 treatment (Figures 1A and 1B). Interestingly, the timing of PD-L1 upregulation was significantly different from that of CD8 + This was immediately followed by a massive influx of TILs (Figure 1C), suggesting the activation of a negative feedback pathway that attempts to suppress T cell-mediated immunity. + Tumor cells, some CD11b + Myeloid cells also express PD-L1, whereas T cells do not (Figure 1D). Thus, mJX-594 is a potent and durable enhancer of anti-cancer immunity and inhibits cytotoxic CD8 + Recruiting T cells to cold tumors and transient tumor vasculature disruptors.

[0211] To elucidate the cancer immune pathways regulated by mJX-594, we comprehensively analyzed the changes in expression levels of 750 immune-related genes after mJX-594 monotherapy using the PanCancer Immune Profiling panel. Results showed striking differences in immune signature-related genes between control and mJX-594-treated tumors (Figure 33E). Approximately 100 immunoregulatory genes, including genes involved in type I IFN signaling activation, DC maturation, and T cell activation, exhibited statistically significant changes in their expression levels (Figure 33F). In particular, we observed an overall higher expression of both inhibitory (Pd-1, Pd-l1, Ctla-4, and Lag-3) and agonistic (including Icos, Gitr, and Cd27) immune checkpoint molecules in the TME compared to controls (Figure 33G). Further analysis of the TME revealed an increase in genes associated with Th1 and Th2 responses, suggesting immune modulation by mJX-594 monotherapy (Figure 33G). We also found a significant increase in several genes involved in the TME and myeloid cells (Figure 33G). In particular, increased expression of Nos2 and Cd86 indicates polarization of myeloid cells toward M1 macrophages. These results suggest that mJX-594 induces long-term immune activation through dynamic changes in the TME, remodeling non-inflammatory tumors into T cell-inflamed tumors capable of responding to immune checkpoint blockade. mJX-594 is CD8 + Increased T cell infiltration into tumors and repolarization of myeloid cells

[0212] The mJX-594-induced tumor growth delay was dose-dependent (Figures 34A and 34B). In parallel, mJX-594-induced CD8 + The increase in T cell infiltration was also dose-dependent (Figures 34C and 34D). Indeed, flow cytometry subset analysis of lymphoid cell fractions also demonstrated mJX-594-induced intratumoral CD8 + T cells and CD4 +The increase in absolute T cell numbers was found to be dose-dependent (Figures 34E and 34G). + Foxp3 + CD25 + The number of regulatory T cells in the IL-16 / IL-2 cells was also increased after triple administration of mJX-594 (Fig. 34H), but the number of CD8 + The ratio of T cells to regulatory T cells was 5.3-fold higher compared to that of the control-treated group (Figure 34I), suggesting an overall increase in T cell effector function in the TME with mJX-594 treatment. Furthermore, the expression of ICOS and granzyme B (GzB), which are costimulatory markers and markers of T cell activation, also increased after mJX-594 treatment. + Further subset analysis of bone marrow cell fractions revealed that CD11b T cells were increased in tumors treated with mJX-594 therapy. + Gr1 + There was no significant change in bone marrow cell fraction (Fig. 34K). + Ly6G - Ly6C + The monocytic myeloid cell fraction was increased, while CD11b + Ly6G + Ly6C int The granulocytic myeloid cell fraction was decreased, suggesting myeloid cell polarization after mJX-594 administration (Figures 34L and 34M). These findings demonstrate that repeated administration of mJX-594 enhances anti-cancer immunity, leading to increased infiltration of activated T cells and repolarization of myeloid cells. Intratumoral injection of mJX-594 generates a systemic, cancer-specific immune response

[0213] To determine whether local injection of mJX-594 can induce a systemic immune response in distant, uninjected tumors, we implanted Renca tumors into both flanks and administered mJX-594 intratumorally on the right side. This treatment suppressed the growth of Renca tumors on both the right and left sides (the contralateral, uninjected site) (Figure 35A). Consistent with tumor growth inhibition in both sites, CD8 expression in the intratumoral region was significantly increased. + T cell infiltration was also increased by 7.9-fold and 5.5-fold in both right-sided and left-sided Renca tumors (Figures 35B and 35C), suggesting that local mJX-594 virotherapy can potently activate systemic anti-cancer immunity.

[0214] Next, to exclude the possibility that the virus spread directly to distant tumors via the systemic circulation after local virotherapy, we examined viral replication in the left, uninjected Renca tumor and found that there was no detectable vaccinia virus in the left tumor (Figure 41), suggesting that the anticancer activity of mJX-594 was immune-mediated and not the result of systemic viral spread.

[0215] To assess whether the observed systemic immune response was tumor-specific, we performed a similar experiment using mice implanted with Renca tumors in the right flank and CT26 tumors in the left flank. Intratumoral treatment of the right-sided Renca tumor with mJX-594 significantly reduced the growth of the injected tumor, whereas growth of the left-sided untreated CT26 tumor was unaffected (Figure 35D). Microscopic analysis showed consistent results, with Renca tumors accumulating CD8+ T cells, but not CT26 tumors (Figures 35E and 35F). This suggests that mJX-594 virotherapy induces tumor-specific CD8 + These results suggest that local mJX-594 treatment can elicit systemic anti-cancer immunity and generate tumor-specific lymphocytic infiltration even in distant tumors. Anti-cancer immunity plays a key role in the overall therapeutic efficacy of JX

[0216] To determine which components of the immune system contribute to the therapeutic efficacy of mJX-594, we examined its effect on tumors in mice treated with neutralizing antibodies against CD8, CD4, or GM-CSF (Figure 36A). + T cells or CD4 + Depletion of either CD4 T cells abolished the effective inhibition of tumor growth by mJX-594 monotherapy (Figures 36B and 36C), highlighting the importance of immune-mediated mechanisms rather than direct tumor lysis in mJX-594-induced tumor inhibition. Surprisingly, depletion of either CD4 T cells abolished the effective inhibition of tumor growth by mJX-594 monotherapy (Figures 36B and 36C). + Depletion of T cells results in CD8 + Intratumoral infiltration of T cells was reduced (Fig. 36D). + CD4 in T cell activation and recruitment + T cells are suggested to be involved. However, CD8 + T cell depletion is CD4 + This did not significantly alter T cell infiltration (Fig. 36E). + T cells are CD4 + These data indicate that intratumoral treatment with mJX-594 did not affect CD8 T cells. + T cells and CD4 +It has been shown that mJX-594 induces T cell priming, which may interact with each other to mediate anti-cancer immunity. Previous virotherapy treatments based on herpesviruses and vaccinia viruses have utilized GM-CSF as an immune-activating transgene to recruit and activate antigen-presenting cells (APCs), subsequently triggering T cell responses. However, the use of GM-CSF has been controversial due to its potential immunosuppressive role in tumor progression, for example, by inducing the proliferation of myeloid-derived suppressor cells (MDSCs) (Thorne, 2014). Consequently, we investigated whether GM-CSF is required for the therapeutic effect of mJX-594. Interestingly, depletion of GM-CSF abolished the anti-tumor effect of mJX-594, resulting in the downregulation of CD8 + T cells and CD4 + Both levels of CD8 T cells were reduced, suggesting that GM-CSF is important for the immunotherapeutic efficacy of mJX-594 (Figures 36C-36E). + T cells and CD4 + Both T cells are essential mediators in the anti-cancer effects of mJX-594, and GM-CSF may confer immunotherapeutic benefits. The combination of mJX-594 and immune checkpoint blockade induces synergistic anti-cancer effects and enhances T lymphocyte infiltration into tumors.

[0217] To overcome resistance to ICI monotherapy, we evaluated the benefits of combining mJX-594 with ICI. At day 12 after treatment, monotherapy with αPD-1 and mJX-594, respectively, delayed tumor growth by 22.8% and 44%, while the combination of αPD-1 and mJX-594 reduced tumor growth by 70% (Figures 37A and 37B). To corroborate these findings, microscopic analysis revealed that CD8 +We found that T cell recruitment was significantly increased in both the peritumoral (18.8-fold) and intratumoral (21.4-fold) regions of tumors treated with the combination therapy compared to controls (Figures 37C and 37D). + Tumor vasculature was significantly reduced in these regions (1.8-fold and 2.6-fold, respectively; Figures 37C and 37D). Additionally, greater tumor apoptosis was noted in tumors treated with the combination therapy compared with controls (Figures 5C and 5D). PD-L1 expression was minimal in control tumors but highly upregulated after mJX-594 treatment (Figures 37C and 37D). This finding suggests that TME-induced PD-L1 expression is an adaptive negative feedback mechanism that suppresses anticancer immunity after oncolytic virotherapy and therefore provides a rationale for combining mJX-594 with PD-1 / PD-L1 blockade to enhance the immunotherapeutic effects of mJX-594 (Figure 37E).

[0218] Overall, these results suggest that the combination therapy reverses resistance to mJX594 or αPD-1 monotherapy by inhibiting CD8 + This suggests that this can be overcome by strengthening anti-cancer immunity through increased T cell infiltration.

[0219] Similarly, combination treatment with αCTLA-4 and mJX-594 was synergistic. While tumor growth was moderately inhibited by either mJX-594 (42.0%) or αCTLA-4 (20.0%) monotherapy, a more potent inhibition (57.6%) was observed with the combination therapy (Figures 42A and 42B). In addition, CD8+ cells were significantly increased in both the peripheral (27.0-fold) and central (26.4-fold) regions of the tumor after combination therapy compared to controls. + A higher accumulation of T cells was observed (Figures 42C and 42D). + Along with increased T cell levels, CD31 +Blood vessels were also significantly disrupted (2.1-fold and 3.8-fold reductions, respectively; Figures 42C and 42E). + T cells and CD4 + It was also revealed that the infiltration of T cells into the tumor was increased by the combination therapy of mJX-594 and αCTLA-4 (Figures 42F to 42H).

[0220] Collectively, these results indicate that combination therapy with mJX-594 and ICIs can overcome immunotherapy resistance in the immunosuppressive TME, thereby resulting in synergistic anticancer effects. The efficacy of combined immunotherapy using intratumoral mJX-594 and ICI is not significantly affected by the treatment schedule

[0221] Because ICIs can negatively affect viral replication and potentially lead to premature OV clearance, several studies have explored the optimal treatment schedule using systemic oncolytic virotherapy in combination with ICIs, and have reported that some combination schedules may antagonize therapeutic efficacy (Liu et al., 2017; Rojas et al., 2015). However, no similar studies have been reported examining local oncolytic virotherapy. To establish the optimal combination schedule for intratumoral mJX-594 and ICIs, we compared the following: (1) simultaneous administration of mJX-594 and ICIs (Schedule I), (2) administration of mJX-594 followed by ICI initiation 3 days later (Schedule II), and (3) administration of mJX-594 followed by ICI initiation 3 days later (Schedule III) (Figure 38A). All combination schedules delayed tumor growth by approximately 40% (Figures 38B and 38C). Similarly, tumor-infiltrating CD8 + T cells and CD4 + T cell levels were also increased by more than 8-fold and more than 4.0-fold, respectively, and CD8 + We also show a significant increase in the expression levels of ICOS and GzB in T cells (Figures 38D-38F).

[0222] Similar to the combination therapy with mJX-594 and αPD-1, the combination of mJX-594 and αCTLA-4 also inhibited tumor growth by approximately 40% regardless of the treatment schedule (Figures 43A and 43B). + T lymphocytes and CD4 + Intratumoral infiltration of T lymphocytes (>7-fold and >7-fold increase, respectively) and CD8 + Expression of GzB and ICOS in T cells was also higher regardless of treatment schedule (Figures 43C-43E).

[0223] Taken together, these findings suggest that intratumoral injection of mJX-594 and combination therapy with systemic immune checkpoint blockade elicit effective anticancer immune responses regardless of the treatment schedule, and that intratumoral administration of mJX-594 is not significantly affected by altered ICI administration schedules. The triple combination of mJX-594, αPD1, and αCTLA4 can induce complete tumor regression in transplanted renal cell carcinoma and provides long-term survival benefit

[0224] Because the dual combination of mJX-594 and ICI did not induce complete tumor regression, we investigated the triple combination of mJX-594, αPD-1, and αCTLA-4. The dual combination of αPD-1 and αCTLA-4 delayed tumor growth by 14.5%, mJX594 monotherapy inhibited tumor growth by 36.9%, and the triple combination inhibited tumor growth by 76.5% (Figures 39A and 39B). Notably, a portion of this triple combination group (approximately 40%) achieved complete tumor regression. This phenomenon was not observed in any of the other groups (Figure 39C).

[0225] To confirm whether this potent anti-cancer effect induced by the triple combination therapy resulted in a long-term survival benefit, we performed a survival analysis of tumor-bearing mice. Indeed, mice treated with the triple combination therapy exhibited a significant survival benefit compared with other treatments (Figure 39D). Furthermore, mice with complete tumor regression remained tumor-free for more than 12 weeks after the end of treatment and were completely protected against rechallenge with tumor cells, suggesting the establishment of effective, long-term immune memory (Figure 39E).

[0226] These findings demonstrate that triple combination immunotherapy has the potential to induce complete tumor regression and long-term survival. Triple combination therapy enhances anti-cancer immune responses in a spontaneous breast cancer model.

[0227] To rigorously demonstrate the long-term immunotherapeutic efficacy of triple combination therapy in immunoresistant tumors, we employed the MMTV-PyMT transgenic mouse model. This model is a spontaneous breast cancer model with intrinsic resistance to cancer immunotherapy (Schmittnaegel et al., 2017). After 4 weeks of treatment, mice treated with the triple combination of mJX-594, αPD-1, and αCTLA-4 exhibited a significant 38.7% reduction in total tumor burden and a significant reduction in the number of palpable mammary tumor nodules compared to control mice (Figures 40A-40D). Furthermore, the triple combination therapy resulted in a 48.1% reduction in the average size of each tumor nodule compared to other treatments, improving overall survival (Figures 40E-40F). Histological analysis (Figure 40G, see legend for detailed explanation) revealed less invasive carcinoma and better-preserved tumor margins in the triple combination group. This indicates that the triple combination effectively delayed tumor progression and invasion. On the other hand, tumors treated with the dual combination of αPD-1 and αCTLA-4 showed an invasive cancer phenotype comparable to that of the control group, infiltrating into surrounding tissues and forming solid sheets of tumor cells. As a result, after triple combination therapy, CD8 +Intratumoral T cell recruitment was significantly increased by more than 50-fold compared to all other treatments (Figures 40H and 40I). However, tumor vascular density was comparable between treatment groups (Figure 40J). These findings indicate that enhanced anticancer immunity, rather than vascular disruption, is crucial for the long-term therapeutic efficacy of the triple combination immunotherapy with mJX-594 and ICI. Finally, the number of hematogenous lung metastases was significantly reduced in the triple combination group (Figures 40K and 40L), demonstrating the effective anti-metastatic effect of the triple combination therapy.

[0228] Collectively, these results demonstrate that triple combination immunotherapy with mJX-594 and an ICI can elicit robust anticancer immune responses even in a poorly immunogenic spontaneous breast cancer model. Consideration

[0229] Herein, we demonstrate that combination therapy with mJX-594 and an ICI is an effective therapeutic strategy for immunoresistant tumors. The combination therapy generates an immunological "boiling point" at which cold, non-inflammatory tumors become sufficiently inflamed to allow the host immune system to eliminate tumor cells. The most potent effect was observed with triple immunotherapy using mJX-594, anti-PD-1, and anti-CTLA4, which induced complete regression in approximately 40% of Renca tumors, one of the most immunotherapy-resistant syngeneic tumors. This powerful synergistic effect can be explained by the complementary cooperation between OV and an ICI.

[0230] JX-594 is the most advanced OV in clinical trials and is known to act through various mechanisms (Abou-Alfa et al., 2016). JX-594 can induce rapid and direct tumor lysis and destruction of blood vessels in tumors, but these effects are transient and mostly disappear within one week of injection. Thereafter, CD8 +T cells massively infiltrate tumors and initiate anticancer immune responses. However, at the same time, tumors begin to evolve to evade immune-mediated elimination by upregulating immune-inhibitory checkpoint molecules, such as PD-1, PD-L1, or CTLA-4, in the TME. Because the most potent and durable anticancer effects of OVs are achieved when successful induction and maintenance of antitumor immunity are coupled, it is reasonable to combine ICIs with OVs to prevent the premature shutdown of OV-induced anticancer immunity.

[0231] Although ICI monotherapy has revolutionized the cancer treatment landscape, its dramatic therapeutic responses are limited to a subset of patients. This has led to the concept of immunologically "hot" or "cold" tumors. Hot tumors respond well to ICIs because they are immunologically inflamed with TILs and have high expression of PD-L1. On the other hand, cold tumors respond well to ICIs because they have low expression of CD8 +Tumors are refractory to ICIs due to a paucity of TILs and an immunosuppressive TME (Bell and Ilkow, 2017; Gajewski et al., 2013). Therefore, current research focuses on overcoming ICI resistance by immunologically converting cold tumors to hot tumors. In this context, our results suggest mJX-594 as an ideal combination partner for ICIs. mJX-594 can selectively replicate in tumor cells, destroy them, and release tumor antigens to stimulate the host immune system. Furthermore, our studies demonstrated that mJX-594 can dramatically convert the TME from a cold state to a hot state by inducing intratumoral inflammatory responses, namely, induction of Th1 responses, activation and recruitment of T cells, upregulation of PD-L1, and polarization of myeloid cells toward antitumor activity. Interestingly, OV replication and spread are known to be more active in cold tumors. Cold tumors have few immune cells capable of clearing OVs, whereas hot tumors with abundant resident TILs may induce premature OV clearance and attenuate their therapeutic efficacy (Bell and Ilkow, 2017). Therefore, taken together with the results of this study, mJX-594 is an optimal combination partner for ICIs, especially for cold, non-inflammatory tumors that are intrinsically resistant to immunotherapy.

[0232] GM-CSF is the most commonly used therapeutic gene payload for OVs. The two OVs currently in the most advanced clinical trials are T-Vec and Pexa-Vec (JX-594), both of which are loaded with GM-CSF. GM-CSF is generally known to induce the proliferation of various immune cells, such as DCs, but there are concerns regarding the undesired proliferation of immunosuppressive cells, such as MDSCs (Hou et al., 2016). In this study, we demonstrated that mJX-594 inhibited intratumoral CD11b expression. + Gr1 +In addition, neutralization of GM-CSF abolished the therapeutic effect of mJX-594, which is partly due to the CD8 + This was due to a decrease in TILs, indicating that GM-CSF plays an essential role in cancer immunity induced by mJX-594.

[0233] Previous studies have shown that the combination of OV and ICI elicits striking immune responses, but the therapeutic effect can be significantly affected by the administration route and schedule. In particular, when both OV and ICI are administered systemically simultaneously, the combination can be antagonistic because the antiviral immunity induced by ICI can promote premature viral clearance. This suggests that an appropriate time lag between OV treatment and successful induction of anticancer immunity is important. In this study, local injection of mJX-594 consistently induced anticancer immunity, which was not significantly affected by the administration schedule. We speculate that this is because intratumoral injection provided a sufficient time lag for OV to inflame the TME, which was then cleared by systemic antiviral immunity. Therefore, when designing clinical trials involving the combination of ICI and OV, intratumoral OV therapy may be more practical than systemic OV therapy in terms of administration schedule.

[0234] In addition to our promising results using the combination of mJX-594 and ICI, several clinical trials are already underway investigating the efficacy of JX-594 in combination with αPD-1, αCTLA-4, or αPD-L1 targeting various solid tumors, including liver, renal, and colon cancers (ClinicalTrials.gov: NCT03071094, NCT02977156, NCT03294083, and NCT03206073). Therefore, we expect to be able to validate the findings of this study in clinical settings in the near future.

[0235] In conclusion, this study demonstrated that intratumoral injection of mJX-954 induced extensive remodeling of the TME from a cold to a hot state, and in combination with an ICI elicited robust anticancer immunity, overcoming immunotherapy resistance. Experimental procedure Mice and cell lines

[0236] Male BALB / c mice, 6–8 weeks old, were purchased from Orient Bio Inc. (Seongnam, Gyeonggi-do, Korea). Female MMTV-PyMT transgenic mice (FVB / N) were purchased from Jackson Laboratory (Bar Harbor, Maine, USA, #002374). Mice were maintained in a specific pathogen-free animal facility at CHA University (Seongnam, Gyeonggi-do, Korea). All animal experiments were approved by the CHA University Institutional Animal Care and Use Committee (IACUC, #170025) and performed in accordance with approved protocols. Renca mouse renal carcinoma cell line and CT26 mouse colon carcinoma cell line were obtained from the American Type Culture Collection (Manassas, VA, USA, #CRL-2947) and the Korean Cell Line Bank (Seoul, Korea, #80009). These cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium or Dulbecco's modified Eagle's medium (DMEM), each supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were incubated in an incubator at 37°C and 5% CO2. Virus production and quantification

[0237] mJX-594, a Western Reserve (WR) strain of vaccinia virus encoding mouse GM-CSF in the vaccinia thymidine kinase locus under the control of the p7.5 promoter, was provided by Sillajen, Inc. (Seoul, Korea) and used throughout this study. This virus was amplified in HeLaS3 cells and subsequently purified. Briefly, HeLaS3 cells were infected with the recombinant vaccinia virus for 3 days, collected by centrifugation, homogenized, and centrifuged again. The virus-containing supernatant was layered on a 36% sucrose cushion and centrifuged at 32,900 g, and the purified virus pellet was resuspended in 1 mM Tris, pH 9.0. To determine the virus titer, serially diluted virus in serum-free DMEM was plated on a monolayer of U-2 OS cells for 2 hours, followed by the addition of 1.5% carboxymethylcellulose in DMEM supplemented with 2% FBS. After 72 hours, cells were stained with 0.1% crystal violet and plaques were counted. Tumor models and treatment regimens

[0238] Tumors were grown in the right flank of wild-type BALB / c mice at a density of 2 × 10 5 Renca cells were implanted by subcutaneous injection. The tumors were 50 mm 3 When the tumor size exceeded 100 μg / mL, mice were treated with PBS or 1×10 HCl by intratumoral injection every 3 days. 7 Treatment was with either 2 × 10 plaque-forming units (pfu) of mJX-594 for the bilateral tumor model or 2 × 10 5 Renca cells were subcutaneously implanted into the right flank, and 4 days later, 1 × 10 5Renca or CT26 cells were subcutaneously implanted into the left flank. For cell depletion experiments, antibodies against CD4 (200 μg, clone GK1.5, BioXCell), CD8 (200 μg, clone 53-6.72, BioXCell), or GM-CSF (200 μg, clone MP1-22E9, BioXCell) were injected intraperitoneally along with mJX-594. For immune checkpoint blockade, anti-PD-1 antibody (10 mg / kg, clone J43, BioXCell) and / or anti-CTLA-4 antibody (4 mg / kg, clone 9D9, BioXCell) were injected intraperitoneally with or without mJX-594 every 3 days, depending on the administration schedule. Tumors were measured every 2 or 3 days using digital calipers. Tumor volume was calculated using the modified ellipse formula (1 / 2 × (length × width) 2 On day 50, 2 × 10 6 cells were injected into the left flank of surviving mice that had completely regressed. 5 Mice were re-challenged with Renca cells and tumor growth and survival were monitored. Mice were euthanized when tumors reached 1.5 cm in diameter or when the mice became moribund.

[0239] Female MMTV-PyMT transgenic mice were purchased from Jackson Laboratory. At 9 weeks of age, all palpable tumor nodules (>20 mm) were removed. 3 The volume of each tumor was measured. The combined total volume of all tumors was used to calculate the tumor burden per mouse. MMTV-PyMT mice were randomized according to their initial tumor burden and administered 1 × 10 tumors in the presence or absence of immune checkpoint inhibitors PD-1 (10 mg / kg) or CTLA-4 (4 mg / kg) at the indicated time points. 7 Mice were treated with pfu of mJX-594. Four weeks after treatment, mice were anesthetized and tissues were harvested for further analysis. Analysis of MMTV-PyMT was performed as previously reported (Kim et al., 2014; Park et al., 2016). Histological analysis

[0240] For hematoxylin and eosin (H&E) staining, tumors were fixed overnight in 4% paraformaldehyde (PFA). After tissue processing using standard procedures, samples were paraffin-embedded, cut into 3 μm sections, and then H&E stained. Immunofluorescence was performed on frozen tissue sections. Tumors were fixed in 1% PFA at room temperature, rinsed several times with PBS, infiltrated with 30% sucrose, and frozen in OCT compound. Frozen sections (50 μm thick) were blocked in 5% normal goat serum in PBS-T (0.1% Triton X-100 in PBS) and then incubated overnight with the following primary antibodies: anti-vaccinia virus (rabbit, Abcam), anti-CD31 (hamster, clone 2H8, Millipore; rabbit, Abcam), anti-CD8 (rat, clone 53-6.7, BD Pharmingen), anti-CD11c (hamster, clone HL3, BD Pharmingen), anti-PD-L1 (rabbit, clone 28-8, Abcam), anti-caspase 3 (rabbit, R&D Systems), anti-pan-cytokeratin (mouse, clone AE1 / AE3, DAKO), anti-CD11b (rat, clone M1 / 70, BD Pharmingen), or anti-CD3e (hamster, clone 145-2C11, BD Pharmingen). After several washes, the samples were incubated with the following secondary antibodies for 2 hours at room temperature: FITC-, Cy3-, or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch), FITC-conjugated anti-rat IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-hamster IgG (Jackson ImmunoResearch), or FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen). Finally, the samples were mounted using fluorescent mounting medium (DAKO), and images were captured using a Zeiss LSM880 microscope (Carl Zeiss). Morphological analysis

[0241] Densitometry of vaccinia virus, blood vessels, T lymphocytes, and dendritic cells, as well as bone marrow cell area, was performed using ImageJ software (http: / / rsb.info.nih.gov / ij). To determine the level of vaccinia virus infection, random 0.49 mm 2 VV per field of view + The area was calculated. For vascular density, random 0.49 mm2 areas in the peritumoral and intratumoral regions were used. 2 The CD31+ area per field of view was calculated. The extent of cytotoxic T lymphocyte infiltration was assessed by randomly dividing the tumor into 0.49 mm 2 Dendritic cell levels were measured in random 0.49 mm 2 The degree of apoptosis was measured by calculating the percentage of CD11c+ area in a random 0.49 mm 2 The colocalization of PD-L1+ with Pan-CK+, CD11b+, and CD3+ cells was measured in random 0.01 mm fields. 2 Lung metastases in MMTV-PyMT mice were quantified by measuring tumor colonies exceeding 100 μm in diameter. All measurements were performed in at least five fields per mouse. Flow cytometry analysis of tumor-infiltrating immune cells

[0242] Tumors from each treatment group were minced and then incubated with collagenase D (20 mg / ml, Roche) and DNase I (2 mg / ml, Roche) for 1 hour at 37°C with shaking. Cell suspensions were generated by repeated pipetting followed by filtration through a 70 μm cell strainer to lyse and remove red blood cells. After washing with PBS, the resuspended cells were filtered through a nylon mesh. Single-cell suspensions from tumor tissue were blocked with an antibody against CD16 / 32 (clone 2.4G2, BD Pharmingen) and stained with an immobilized viability dye (eFlour450, eBioscience) to identify live cells. For surface marker analysis, cells were incubated in PBS containing 1% FBS for 10 min at 4°C for 1 min at 4°C for CD45 (30-F11, BD Pharmingen), CD4 (RM4-5, BD Pharmingen), CD8 (53-6.7, BD Pharmingen), CD3 (17A2 or 145-2C11, eBioscience), ICOS (7E.17G9 or 15F9, eBioscience), CDllb (M1 / 70, BD Pharmingen), F4 / 80 (BM8, eBioscience), MHC Cells were stained with antibodies targeting Lys II (M5 / 114.15.2, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8-Ly6g or RB6-8C5, eBioscience), or CD206 (MR5D3, eBioscience) for 30 minutes on ice. Cells were then permeabilized using a FoxP3 Fixation and Permeabilization Kit (eBioscience) and stained for FoxP3 (FJK-16s, eBioscience), CD25 (PC61.5, eBioscience), or Granzyme B (NGZB, eBioscience). Labeled cells were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR). RNA isolation and NanoString gene expression analysis

[0243] Total RNA was extracted from whole tumor lysates using TRIzol (Invitrogen) and purified with ethanol. RNA quality was confirmed using a Fragment Analyzer instrument (Advanced Analytical Technologies, Iowa, USA). Immune profiling was performed using 100 ng of total RNA isolated from tumor tissue using the digitally multiplexed NanoString nCounter PanCancer Immune Profiling Mouse Panel (NanoString Technologies). Hybridization was performed for 16–30 h at 65°C by combining 5 μl of each RNA sample with nCounter Reporter probe and 2 μl of nCounter Capture probe in 8 μl of hybridization buffer, for a total reaction volume of 15 μl. Excess probe was removed by two-step magnetic bead-based purification using an nCounter Prep Station (NanoString Technologies). The abundance of specific target molecules was quantified using an nCounter Digital Analyzer by counting individual fluorescent barcodes and assessing the corresponding target molecules. For each assay, a high-density scan covering 280 fields of view was performed. Data were collected using an nCounter Digital Analyzer after capturing images of the fluorescent reporters immobilized in the sample cartridge using a CCD camera. Data analysis was performed using nSolver software (NanoString Technologies). mRNA profiling data were normalized to housekeeping genes and analyzed using R software (www.r-project.org). statistical analysis

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[0245] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods described herein, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the concept, spirit, and scope of the invention as defined by the appended claims.

[0246] The above examples are provided to provide those skilled in the art with a complete disclosure of embodiments of the compositions, systems, and methods of the present invention, as well as an explanation of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0247] All heading and section designations are used for clarity and reference purposes only and are not to be construed as limiting in any way. For example, one of ordinary skill in the art will recognize the utility of combining various aspects of different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0248] All references cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0249] As will be apparent to those skilled in the art, many variations and modifications of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered for illustrative purposes only, and this application is limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. The present invention provides, for example, the following items. (Item 1) A method for treating and / or preventing cancer in a subject in need of such treatment, comprising concurrently administering to the subject effective amounts of a combination comprising (a) a replication-competent oncolytic vaccinia virus and (b) one or more immune checkpoint inhibitors. (Item 2) 2. The method of claim 1, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally. (Item 3) 3. The method of claim 2, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally. (Item 4) 4. The method of any one of items 1 to 3, wherein the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in a tumor. (Item 5) 5. The method of any one of items 1 to 4, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level before administration of the replication-competent oncolytic vaccinia virus. (Item 6) 6. The method according to any one of items 1 to 5, wherein the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to the immune checkpoint protein, and is preferably a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. (Item 7) The checkpoint inhibitors are selected from the group consisting of cytotoxic T-lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD-1), PD-L1, PD-L2, B7-H3, B7-H4, herpesvirus entry mediator (HVEM), T-cell membrane protein 3 (TEM3), galectin 9 (GAL9), lymphocyte activation gene 3 (LAG3), V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), and IL-11. 7. The method according to any one of items 1 to 6, wherein the method inhibits an immune checkpoint protein selected from the group consisting of B and T lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase (IDO), or a combination thereof. (Item 8) 8. The method of item 7, wherein the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1 or PD-L1, and is preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. (Item 9) Item 8. The method of item 7, wherein the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to CTLA4, preferably selected from the group consisting of ipilimumab and tremelimumab. (Item 10) 10. The method of any one of items 1 to 9, wherein the multiple checkpoint inhibitors are administered to the subject in parallel with the oncolytic vaccinia virus. (Item 11) The object is (a) CTLA4 inhibitor, PD-1 inhibitor, and replication-competent oncolytic vaccinia virus; (b) a CTLA4 inhibitor, an IDO inhibitor, and a replication-competent oncolytic vaccinia virus; (c) a PD-1 inhibitor, an IDO inhibitor, and a replication-competent oncolytic vaccinia virus, or (d) PD-1 inhibitor, CTLA4 inhibitor, IDO inhibitor, and replication-competent oncolytic vaccinia virus; (e) a LAG3 inhibitor, a PD-1 inhibitor, and a replication-competent oncolytic vaccinia virus; or (f) The method of item 10, wherein the TIGIT inhibitor, the PD-1 inhibitor, and the replication-competent oncolytic vaccinia virus are administered in parallel. (Item 12) 12. The method of any one of items 1 to 11, wherein the replication-competent oncolytic vaccinia virus is a Wyeth strain, a Western Reserve strain, a Lister strain, or a Copenhagen strain. (Item 13) 13. The method of any one of items 1 to 12, wherein the vaccinia virus comprises one or more genetic modifications that increase selectivity of the virus for cancer cells, preferably the virus is engineered to lack a functional thymidine kinase and / or to lack a functional vaccinia growth factor. (Item 14) 14. The method of any one of items 1 to 13, wherein the vaccinia virus comprises a functional 14L gene and / or F4L gene. (Item 15) The vaccinia virus is capable of producing (i) a cytokine, preferably selected from GM-CSF, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21, IL-24, IFN-γ, TNF-α, and / or (ii) a cytokine, preferably selected from BAGE, GAGE-1, GAGE-2, CEA, AIM2, CDK4, BMI1, COX-2, MUM-1, MUC-1, TRP-1 TRP-2, GP100, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, SOX2, human papillomavirus E6, human papillomavirus E7, ARTl, ART4, SARTl, SART2, SART3, B-cyclin, β-catenin, Gli1, Cav-1, cathepsin B, CD74, E-cadherin, EphA2 / Eck, Fra-1 / Fosl 1, ganglioside / GD2, GnT-V, β1,6-Ν, Her2 / neu, Ki67, Ku70 / 80, IL-13Ra2, MAGE-1, MAGE-3, NY-ESO-1, MART-1, PROX1, PSCA, SOX10, SOX11, survivin, caspase-8, UPAR, CA-125, PSA, pl85HER2, CD5, IL-2R, Fap-α, tenascin, melanoma-associated antigen p97, and WT-1, regulator of G-protein signaling 5 (RGS5). 5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible lipid droplet-associated protein 2 (HIG2),hypoxial inducible lipid droplet associated), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Ql-like: RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFIl), lysosomal protein transmembrane 4 alpha (LAPTM4A); RAB1B, RAS 15. The method of any one of items 1 to 14, wherein the tumor is engineered to express a tumor antigen selected from the group consisting of oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium-binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin 2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor 1 (ATPIF1), stromal antibody 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). (Item 16) The vaccinia virus is about 10 7 ~about 10 11 Amount of pfu, preferably about 10 8 ~10 10 pfu, more preferably about 10 9 ~10 10 16. The method of any one of items 1 to 15, wherein the amount of the antibody administered is in an amount of pfu. (Item 17) 17. The method of any one of items 1 to 16, wherein the checkpoint inhibitor is administered in an amount of about 2 mg / kg to 15 mg / kg. (Item 18) 18. The method according to any one of items 1 to 17, wherein the subject has a cancer selected from hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, and liver cancer. (Item 19) 19. The method of claim 18, wherein the subject has renal cell carcinoma. (Item 20) 20. The method of any one of items 1 to 19, wherein the subject has previously failed at least one chemotherapy or immunotherapy. (Item 21) 21. The method of item 20, wherein the subject has cancer that is refractory to immune checkpoint inhibitor therapy, preferably the cancer is resistant to treatment with an anti-PD-1 antibody and / or an anti-CTLA-4 antibody. (Item 22) 22. The method of claim 21, wherein the subject is identified as a candidate for immune checkpoint inhibitor therapy. (Item 23) 23. The method of any one of items 1 to 22, comprising administering to the subject an additional therapy selected from chemotherapy (alkylating agents, nucleoside analogues, cytoskeleton-modifying agents, cytostatic agents) and radiation therapy. (Item 24) 24. The method of any one of items 1 to 23, comprising administering to the subject an additional oncolytic virus therapy (e.g., rhabdovirus, Semliki Forest Virus). (Item 25) 25. The method according to any one of items 1 to 24, wherein the subject is a human. (Item 26) 26. The method of any one of items 1 to 25, wherein a first dose of the replication-competent oncolytic vaccinia virus and a first dose of the immune checkpoint inhibitor are administered to the subject simultaneously, followed by at least one subsequent sequential co-administration of the virus and the checkpoint inhibitor to the subject. (Item 27) 27. The method of claim 26, comprising at least first, second, and third sequential co-administrations of a replication-competent oncolytic vaccinia virus and a checkpoint inhibitor to the subject. (Item 28) 28. The method of claim 27, comprising at least a first, second, third, and fourth sequential co-administration of a replication-competent oncolytic vaccinia virus and a checkpoint inhibitor to the subject. (Item 29) 29. The method of any one of items 26-28, wherein the simultaneous administration to the subject of a first dose of a replication-competent oncolytic vaccinia virus and a first dose of an immune checkpoint inhibitor, and at least one subsequent sequential simultaneous administration of the virus and checkpoint inhibitor, is followed by administration to the subject of at least one dose of the checkpoint inhibitor alone. (Item 30) 30. The method of any one of items 26 to 29, comprising an interval of 1 to 3 weeks between consecutive co-administrations of said agents, preferably comprising an interval of about 1 week, about 2 weeks, or about 3 weeks. (Item 31) A method for treating a tumor in a human, comprising concurrently administering to the human a combination containing (a) a replication-competent oncolytic vaccinia virus and (b) an inhibitor of an immune checkpoint protein. (Item 32) 32. The method of claim 31, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally. (Item 33) 32. The method of claim 31, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally. (Item 34) 32. The method of claim 31, wherein the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. (Item 35) 32. The method of claim 31, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administration of the replication-competent oncolytic vaccinia virus. (Item 36) 36. The method of item 34 or 35, comprising measuring the expression level of the immune checkpoint protein in the tumor prior to administering the combination. (Item 37) 37. The method of any one of items 31 to 36, wherein the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. (Item 38) 32. The method of claim 31, wherein the immune checkpoint protein is CTLA-4. (Item 39) 32. The method of claim 31, wherein the immune checkpoint protein is PD-L1. (Item 40) 32. The method of claim 31, wherein the immune checkpoint protein is LAG3. (Item 41) 32. The method of claim 31, wherein the immune checkpoint protein is TIGIT. (Item 42) 32. The method of claim 31, wherein the immune checkpoint protein is PD-1. (Item 43) 32. The method of claim 31, wherein the immune checkpoint protein is TIM3. (Item 44) 44. The method according to any one of items 31 to 43, wherein the tumor is a solid cancer. (Item 45) 44. The method according to any one of items 31 to 43, wherein the tumor is colon cancer. (Item 46) 44. The method according to any one of items 31 to 43, wherein the tumor is renal cell carcinoma. (Item 47) 43. The method of item 39 or 42, wherein the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1, and is preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. (Item 48) 39. The method of claim 38, wherein the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4, preferably selected from the group consisting of ipilimumab and tremelimumab. (Item 49) A method of treating a tumor in a human, comprising concurrently administering to the human a combination comprising: (a) a replication-competent oncolytic vaccinia virus, (b) an inhibitor of PD-1 and / or PD-L1, and (c) an inhibitor of an immune checkpoint protein. (Item 50) 50. The method of claim 49, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administration of the replication-competent oncolytic vaccinia virus. (Item 51) 50. The method of claim 49, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally. (Item 52) 50. The method of claim 49, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally. (Item 53) 50. The method of claim 49, wherein the replication-competent oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. (Item 54) 50. The method of claim 49, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administration of the replication-competent oncolytic vaccinia virus. (Item 55) 55. The method of claim 53 or 54, comprising measuring the expression level of the checkpoint protein in the tumor prior to administering the combination. (Item 56) 56. The method of any one of items 49 to 55, wherein the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3, and TIGIT. (Item 57) 57. The method of claim 56, wherein the immune checkpoint protein is CTLA-4. (Item 58) 57. The method of claim 56, wherein the immune checkpoint protein is LAG3. (Item 59) 57. The method of claim 56, wherein the immune checkpoint protein is TIGIT. (Item 60) 57. The method of claim 56, wherein the immune checkpoint protein is TIM3. (Item 61) 61. The method according to any one of items 49 to 60, wherein the tumor is a solid cancer. (Item 62) 62. The method according to any one of items 49 to 61, wherein the tumor is colon cancer. (Item 63) 63. The method according to any one of items 49 to 62, wherein the tumor is renal cell carcinoma. (Item 64) 50. The method of item 49, wherein the inhibitor of an immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1, and is preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. (Item 65) 58. The method of claim 57, wherein the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4, preferably selected from the group consisting of ipilimumab and tremelimumab.

Claims

1. 1. A combination comprising, in synergistic amounts, (a) a replication-competent oncolytic vaccinia virus that contains a functional F4L gene, does not express CXCL-11, is thymidine kinase-deficient, and is engineered to express human GM-CSF, and (b) one or more immune checkpoint inhibitors, for use in treating renal cell carcinoma, colon cancer, hepatocellular carcinoma, breast cancer, melanoma, prostate cancer, pancreatic cancer, or ovarian cancer in a human subject in need of such treatment; a combination wherein the one or more immune checkpoint inhibitors inhibit one or more immune checkpoint proteins selected from cytotoxic T-lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD-1), and PD-L1, (a) and (b) are administered concurrently, and the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to the immune checkpoint protein, and is preferably a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein comprising the antibody or a fragment thereof, or a combination thereof.

2. The combination for use according to claim 1, wherein the replication-competent oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally.

3. 3. The combination for use according to claim 1 or 2, wherein the immune checkpoint inhibitor is a monoclonal antibody.

4. The combination for use according to claim 3, wherein the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1.

5. The combination for use according to claim 3, wherein the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to CTLA4.

6. (a) a CTLA4 inhibitor, a PD-1 inhibitor, and a replication-competent oncolytic vaccinia virus; (b) a CTLA4 inhibitor, a PD-L1 inhibitor, and a replication-competent oncolytic vaccinia virus; or (c) a PD-1 inhibitor, a PD-L1 inhibitor, and a replication-competent oncolytic vaccinia virus A combination for use according to any one of claims 1 to 5, comprising:

7. The combination for use according to any one of claims 1 to 6, wherein the replication-competent oncolytic vaccinia virus is a Wyeth, Western Reserve, Lister, or Copenhagen strain.

8. The combination for use according to any one of claims 1 to 7, wherein said vaccinia virus lacks a functional vaccinia growth factor.

9. The combination for use according to any one of claims 1 to 8, wherein said vaccinia virus comprises functional 14L and F4L genes.

10. The vaccinia virus is ７ ~10 １１ The combination for use according to any one of claims 1 to 9, administered in an amount of pfu.

11. The vaccinia virus is ８ ~10 １０ 11. The combination for use according to claim 10, administered in an amount of pfu.

12. The vaccinia virus is ９ ~10 １０ 11. The combination for use according to claim 10, administered in an amount of pfu.

13. The combination for use according to any one of claims 1 to 12, wherein the checkpoint inhibitor is administered in an amount of 2 mg / kg to 15 mg / kg.

14. A combination for use according to any one of claims 1 to 13 for treating metastatic renal cell carcinoma and / or for treating metastatic pancreatic cancer.

15. The combination for use according to any one of claims 1 to 14, wherein the subject has previously failed at least one chemotherapy or immunotherapy.

16. The combination for use according to claim 15, wherein the subject has a cancer that is refractory to treatment with an anti-PD-1 antibody and / or an anti-CTLA-4 antibody.

17. 17. The combination for use according to claim 16, wherein the subject is identified as a candidate for immune checkpoint inhibitor therapy.

18. The combination for use according to any one of claims 1 to 17, which is administered to the subject in combination with an additional therapy selected from chemotherapy and radiotherapy.

19. The combination for use according to any one of claims 1 to 18, which is administered to the subject in combination with an additional oncolytic virus therapy.

20. The combination for use according to any one of claims 1 to 19, wherein at least one dose of said replication-competent oncolytic vaccinia virus is administered simultaneously with at least one dose of an immune checkpoint inhibitor.

21. 21. The combination for use according to claim 20, wherein at least one dose of said replication-competent oncolytic vaccinia virus is administered simultaneously with a first dose of an immune checkpoint inhibitor.

22. 22. The combination for use according to claim 21, wherein at least one dose of the replication-competent oncolytic vaccinia virus is administered to the subject within 24 hours of administration of a first dose of the immune checkpoint inhibitor.

23. 23. The combination for use according to any one of claims 1 to 22, wherein the replication-competent oncolytic vaccinia virus is a Wyeth strain or a Western Reserve strain, and the one or more immune checkpoint inhibitors comprise an anti-PD-1 antibody.

24. 24. The combination for use according to any one of claims 1 to 23, wherein the oncolytic vaccinia virus is administered once a week and / or the one or more immune checkpoint inhibitors are administered once every three weeks.

25. A combination for use according to any one of claims 1 to 24, wherein the parallel administration comprises administration of (a) within 12 hours of administration of (b).

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