Oncolytic viruses expressing IRF modulators for the treatment of cancer

Abstract

The present invention provides oncolytic viruses that express modulators of interferon regulatory factors (IRFs), and compositions comprising the same. The present invention further provides methods of using the oncolytic viruses and compositions to treat cancer and to improve a subject's responsiveness to immunomodulatory drugs (e.g., immune checkpoint inhibitors).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 985,979, filed March 6, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Grant Notification This invention was made with government support under Grant No. CA178766 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on March 5, 2021, is named 072396_0849_ST25.txt and is 6,375 bytes in size.

[0004] Technical Field The present invention provides oncolytic viruses that express modulators of interferon regulatory factors (IRFs) (i.e., IRF modulators), and compositions comprising the same. The present invention further provides methods of using the oncolytic viruses and compositions to treat cancer and to improve a subject's responsiveness to immunomodulatory agents (e.g., immune checkpoint inhibitors). [Background technology]

[0005] background Immunotherapies, such as immune checkpoint inhibitors (e.g., anti-PD-1 or anti-CTLA-4 antibodies), have become a mainstay of cancer treatment. However, these therapies, both as monotherapy and in combination, are effective in only a subset of patients. There is a growing body of literature demonstrating that patients who previously responded to immune checkpoint inhibitors can subsequently develop resistance to these drugs.

[0006] Oncolytic virus (OV) cancer therapy is a type of immunotherapy that uses viruses that can selectively infect and lyse tumor cells but have no or minimal pathogenicity to normal, non-tumor host cells. In addition to their direct killing (oncolysis) ability, oncolytic viruses can also induce host antitumor immune responses. However, OV cancer therapy has limited efficacy in clinical applications.

[0007] Thus, there remains a need for methods and compositions to improve cancer patient responsiveness to immunotherapy (e.g., immune checkpoint inhibitors) and to improve the efficacy of cancer treatment with OVs. Summary of the Invention

[0008] The present invention provides oncolytic viruses expressing modulators of interferon regulatory factors (IRFs), and compositions comprising same, based at least in part on the discovery that delivery of an oncolytic virus expressing an IRF1 inhibitor to a tumor inhibited tumor growth in vivo.

[0009] In some embodiments, provided herein is an oncolytic virus comprising a nucleic acid molecule encoding a modulator of an interferon regulatory factor (IRF).

[0010] In certain embodiments, the IRF is IRF1, IRF3, IRF7, or a combination thereof. In certain embodiments, the IRF is IRF1. In certain embodiments, the modulator inhibits the activity of an IRF. In certain embodiments, the modulator inhibits the activity of IRF1.

[0011] In certain embodiments, the modulator is IRF2. In certain embodiments, the IRF2 is human IRF2 or mouse IRF2.

[0012] In certain embodiments, the modulator decreases IRF-mediated gene expression, hi certain embodiments, the modulator decreases CD274 gene expression.

[0013] In certain embodiments, the nucleic acid molecule is a foreign nucleic acid molecule, hi certain embodiments, the nucleic acid molecule is integrated into the genome of an oncolytic virus.

[0014] In certain embodiments, the oncolytic virus is an oncolytic vaccinia virus. In certain embodiments, the oncolytic vaccinia virus lacks expression of a functional thymidine kinase (TK).

[0015] In another aspect, the present invention provides a method of treating a subject having cancer, comprising administering to the subject an oncolytic virus of the present invention. In certain embodiments, the subject is a human subject.

[0016] In certain embodiments, the methods of the present invention further comprise administering an immunomodulatory agent to the subject. In certain embodiments, the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof. In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0017] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof. In certain embodiments, the cancer is melanoma or renal cancer.

[0018] In another aspect, the invention provides a method for improving a subject's responsiveness to an immunomodulatory drug, the method comprising administering an oncolytic virus of the invention to the subject, the subject having cancer. In certain embodiments, the subject is a human subject.

[0019] In certain embodiments, the subject has previously been treated with an immunomodulatory agent. In certain embodiments, the subject has developed resistance to the immunomodulatory agent. In certain embodiments, the method of the present invention further comprises administering an immunomodulatory agent to the subject. In certain embodiments, the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof. In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0020] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof. In certain embodiments, the cancer is melanoma or renal cancer.

[0021] In another aspect, the present invention provides a pharmaceutical composition comprising an oncolytic virus of the present invention.

[0022] In certain embodiments, the pharmaceutical composition of the present invention further comprises an immunomodulatory agent. In certain embodiments, the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof. In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0023] In certain embodiments, a pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0024] In certain embodiments, the pharmaceutical compositions of the present invention are intended to treat a subject with cancer or to improve a subject's responsiveness to an immunomodulatory agent.

[0025] In another aspect, the present specification provides a kit comprising an oncolytic virus of the present invention or a pharmaceutical composition of the present invention. In certain embodiments, the kit of the present invention further comprises an immunomodulatory agent. In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0026] In certain embodiments, the kit further comprises instructions for treating a subject with cancer or for improving a subject's responsiveness to an immunomodulatory agent. [Brief explanation of the drawings]

[0027] [Figure 1A] Figures 1A-1D show that IRF2 promoted tumor regression. Figures 1A-1B show quantification of PD-L1 expression by flow cytometry in human MEL-285 melanoma cells (Figure 1A) and mouse B16 melanoma cells (Figure 1B). Human MEL-285 melanoma cells were transfected with a human IRF2 expression vector or a control vector and then stimulated with IFN-γ. Mouse B16 melanoma cells were transfected with a mouse Irf2 (mIrf2) expression vector or a control vector and then stimulated with IFN-γ. [Figure 1B] Figures 1A-1B show quantification of PD-L1 expression by flow cytometry in human MEL-285 melanoma cells (Figure 1A) and murine B16 melanoma cells (Figure 1B). Human MEL-285 melanoma cells were transfected with a human IRF2 expression vector or a control vector and then stimulated with IFN-γ. Murine B16 melanoma cells were transfected with a murine Irf2 (mIrf2) expression vector or a control vector and then stimulated with IFN-γ. [Figure 1C]Figure 1C shows tumor volumes measured from days 0 to 22 in mice implanted with murine B16 melanoma cells and treated with oncolytic vaccinia virus carrying murine Irf2 (VV-mIrf2) or control vaccinia virus (VV-control). [Figure 1D] FIG. 1D shows tumor volumes measured in BALB / C mice injected with RENCA tumors followed by treatment with VV-mIrf2 or VV-control.

[0028] Detailed Description Non-limiting embodiments of the present invention are illustrated herein and by way of example. For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. Oncolytic viruses expressing IRF modulators; 5.3. Pharmaceutical compositions; 5.4. Treatment methods; and 5.5. Kits.

[0029] 5.1. Definition The terms used herein generally have their ordinary meanings in the art, within the context of this invention, as well as in the particular context in which each term is used. Certain terms are explained below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of the invention, and how to make and use them.

[0030] As used herein, the use of the words "a" or "an," when used in conjunction with the term "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." Furthermore, the terms "having," "including," "containing," and "comprising" are interchangeable, and those of skill in the art will recognize that these terms are open-ended.

[0031] The terms "about" or "approximately" mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value.

[0032] The term "modulator," as used herein as "modulator of interferon regulatory factor (IRF)" or synonymously "IRF modulator," refers to a molecule that can regulate the activity of an IRF. In certain embodiments, the modulator can inhibit the activity of an IRF. In certain embodiments, the modulator is a protein molecule (e.g., IRF2).

[0033] As used herein, the term "oncolytic virus" or "OV" refers to a virus that selectively replicates in cancer cells, has no or minimal effect on normal cells, but has the ability to slow the growth of cancer cells or induce cancer cell death in vitro or in vivo. In certain embodiments, oncolytic viruses propagate into tumors without damaging non-cancerous tissues. In certain embodiments, oncolytic viruses do not replicate or replicate at a slower rate in non-cancerous cells than in cancer cells. Non-limiting examples of oncolytic viruses include coxsackievirus, Maraba virus (rhabdovirus), parvovirus, Seneca Valley virus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), retrovirus, reovirus, measles virus, Sindbis virus, influenza virus, herpes simplex virus (HSV), Sendai virus, vaccinia virus (VV), and adenovirus, as well as their mutant strains.

[0034] As used herein, the term "vaccinia virus" or "VV" refers to an enveloped DNA virus belonging to the Poxviridae family. In certain embodiments, VV comprises a linear, double-stranded DNA genome of approximately 200 kb. Non-limiting examples of vaccinia virus strains include Western Reserve (WR), Tashkent, Lister (also known as Elstree), Dryvax (also known as Wyeth), IHD-J, and IHD-W, Brighton, Ankara, modified vaccinia Ankara (MVA), Dairen (e.g., Dairen I (DIs)), LIPV, Lister clone 16m8 (LC16m8), LC16MO, LIVP, WR 65-16, Connaught, New York City Board of Health (NYCBH), EM63, ACAM2000™, CV-1, Paris, Copenhagen (Cop), Bern, and Tian Tan (VTT).

[0035] The term "mutation" as used herein refers to a mutation in an amino acid sequence or a nucleotide sequence. In certain embodiments, a mutation in an amino acid sequence can be a substitution (replacement), insertion (addition), or deletion (truncation) of at least one amino acid in the amino acid sequence. In certain embodiments, a mutation in a nucleotide sequence can be a substitution (substitution), insertion (addition), or deletion (truncation) of at least one nucleotide in the nucleotide sequence.

[0036] As used herein, an "individual" or "subject" refers to a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0037] As used herein, the term "disease" means any abnormality or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0038] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an oncolytic virus composition sufficient to reduce, inhibit, or eliminate tumor cell proliferation in vitro or in vivo. In certain embodiments, the reduction, inhibition, or elimination of tumor cell proliferation may be the result of necrosis, apoptosis, or an immune response. The amount of an oncolytic virus composition that is therapeutically effective or efficacious may vary depending on the circumstances. An effective amount may be administered in one or more doses.

[0039] As used herein and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, prevention of disease, delay or slowing of disease progression, and / or improvement or alleviation of disease state. A reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of a complication or symptom. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0040] 5.2. Oncolytic viruses expressing IRF modulators Provided herein are oncolytic viruses that express a modulator of IRF (i.e., an IRF modulator). In certain embodiments, the oncolytic virus comprises a nucleic acid molecule encoding the IRF modulator. In certain embodiments, the nucleic acid molecule is an exogenous nucleic acid molecule. In certain embodiments, the nucleic acid molecule is integrated into the genome of the oncolytic virus. The nucleic acid molecule encoding the IRF modulator can be a DNA molecule, an RNA molecule, or a cDNA molecule, so as to match the nucleic acid of the oncolytic virus genome into which it is integrated.

[0041] Interferon regulatory factors (IRFs) are a family of transcription factors that can control the expression of proteins involved in innate and adaptive immunity. Currently, nine IRFs exist in mammals, including IRF1, IRF2, IRF3, IRF4 (i.e., PIP, ICSAT), IRF5, IRF6, IRF7, IRF8 (i.e., ICSBP), and IRF9 (i.e., p48, ISGF3γ). The present disclosure has discovered that administering a drug (e.g., an IRF modulator) that inhibits IRF activity in tumors in vivo can promote anti-tumor immune responses and suppress tumor growth. The present disclosure has also discovered that IRF inhibitors can reduce the expression of programmed death-ligand 1 (PD-L1). PD-L1 plays an essential role in physiological immune homeostasis and is involved in immune evasion strategies employed by cancer cells. Reducing PD-L1 expression can improve the host's anti-tumor immune response and enhance the responsiveness of cancer cells to immunotherapy.

[0042] In certain embodiments, the oncolytic viruses of the invention express an IRF modulator that modulates the activity of an IRF, which suppresses anti-tumor immunity. IRFs that can suppress anti-tumor immunity include, but are not limited to, IRF1, IRF3, and IRF7.

[0043] In certain embodiments, the IRF modulator (e.g., IRF2) inhibits (e.g., reduces or eliminates) the activity of IRF1, IRF3, IRF7, or a combination thereof. In certain embodiments, the IRF modulator inhibits the activity of IRF1. In certain embodiments, the IRF modulator inhibits (e.g., reduces or eliminates) the expression of a gene regulated by IRF1. In certain embodiments, the IRF modulator inhibits, reduces, and / or eliminates the expression of the CD274 gene (encoding PD-L1), ITGA8 gene, ENAH gene, PMP22 gene, SULF2 gene, CIITA gene, PGF gene, COL4A1 gene, ERAP1 gene, NNMT gene, AXL gene, or a combination thereof. In certain embodiments, the IRF modulator inhibits, reduces, and / or eliminates the level of a protein expressed by the CD274 gene, the ITGA8 gene, the ENAH gene, the PMP22 gene, the SULF2 gene, the CIITA gene, the PGF gene, the COL4A1 gene, the ERAP1 gene, the NNMT gene, the AXL gene, or a combination thereof. In certain embodiments, the IRF modulator reduces the expression of the CD274 gene. In certain embodiments, the IRF modulator reduces the level of the PD-L1 protein.

[0044] In certain embodiments, the IRF modulator is IRF2. IRF2 can competitively inhibit IRF-mediated (e.g., IRF1-mediated) transcriptional activation of interferon α and β, as well as other genes that utilize IRFs for transcriptional activation. In certain embodiments, the oncolytic viruses of the invention comprise a nucleic acid molecule encoding IRF2.

[0045] In certain embodiments, the nucleic acid molecule encodes human IRF2. In certain embodiments, the nucleic acid molecule encodes human IRF2 having the amino acid sequence set forth in SEQ ID NO:1.

[0046] MPVERMRMRPWLEEQINSNTIPGLKWLNKEKKIFQIPWMHAARHGWDVEKDAPLFRNWAIHTGKHQPGVDKPDPKTWKANFRCAMNSLPDIEEVKDKSIKKGNNAFRVYRMLPLSERPSKKGKKPKTEKEDKVKHIKQEPVESSLGLSNGVSDLSPEYAVLTSTIKNEVDSTVNIIVVGQSHLDSNIENQEIVTNPPDICQVVEVTTESDEQPVSMSELYPLQISPVSSYAESETTDSVPSDEESAEGRPHWRKRNIEGKQYLSNMGTRGSYLLPGMASFVTSNKPDLQVTIKEESNPVPYNSSWPPFQDLPLSSSMTPASSSSRPDRETRASVIKKTSDITQARVKSC [SEQ ID NO: 1]

[0047] In certain embodiments, human IRF2 has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to the amino acid sequence set forth in GenBank / NCBI database accession number NP_002190. In certain embodiments, a nucleic acid molecule encodes human IRF2, which may include substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank / NCBI database accession number NP_002190 that do not significantly alter the function or activity of human IRF2.

[0048] In certain embodiments, the nucleic acid molecule encodes mouse IRF2. In certain embodiments, the nucleic acid molecule encodes mouse IRF2 having the amino acid sequence set forth in SEQ ID NO:2.

[0049] MPVERMRMRPWLEEQINSNTIPGLKWLNKEKKIFQIPWMHAARHGWDVEKDAPLFRNWAIHTGKHQPGIDKPDPKTWKANFRCAMNSLPDIEEVKDRSIKKGNNAFRVYRMLPLSERPSKKGKKPKTEKEERVKHIKQEPVESSLGLSNGVSGFSPEYAVLTSAIKNEVDSTVNIIVVGQSHLDSNIEDQEIVTNPPDICQVVEVTTESDDQPVSMSELYPLQISPVSSYAESETTDSVASDEENAEGRPHWRKRSIEGKQYLSNMGTRNTYLLPSMATFVTSNKPDLQVTIKEDSCPMPYNSSWPPFTDLPLPAPVTPTPSSSRPDRETRASVIKKTSDITQARV [SEQ ID NO: 2]

[0050] In certain embodiments, the mouse IRF2 has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to the amino acid sequence set forth in GenBank / NCBI database accession number NP_032417. In certain embodiments, the nucleic acid molecule encodes a mouse IRF2 that may include substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank / NCBI database accession number NP_032417 that do not significantly alter the function or activity of the mouse IRF2.

[0051] In certain embodiments, conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, only one, no more than two, no more than three, no more than four, or no more than five residues in a designated sequence are changed. Examples of conservative amino acid substitutions are shown in Table 1 below.

[0052] [Table 1]

[0053] As used herein, the homology percentage between two amino acid sequences is equivalent to the identity percentage between two sequences.The identity percentage between two sequences is a function of the number of identical positions that the sequences share, taking into account the number of gaps that need to be introduced for optimal alignment of two sequences and the length of each gap (i.e., homology percentage=number of identical positions / total number of positions×100).Comparing sequences and determining the identity percentage between two sequences can be achieved using mathematical algorithms.

[0054] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol., 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0055] Any suitable oncolytic virus can be used in the present disclosure.Non-limiting examples of the oncolytic virus that can be used in the present disclosure include Coxsackievirus, Myxoma virus, Maraba virus (rhabdovirus), Parvovirus, Seneca Valley virus, Vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), retrovirus, Reovirus, Measles virus, Sindbis virus, Influenza virus, Herpes simplex virus (HSV), Sendai virus, Vaccinia virus (VV) and Adenovirus, and their mutant strains (variants).

[0056] In certain embodiments, the oncolytic virus described herein is an oncolytic vaccinia virus. Any suitable strain of vaccinia virus can be used in accordance with the presently disclosed subject matter. Non-limiting examples of vaccinia virus strains that can be used in connection with the presently disclosed subject matter include the Western Reserve (WR) strain, the Tashkent strain, the Lister strain (also known as Elstree), the Dryvax strain (also known as the Wyeth strain), the IHD-J strain, and the IHD-W strain, the Brighton strain, the Ankara strain, the modified vaccinia Ankara (MVA) strain, the Dairen strain (e.g., the Dairen I strain (DIs)), the LIPV strain, the Lister clone 16m8 (LC16m8) strain, the LC16MO strain, the LIVP strain, the WR 65-16 strain, the Connaught strain, the New York City Board of Health (NYCBH) strain, the EM63 strain, the ACAM2000™ strain, the CV-1 strain, the Paris strain, the Copenhagen (Cop) strain, the Bern strain, the USSR strain, the Evans strain, and the Tian Tan (VTT) strain, derivatives thereof, or modified versions thereof.

[0057] Further non-limiting examples of oncolytic viruses that can be used herein include Talimogene Laherparepvec (T-Vec) (Amgen), TBI-1401 (HF10) (Takara), HSV1716 (Virtu Biologics), ADV / HSV-tk (Merk), LOAd703 (Loken), CG0070 (Cold Genesys), ColoAd1 (Enadenotucirev) (PsiOxus), ONCOS-102 (Targovax Oy), DNX-2401 (DNAtrix), VCN-01 (VCN), Ad-MAGEA3 and MG1-MAGEA3 (Turnstone), NSC-CRAd-Survivin-pk7 (Northwestern), Ad5-yCD / mutTKSR39rep-hIL12 (Henry Ford), Ad5-yCD / mutTKSR39rep-ADP (Henry Ford), MV-NIS (Mayo), MV-NIS (University of Arkansas), GL-ONC1 (Genelux), Pexastimogene Devacirepvec (Pexa-Vec) (Jennerx), REOLYSIN (Oncolytics), CVA21 (CAVATAK) (Viralytics), H-1PV (ParvOryx) (Oryx GmbH), PVSRIPO (Duke), vvDD (NIH), TBio-6517 (Turnstone), and VSV-hIFNbeta-NIS (Mayo).

[0058] In certain embodiments, a nucleic acid molecule encoding an IRF modulator is integrated into the genome of an oncolytic virus, and expression of the nucleic acid molecule is operably linked to a promoter that is active or activatable in an oncolytic virus-infected cell, e.g., an oncolytic virus promoter. As used herein, "operably linked" means that the promoter is in the correct functional location and / or orientation relative to the locus of the nucleic acid so that it can regulate transcription initiation and / or expression of that locus.

[0059] In certain embodiments, the promoter is a vaccinia virus promoter. In certain embodiments, the vaccinia virus promoter is a synthetic vaccinia promoter. Non-limiting examples of vaccinia promoters that can be used in connection with the presently disclosed subject matter include pSE / L and p7.5.

[0060] In certain embodiments, the oncolytic virus is attenuated to reduce viral pathogenicity and improve the safety of therapeutic use of the oncolytic virus. In certain embodiments, the oncolytic virus is a naturally attenuated strain. In certain embodiments, the oncolytic virus is genetically modified to reduce viral pathogenicity.

[0061] In certain embodiments, the oncolytic vaccinia viruses described herein lack expression of functional thymidine kinase (TK). In certain embodiments, the oncolytic vaccinia viruses described herein are TK-negative. TK is encoded by the J2R gene (also known as the tk gene) and forms part of the salvage pathway for pyrimidine deoxyribonucleotide synthesis. The lack of functional TK expression can improve the safety of the oncolytic vaccinia virus. In certain embodiments, the oncolytic vaccinia virus comprises a mutation in the J2R gene. In certain embodiments, the mutation in the J2R gene can be a deletion, substitution, and / or insertion of at least one nucleotide in the J2R gene nucleotide sequence. In certain embodiments, the mutation in the J2R gene comprises an insertion of a nucleic acid molecule into the J2R gene locus.

[0062] In certain embodiments, the mutation in the gene (e.g., the J2R gene) is an inactivating mutation that significantly reduces expression of the gene or renders the product encoded by the gene (e.g., TK) non-functional or significantly reduces its ability to function. In certain embodiments, a nucleic acid molecule encoding an IRF modulator (e.g., IRF2) is integrated into the J2R gene locus.

[0063] In addition to modifying the expression of TK, other approaches can be used to create attenuated oncolytic viruses, and improve the safety of oncolytic virus therapeutic use.Non-limiting examples of attenuated oncolytic viruses include vSP virus (Guo et al., Cancer Res. 2005 Nov 1;65(21):9991-8), modified vaccinia Ankara (MVA) (Harrop et al., Clin Cancer Res. 2006 Jun 1;12(11 Pt 1):3416-24), vvDD, double viral gene deletion (tk- and vgf-) vaccinia virus described in McCart et al., Cancer Res 2001;61:8751-7, and ACAM200 (Osborne et al., Vaccine. 2007 Dec 17;25(52):8807-32), the contents of which are incorporated herein by reference in their entirety.

[0064] 5.3. Pharmaceutical Compositions Provided herein are pharmaceutical compositions containing an oncolytic virus (e.g., an oncolytic virus described in Section 5.2) comprising a nucleic acid molecule encoding an IRF modulator. In certain embodiments, the pharmaceutical composition comprises an effective amount of an oncolytic virus of the invention.

[0065] In certain embodiments, the pharmaceutical composition comprises about 10 3 Plaque-forming units (PFU) to approximately 10 13 In certain embodiments, the pharmaceutical composition comprises an oncolytic virus in an amount of up to about 10 PFU. 5Approximately 10 minutes from PFU 13 PFU, approx. 10 5 Approximately 10 minutes from PFU 12 PFU, approx. 10 5 Approximately 10 minutes from PFU 11 PFU, approx. 10 5 Approximately 10 minutes from PFU 10 PFU, approx. 10 5 Approximately 10 minutes from PFU 9 PFU, approx. 10 5 Approximately 10 minutes from PFU 8 PFU, approx. 10 5 Approximately 10 minutes from PFU 7 PFU, approx. 10 5 Approximately 10 minutes from PFU 6 PFU, approx. 10 6 Approximately 10 minutes from PFU 13 PFU, approx. 10 6 Approximately 10 minutes from PFU 12 PFU, approx. 10 6 Approximately 10 minutes from PFU 11 PFU, approx. 10 6 Approximately 10 minutes from PFU 10 PFU, approx. 10 6 Approximately 10 minutes from PFU 9 PFU, approx. 10 6 Approximately 10 minutes from PFU 8 PFU, approx. 10 6 Approximately 10 minutes from PFU 7 PFU, approx. 10 7 Approximately 10 minutes from PFU 13 PFU, approx. 10 7 Approximately 10 minutes from PFU 12 PFU, approx. 10 7 Approximately 10 minutes from PFU 11 PFU, approx. 10 7 Approximately 10 minutes from PFU 10 PFU, approx. 10 7 Approximately 10 minutes from PFU 9 PFU, approx. 10 7 Approximately 10 minutes from PFU 8 PFU, approx. 10 8 Approximately 10 minutes from PFU 13 PFU, approx. 10 8 Approximately 10 minutes from PFU 12 PFU, approx. 10 8 Approximately 10 minutes from PFU 11 PFU, approx. 10 8 Approximately 10 minutes from PFU10 PFU, approx. 10 8 Approximately 10 minutes from PFU 9 PFU, or approximately 10 9 Approximately 10 minutes from PFU 10 In certain embodiments, the pharmaceutical composition comprises at least about 1 x 10 PFU of an oncolytic virus. 5 PFU, at least approximately 5 × 10 5 PFU, at least approximately 1 × 10 6 PFU, at least approximately 5 × 10 6 PFU, at least approximately 1 × 10 7 PFU, at least approximately 5 × 10 7 PFU, at least approximately 1 × 10 8 PFU, at least approximately 5 × 10 8 PFU, at least approximately 1 × 10 9 PFU, at least approximately 5 × 10 9 PFU, at least approximately 1 × 10 10 PFU, at least approximately 5 × 10 10 PFU, at least approximately 1 × 10 11 PFU, at least approximately 5 × 10 11 PFU, at least approximately 1 × 10 12 PFU, at least approximately 5 × 10 12 PFU, or at least about 1 × 10 13 In certain embodiments, the pharmaceutical composition comprises about 1 x 10 PFU of an oncolytic virus. 5 PFU, approx. 5×10 5 PFU, approx. 1×10 6 PFU, approx. 5×10 6 PFU, approx. 1×10 7 PFU, approx. 5×10 7 PFU, approximately 1×10 8 PFU, approx. 5×10 8 PFU, approx. 1×10 9 PFU, approx. 5×10 9 PFU, approx. 1×10 10 PFU, approx. 5×10 10 PFU, approx. 1×10 11 PFU, approx. 5×10 11 PFU, approx. 1×10 12 PFU, approx. 5×10 12 PFU, or approximately 1 x 1013 In certain embodiments, the pharmaceutical composition comprises about 1 x 10 PFU of an oncolytic virus. 6 Approximately 3 × 10 PFU 9 In certain embodiments, the pharmaceutical composition comprises an oncolytic virus in an amount of up to about 10 PFU. 8 Approximately 10 minutes from PFU 9 PFU, approx. 10 9 Approximately 10 minutes from PFU 10 PFU, or approximately 10 6 Approximately 10 minutes from PFU 7 In certain embodiments, the pharmaceutical composition comprises up to about 2.5 x 10 PFU of an oncolytic virus. 6 PFU, approx. 1×10 7 PFU, approx. 5×10 8 PFU, approx. 6×10 8 PFU, approx. 2×10 9 , about 2.5×10 9 , or approximately 3 × 10 9 PFU of oncolytic virus.

[0066] In certain embodiments, the pharmaceutical composition can be prepared as a solution, a dispersion (in glycerol, in liquid polyethylene glycol), and any combination thereof in oil, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a nanoparticle-containing dosage form, a microparticle-containing dosage form, a polymeric dosage form, or any combination thereof.

[0067] In certain embodiments, the pharmaceutical compositions described herein further comprise a pharmaceutically acceptable carrier, e.g., a pharmaceutical excipient. In certain embodiments, a pharmaceutically acceptable carrier includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to whom it is administered. Non-limiting examples of suitable drug carriers include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, indwelling elements containing oncolytic viruses, and any other suitable solvents, delivery or formulation means or materials.

[0068] In certain embodiments, the pharmaceutically acceptable carrier can be a buffer. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. Buffers include sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, other calcium salts, and combinations thereof.

[0069] In certain embodiments, the oncolytic viruses described herein can be propagated in suitable host cells, separated from the host cells, and stored under conditions that enhance the stability and integrity of the virus so as to minimize loss of infectivity over time. In certain embodiments, the oncolytic viruses described herein can be stored by freezing or drying, for example, lyophilization. In certain embodiments, prior to administration, the stored oncolytic viruses can be reconstituted (if dried for storage) and diluted with a pharmaceutically acceptable carrier for administration.

[0070] In certain embodiments, the pharmaceutical compositions described herein can further comprise an immunomodulatory agent (eg, an immunomodulatory agent described in Section 5.4).

[0071] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus (e.g., an oncolytic virus described in Section 5.2) comprising a nucleic acid molecule encoding a modulator of an IRF and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus (e.g., an oncolytic virus described in Section 5.2) comprising a nucleic acid molecule encoding a modulator of an IRF, and an excipient and / or buffer.

[0072] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), and a pharmaceutical excipient and / or buffer.

[0073] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2 and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2, and a pharmaceutical excipient and / or buffer.

[0074] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding a modulator of an IRF (e.g., an oncolytic virus described in Section 5.2) and an immunomodulatory agent (e.g., an immunomodulatory agent described in Section 5.4). In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), and an immune checkpoint inhibitor. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2, and an immune checkpoint inhibitor.

[0075] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2 and an immune checkpoint inhibitor selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2 and an anti-PD-L1 antibody. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2 and an anti-CTLA-4 antibody.

[0076] In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding a modulator of IRF (e.g., an oncolytic virus described in Section 5.2), an immunomodulatory agent (e.g., an immunomodulatory agent described in Section 5.4), and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions described herein comprise an oncolytic virus comprising a nucleic acid molecule encoding IRF2, an anti-PD-L1 antibody or an anti-CTLA-4 antibody, and a pharmaceutically acceptable carrier.

[0077] 5.4 Treatment method Provided herein are methods for treating a subject with cancer. In certain embodiments, the methods comprise administering to the subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator (e.g., an oncolytic virus described in Section 5.2), or a pharmaceutical composition comprising the oncolytic virus (e.g., a pharmaceutical composition described in Section 5.3). In certain embodiments, the methods comprise administering to the subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), or a pharmaceutical composition comprising the oncolytic virus. In certain embodiments, the methods comprise administering to the subject an oncolytic virus comprising a nucleic acid molecule encoding IRF2, or a composition comprising the oncolytic virus.

[0078] In certain embodiments, the methods described herein reduce aggregated cancer cell mass, reduce cancer cell proliferation rate, reduce cancer cell proliferation, reduce tumor mass, reduce tumor volume, reduce tumor weight, reduce tumor cell proliferation, reduce tumor growth rate, and / or reduce tumor metastasis in a subject.

[0079] The methods described herein can be used to treat any suitable cancer. Non-limiting examples of cancers that can be treated with the methods described herein include adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof.

[0080] In certain embodiments, the methods described herein can be used to treat solid tumors. In certain embodiments, the methods described herein can be used to treat melanoma. In certain embodiments, the methods described herein can be used to treat renal cancer.

[0081] In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a non-human subject, such as, but not limited to, a non-primate, dog, cat, horse, rabbit, mouse, rat, guinea pig, poultry, cow, goat, or sheep.

[0082] In certain embodiments, the methods described herein comprise a method for producing a medicament comprising administering a medicament comprising administering a medicament comprising administering a medicament to a subject in need thereof, the method ... 3 from about 10 13 In certain embodiments, the methods described herein include administering to a subject an oncolytic virus in an amount of up to about 10 PFU. 5 from about 10 13 In certain embodiments, the methods described herein include administering to a subject an oncolytic virus in an amount of up to about 10 PFU. 5 Approximately 10 minutes from PFU 13 PFU, approx. 10 5 Approximately 10 minutes from PFU 12 PFU, approx. 10 5 Approximately 10 minutes from PFU 11 PFU, approx. 10 5 Approximately 10 minutes from PFU 10 PFU, approx. 10 5 Approximately 10 minutes from PFU 9 PFU, approx. 10 5Approximately 10 minutes from PFU 8 PFU, approx. 10 5 Approximately 10 minutes from PFU 7 PFU, approx. 10 5 Approximately 10 minutes from PFU 6 PFU, approx. 10 6 Approximately 10 minutes from PFU 13 PFU, approx. 10 6 Approximately 10 minutes from PFU 12 PFU, approx. 10 6 Approximately 10 minutes from PFU 11 PFU, approx. 10 6 Approximately 10 minutes from PFU 10 PFU, approx. 10 6 Approximately 10 minutes from PFU 9 PFU, approx. 10 6 Approximately 10 minutes from PFU 8 PFU, approx. 10 6 Approximately 10 minutes from PFU 7 PFU, approx. 10 7 Approximately 10 minutes from PFU 13 PFU, approx. 10 7 Approximately 10 minutes from PFU 12 PFU, approx. 10 7 Approximately 10 minutes from PFU 11 PFU, approx. 10 7 Approximately 10 minutes from PFU 10 PFU, approx. 10 7 Approximately 10 minutes from PFU 9 PFU, approx. 10 7 Approximately 10 minutes from PFU 8 PFU, approx. 10 8 Approximately 10 minutes from PFU 13 PFU, approx. 10 8 Approximately 10 minutes from PFU 12 PFU, approx. 10 8 Approximately 10 minutes from PFU 11 PFU, approx. 10 8 Approximately 10 minutes from PFU 10 PFU, approx. 10 8 Approximately 10 minutes from PFU 9 In certain embodiments, the methods described herein comprise administering to a subject an oncolytic virus in an amount of at least about 1 x 10 PFU. 5 PFU, at least approximately 5 × 10 5 PFU, at least approximately 1 × 10 6 PFU, at least approximately 5 × 10 6PFU, at least approximately 1 × 10 7 PFU, at least approximately 5 × 10 7 PFU, at least approximately 1 × 10 8 PFU, at least approximately 5 × 10 8 PFU, at least approximately 1 × 10 9 PFU, at least approximately 5 × 10 9 PFU, at least approximately 1 × 10 10 PFU, at least approximately 5 × 10 10 PFU, at least approximately 1 × 10 11 PFU, at least approximately 5 × 10 11 PFU, at least approximately 1 × 10 12 PFU, at least approximately 5 × 10 12 PFU, or at least about 1 × 10 13 In certain embodiments, the methods described herein involve administering to a subject an oncolytic virus in an amount of about 1 x 10 PFU. 5 PFU, approx. 5×10 5 PFU, approx. 1×10 6 PFU, approx. 5×10 6 PFU, approx. 1×10 7 PFU, approx. 5×10 7 PFU, approx. 1×10 8 PFU, approx. 5×10 8 PFU, approx. 1×10 9 PFU, approx. 5×10 9 PFU, approx. 1×10 10 PFU, approx. 5×10 10 PFU, approx. 1×10 11 PFU, approx. 5×10 11 PFU, approx. 1×10 12 PFU, approx. 5×10 12 PFU, or approximately 1 x 10 13 In certain embodiments, the methods described herein involve administering to a subject an oncolytic virus in an amount of about 1 x 10 PFU. 6 Approximately 3 × 10 PFU 9 PFU, approx. 10 8 Approximately 10 minutes from PFU 9 PFU, approx. 10 9 Approximately 10 minutes from PFU 10 PFU, or approximately 10 6 Approximately 10 minutes from PFU 7In certain embodiments, the methods described herein involve administering to a subject an oncolytic virus in an amount up to about 2.5 x 10 PFU. 6 PFU, approx. 1×10 7 PFU, approx. 5×10 8 PFU, approx. 6×10 8 PFU, approx. 2×10 9 PFU, approximately 2.5×10 9 PFU, or approximately 3 x 10 9 The method includes administering to a subject an amount of an oncolytic virus in an amount of PFU.

[0083] In certain embodiments, the methods described herein include administering an oncolytic virus to a subject in a single dose or multiple doses. In certain embodiments, when an oncolytic virus is administered to a subject in multiple doses, the doses can be administered continuously, for example, daily, weekly, or at monthly intervals, or according to the individual needs of the subject.

[0084] Any suitable administration method can be used in accordance with the presently disclosed subject matter to administer an oncolytic virus to a subject. In certain embodiments, the oncolytic viruses described herein are administered systemically. In certain embodiments, the oncolytic viruses described herein can be administered directly to the tumor site, for example, by direct intratumoral injection.

[0085] For example, but not limited to, the route of administration can be inhalation, intranasal, intravenous, intraarterial, intrathecal, intratumoral, intraperitoneal, intramuscular, subcutaneous, topical, intradermal, local site, oral administration, or a combination thereof. In certain embodiments, the oncolytic viruses described herein are administered to the subject from a source implanted in the subject. In certain embodiments, the oncolytic viruses described herein are administered to the subject by continuous infusion over a selected period of time.

[0086] The present specification further provides methods for improving a subject's responsiveness to an immunomodulatory drug. In certain embodiments, the method comprises administering to the subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator (e.g., an oncolytic virus described in Section 5.2) or a pharmaceutical composition comprising the oncolytic virus (e.g., a pharmaceutical composition described in Section 5.3). In certain embodiments, the subject has previously been treated with an immunomodulatory drug. In certain embodiments, the subject has developed resistance to the immunomodulatory drug. In certain embodiments, the method further comprises administering to the subject an immunomodulatory drug in combination with an oncolytic virus described herein.

[0087] Also provided herein are methods of treating a subject with cancer, comprising administering to the subject an oncolytic virus (e.g., an oncolytic virus described in Section 5.2) comprising a nucleic acid molecule encoding an IRF modulator, in combination with an immunomodulatory agent.

[0088] Any suitable immunomodulatory agent that targets components of the immune system to combat cancer can be used with the methods of the present invention. Non-limiting examples of immunomodulatory agents include immune checkpoint inhibitors, T cells, dendritic cells, therapeutic antibodies (e.g., anti-CD33 antibodies, anti-CD11b antibodies), cancer vaccines, cytokines (e.g., IL-12, GM-CSF, IL-2, IFNβ, IFNγ, MIP-1, MCP-1, IL-8), Bacillus Calmette-Guerin (BCG), and any combination thereof. In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. Non-limiting examples of anti-PD1 antibodies include pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), and combinations thereof. Non-limiting examples of anti-PD-L1 antibodies include atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), and combinations thereof. Non-limiting examples of anti-CTLA-4 antibodies include ipilimumab (Yervoy). In certain embodiments, the immunomodulatory agent is an anti-PD-L1 antibody. In certain embodiments, the immunomodulatory agent is an anti-CTLA-4 antibody.

[0089] In certain embodiments, a method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), in combination with an immunomodulatory agent. In certain embodiments, a method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), in combination with an immune checkpoint inhibitor. In certain embodiments, a method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), in combination with an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0090] In certain embodiments, the method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding IRF2 in combination with an immunomodulatory agent. In certain embodiments, the method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding IRF2 in combination with an immune checkpoint inhibitor. In certain embodiments, the method comprises administering to a subject an oncolytic virus comprising a nucleic acid molecule encoding IRF2 in combination with an anti-PD-L1 antibody or an anti-CTLA-4 antibody. In certain embodiments, the oncolytic virus and the immunomodulatory agent can be administered to a subject as part of a treatment regimen. In certain embodiments, the oncolytic virus and the immunomodulatory agent can be administered concomitantly to a subject. In certain embodiments, the oncolytic virus and the immunomodulatory agent can be administered simultaneously. In certain embodiments, the oncolytic virus and immunomodulatory agent can be administered sequentially in any order (e.g., the oncolytic virus is administered to the subject before the immunomodulatory agent is administered; or the oncolytic virus is administered to the subject after the immunomodulatory agent is administered), but can also be administered at different times (e.g., the oncolytic virus and immunomodulatory agent are administered to the subject on the same day but at different times; the oncolytic virus and immunomodulatory agent are administered to the subject on different days within the same week).

[0091] 5.5 Kit The present invention further provides kits comprising an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator (e.g., an oncolytic virus described in Section 5.2) or a pharmaceutical composition comprising said oncolytic virus (e.g., a pharmaceutical composition described in Section 5.3). In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), or a pharmaceutical composition comprising said oncolytic virus. In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding IRF2, or a composition comprising said oncolytic virus.

[0092] In certain embodiments, the kits described herein can further include instructions. In certain embodiments, the instructions include a description of the oncolytic virus and, if necessary, a description of other components included in the kit. In certain embodiments, the kit includes instructions for treating a subject with cancer or for improving a subject's responsiveness to an immunomodulatory drug. In certain embodiments, the instructions further include instructions for administration, including methods for determining the appropriate subject condition, appropriate dosage, and / or appropriate administration method for administering the modified virus. In certain embodiments, the instructions further include guidance for monitoring the subject throughout the treatment period.

[0093] In certain embodiments, the kits described herein include a device for administering an oncolytic virus or pharmaceutical composition to a subject. Any suitable device known in the art for administering drugs and pharmaceutical compositions can be included in the kits described herein. For example, but not limited to, suitable devices include hypodermic needles, intravenous needles, catheters, needleless syringes, inhalers, and liquid dispensers such as eyedroppers. In certain embodiments, an oncolytic virus to be delivered systemically, for example, by intravenous injection, can be included in the kit along with a hypodermic syringe with a needle.

[0094] In certain embodiments, the kits described herein may further comprise an immunomodulatory agent (e.g., an immunomodulatory agent described in Section 5.4). In certain embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof. In certain embodiments, the immunomodulatory agent is an anti-PD-L1 antibody. In certain embodiments, the immunomodulatory agent is an anti-CTLA-4 antibody.

[0095] In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator (e.g., an oncolytic virus described in Section 5.2) and an immune checkpoint inhibitor. In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding an IRF modulator that inhibits the activity of an IRF (e.g., IRF1), and an immune checkpoint inhibitor. In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding IRF2, and an immune checkpoint inhibitor. In certain embodiments, the kit comprises an oncolytic virus comprising a nucleic acid molecule encoding IRF2, and an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[0096] 6. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are offered by way of illustration, not limitation, of the presently disclosed subject matter. Example 1

[0097] Targeting IRF: Targeted expression of IRF2 inhibited tumor growth One factor that can influence a subject's responsiveness to immunotherapy is the interferon (IFN) response in the tumor microenvironment. IFNs can play opposing roles in tumor cells compared to immune cells. The present disclosure aims to exploit this opposing IFN response by modulating molecules (e.g., IRFs) that control the IFN response to improve the efficacy and responsiveness of immunotherapy. We generated a large number of CRISPR / Cas9-based gene-edited syngeneic tumor cells and demonstrated that the tumor-specific functions of specific IRFs, such as IRF1, IRF3, and IRF7, may underlie the opposing IFN responses in tumor cells and host non-tumor immune cells. We also found that targeting IRFs in the tumor microenvironment using oncolytic viruses is therapeutically useful. We further developed an IRF2-based transcriptional regulator that modulates IRF function in the tumor microenvironment using a modified oncolytic virus. We found that an oncolytic vaccinia virus expressing IRF2 successfully reduced tumor burden in a preclinical mouse model.

[0098] IRF2 has been found to be deficient in primary human cancers, including lung, colon, breast, and prostate cancers. The present disclosure demonstrates that IRF2 can inhibit IRF1-mediated gene induction (e.g., PD-L1) and promote anti-tumor immune responses.

[0099] In vitro overexpression of IRF2 in cancer cells To assess whether IRF2 can regulate IRF1 activity and IRF1-mediated gene expression, we overexpressed IRF2 in human melanoma cells (MEL-285) and mouse melanoma cells (B16). Viral vectors carrying the human IRF2 gene or the mouse Irf2 gene were constructed. MEL-285 and B16 tumor cells were transfected with the IRF2- or Irf2-carrying vector, respectively. The transfected cells were then stimulated with IFNγ. PD-L1 protein expression in MEL-285 and B16 tumor cells was assessed by flow cytometry. Overexpression of IRF2 in human MEL-285 and mouse B16 melanoma cells reduced PD-L1 expression in both cell lines (Figures 1A-1B).

[0100] In vivo administration of mIrf2-expressing oncolytic vaccinia virus in a preclinical mouse model An oncolytic vaccinia virus expressing IRF-2 was generated by inserting the mouse Irf2 gene into the TK locus of the oncolytic vaccinia virus genome. This insertion disrupted the TK gene. In vivo studies were performed to examine the antitumor activity of the mIrf2-expressing oncolytic virus in two mouse tumor models. Mice were implanted with B16 tumor cells (melanoma tumor cells) on day 0. Ten days after implantation, tumor-bearing mice were treated with PBS, 2.5 × 10 6 PFU thymidine kinase-deficient (TK-) vaccinia virus (VV control), or 2.5 × 10 6 PFU mIrf2-expressing oncolytic vaccinia virus (VV-mIrf2) was injected intratumorally. Tumor volume was monitored and measured for 22 days. Intratumoral injection of mIrf2-expressing oncolytic vaccinia virus significantly inhibited B16 tumor growth compared with PBS and VV controls (Figure 1C).

[0101] Furthermore, the antitumor activity of the mIrf2-expressing oncolytic virus was evaluated in a preclinical RENCA tumor (renal carcinoma) mouse model. RENCA tumors were engrafted into BALB / C mice by subcutaneous injection. Tumor-bearing mice received PBS, 1 × 10 7 PFU thymidine kinase-deficient (TK-) vaccinia virus (VV control), or 1 × 10 7 PFU mIrf2-expressing oncolytic vaccinia virus (VV-mIrf2) was injected intratumorally. Tumor growth was monitored and measured. Intratumoral injection of VV-mIrf2 significantly inhibited RENCA tumor growth compared with the PBS control (Figure 1D). Furthermore, the antitumor effect of the VV control was significantly improved by the expression of mIrf-2.

[0102] The PD-L1 / PD-1 system is an essential immune checkpoint that cancer cells can exploit to evade immune detection and elimination. Efforts have been made to block immune checkpoint proteins, including PD-L1 and PD-1, to overcome cancer's ability to evade immune responses and promote host immune responses in defense against cancer. This disclosure demonstrates that IRF2 can effectively downregulate PD-L1 protein expression in cancer cells, thus suppressing the activation of the PD-L1 / PD-1 pathway. The present invention further suggests that overexpression of IRF2 in cancer cells may improve host immune responses in attacking cancer cells and may enhance the responsiveness of cancer cells to immunotherapy, such as immune checkpoint inhibitors (e.g., anti-PD-L1 antibodies).

[0103] Oncolytic viruses can selectively infect and lyse tumor cells, inducing antitumor immune responses. The present invention demonstrated that the antitumor activity of oncolytic viruses was significantly enhanced by incorporating the immunomodulatory gene IRF2 into the genome of oncolytic viruses. These results demonstrate that IRF proteins have diverse functions in the tumor microenvironment.

[0104] While the subject matter of the present disclosure and some of its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present invention. Furthermore, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufactures, compositions, and methods described herein. As those skilled in the art can readily appreciate from the description of the subject matter of the present disclosure, any currently existing or later-developed process, machine, manufacture, composition, or method that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized in accordance with the subject matter of the present disclosure. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufactures, compositions, or methods.

[0105] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the descriptions of which are incorporated herein by reference in their entireties for all purposes. The present disclosure includes the following embodiments. [1] An oncolytic virus containing a nucleic acid molecule encoding a modulator of an interferon regulatory factor (IRF). [2] The oncolytic virus of embodiment 1, wherein the IRF is IRF1, IRF3, IRF7, or a combination thereof. [3] An oncolytic virus according to embodiment 1 or 2, wherein the IRF is IRF1. [4] An oncolytic virus according to any one of embodiments 1 to 3, wherein the modulator inhibits the activity of IRF. [5] An oncolytic virus according to any one of embodiments 1 to 4, wherein the modulator inhibits the activity of IRF1. [6] An oncolytic virus described in any one of embodiments 1 to 5, wherein the modulator reduces IRF-mediated gene expression. [7] An oncolytic virus described in any one of embodiments 1 to 6, wherein the modulator reduces expression of the CD274 gene. [8] An oncolytic virus described in any one of embodiments 1 to 7, wherein the modulator is IRF2. [9] The oncolytic virus of embodiment 8, wherein the IRF2 is human IRF2 or mouse IRF2.

[10] An oncolytic virus described in any one of embodiments 1 to 9, wherein the nucleic acid molecule is an exogenous nucleic acid molecule.

[11] An oncolytic virus described in any one of embodiments 1 to 10, wherein the nucleic acid molecule is integrated into the genome of the oncolytic virus.

[12] An oncolytic virus described in any one of embodiments 1 to 11, wherein the oncolytic virus is an oncolytic vaccinia virus.

[13] The oncolytic virus of embodiment 12, wherein the oncolytic vaccinia virus lacks expression of a functional thymidine kinase (TK).

[14] A method for treating a subject having cancer, comprising administering to the subject an oncolytic virus described in any one of embodiments 1 to 13.

[15] The method of embodiment 14, wherein the subject is a human subject.

[16] The method of embodiment 14 or 15, further comprising administering to the subject an immunomodulatory agent.

[17] The method of embodiment 16, wherein the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof.

[18] The method of embodiment 17, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

[19] The method of embodiment 17 or 18, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

[20] The method of any one of embodiments 17 to 19, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[21] The method of any one of embodiments 14 to 20, wherein the cancer is a solid tumor.

[22] The method of any one of embodiments 14 to 21, wherein the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof.

[23] The method of any one of embodiments 14 to 22, wherein the cancer is melanoma or renal cancer.

[24] A method for improving a subject's responsiveness to an immunomodulatory drug, the method comprising administering to the subject an oncolytic virus described in any one of embodiments 1 to 13, wherein the subject has cancer.

[25] The method of embodiment 24, wherein the subject is a human subject.

[26] The method of embodiment 24 or 25, wherein the subject has previously been treated with an immunomodulatory agent.

[27] The method of any one of embodiments 24-26, wherein the subject has developed resistance to the immunomodulatory drug.

[28] The method of any one of embodiments 24 to 27, further comprising administering to the subject an immunomodulatory agent.

[29] The method of any one of embodiments 24-28, wherein the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof.

[30] The method of embodiment 29, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

[31] The method of embodiment 29 or 30, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

[32] The method of any one of embodiments 29-31, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[33] The method of any one of embodiments 24 to 32, wherein the cancer is a solid tumor.

[34] The method of any one of embodiments 24 to 33, wherein the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof.

[35] The method of any one of embodiments 24 to 34, wherein the cancer is melanoma or renal cancer.

[36] A pharmaceutical composition comprising an oncolytic virus described in any one of embodiments 1 to 13.

[37] The pharmaceutical composition of embodiment 36, further comprising an immunomodulatory agent.

[38] The pharmaceutical composition of embodiment 37, wherein the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof.

[39] The pharmaceutical composition of embodiment 38, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

[40] The pharmaceutical composition of embodiment 38 or 39, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

[41] The pharmaceutical composition of any one of embodiments 38 to 40, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[42] A pharmaceutical composition described in any one of embodiments 36 to 41, further comprising a pharmaceutically acceptable carrier.

[43] A pharmaceutical composition described in any one of embodiments 36 to 42, for treating a subject with cancer or improving the subject's responsiveness to an immunomodulatory drug.

[44] A kit comprising an oncolytic virus described in any one of embodiments 1 to 13, or a pharmaceutical composition described in any one of embodiments 36 to 43.

[45] The kit of embodiment 44, further comprising an immunomodulatory agent.

[46] The kit of embodiment 45, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

[47] The kit of embodiment 46, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

[48] ​​The kit of embodiment 46 or 47, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

[49] The kit of any of embodiments 44 to 48, wherein the kit further comprises instructions for treating a subject with cancer or improving a subject's responsiveness to an immunomodulatory agent.

Claims

1. An oncolytic virus that contains a nucleic acid molecule encoding interferon regulatory factor 2 (IRF2) and lacks expression of functional thymidine kinase (TK).

2. The oncolytic virus of claim 1, wherein IRF2 inhibits the activity of IRF.

3. The oncolytic virus of claim 1 or 2, wherein IRF2 inhibits the activity of IRF1.

4. The oncolytic virus of any one of claims 1 to 3, wherein IRF2 reduces IRF-mediated gene expression.

5. The oncolytic virus according to any one of claims 1 to 4, wherein IRF2 reduces the expression of the CD274 gene.

6. The oncolytic virus according to any one of claims 1 to 5, wherein IRF2 is human IRF2 or mouse IRF2.

7. The oncolytic virus according to any one of claims 1 to 6, wherein the nucleic acid molecule is a foreign nucleic acid molecule.

8. The oncolytic virus of any one of claims 1 to 7, wherein the nucleic acid molecule is integrated into the genome of the oncolytic virus.

9. The oncolytic virus according to any one of claims 1 to 8, wherein the oncolytic virus is an oncolytic vaccinia virus.

10. A composition for treating a subject with cancer, comprising the oncolytic virus of any one of claims 1 to 9.

11. The composition of claim 10 , wherein the subject is a human subject.

12. 12. The composition of claim 10 or 11, further comprising an immunomodulatory agent.

13. 13. The composition of claim 12, wherein the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof.

14. 14. The composition of claim 13, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

15. 15. The composition of claim 13 or 14, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

16. The composition of any one of claims 13 to 15, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

17. The composition of any one of claims 10 to 16, wherein the cancer is a solid tumor.

18. 18. The composition of any one of claims 10 to 17, wherein the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof.

19. The composition of any one of claims 10 to 18, wherein the cancer is melanoma or renal cancer.

20. 10. A composition for improving a subject's responsiveness to an immunomodulatory drug, the composition comprising an oncolytic virus of any one of claims 1 to 9, wherein the subject has cancer.

21. 21. The composition of claim 20, wherein the subject is a human subject.

22. 22. The composition of claim 20 or 21, wherein the subject has previously been treated with an immunomodulatory drug.

23. The composition of any one of claims 20 to 22, wherein the subject has developed resistance to an immunomodulatory drug.

24. 24. The composition of any one of claims 20 to 23, further comprising an immunomodulatory agent.

25. 25. The composition of any one of claims 20 to 24, wherein the immunomodulatory agent is selected from the group consisting of an immune checkpoint inhibitor, a T cell, a dendritic cell, a therapeutic antibody, a cancer vaccine, a cytokine, Bacillus Calmette-Guerin (BCG), and any combination thereof.

26. 26. The composition of claim 25, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

27. 27. The composition of claim 25 or 26, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

28. The composition of any one of claims 25 to 27, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

29. The composition of any one of claims 20 to 28, wherein the cancer is a solid tumor.

30. 30. The composition of any one of claims 20 to 29, wherein the cancer is selected from the group consisting of adenocarcinoma, osteosarcoma, cervical cancer, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, leukemia, lymphoma, renal cancer, pancreatic cancer, gastric cancer, colon cancer, duodenal cancer, glioblastoma multiforme, astrocytoma, sarcoma, and combinations thereof.

31. The composition of any one of claims 20 to 30, wherein the cancer is melanoma or renal cancer.

32. The composition of any one of claims 10 to 31, further comprising a pharmaceutically acceptable carrier.

33. A kit comprising an oncolytic virus according to any one of claims 1 to 9 or a composition according to any one of claims 10 to 32.

34. 34. The kit of claim 33, further comprising an immunomodulatory agent.

35. 35. The kit of claim 34, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

36. 36. The kit of claim 35, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, and any combination thereof.

37. The kit of claim 35 or 36, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-CTLA-4 antibody.

38. 38. The kit of any one of claims 33 to 37, wherein the kit further comprises instructions for treating a subject with cancer or for improving a subject's responsiveness to an immunomodulatory agent.

39. 39. The kit of any one of claims 33 to 38 for treating a subject with cancer or for improving a subject's responsiveness to an immunomodulatory drug.

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