Oncolytic viruses for modified MHC expression

Abstract

The present disclosure provides recombinant oncolytic viruses with gene deletions or insertions that result in the downregulation of major histocompatibility complex class I and alternatively or additionally the upregulation of major histocompatibility complex class II. Also provided are immunological and pharmaceutical compositions that include these recombinant viruses, and methods of using these compositions.Furthermore, provided herein is a composition that includes an oncolytic virus, wherein the oncolytic virus includes genomic modification, and the genomic modification includes a deletion or functional deletion of the vaccinia virus gene that codes for A35 protein, and an insertion of an exogenous gene that codes for cowpox protein CPXV012 or cowpox protein CPXV203.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 182,243, filed April 30, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Cancer is the second leading cause of death in the United States. Available therapies face challenges in selective targeting of cancer cells, localized gene expression, and reaching and modifying the tumor microenvironment (TME). Therefore, there remains a need for improved compositions and methods of use to address these challenges. Summary of the Invention

[0003] A brief summary Provided herein is a composition comprising an oncolytic virus, wherein the oncolytic virus comprises a genomic modification, and wherein the genomic modification comprises an exogenous nucleic acid encoding an MHC I inhibitor. Provided herein further is a composition wherein the genomic modification further comprises a deletion or functional deletion of an endogenous nucleic acid encoding an MHC II inhibitor; or an exogenous nucleic acid that results in activation or enhanced activation of MHC II presentation. Provided herein further is a composition wherein the genomic modification further comprises a deletion or functional deletion of an endogenous nucleic acid encoding an MHC II inhibitor; and an exogenous nucleic acid that results in activation or enhanced activation of MHC II presentation. Provided herein further is a composition wherein the deletion or functional deletion of an endogenous nucleic acid encoding an MHC II inhibitor comprises a deletion or functional deletion of a vaccinia virus gene encoding protein A35. Provided herein further is a composition wherein the deletion or functional deletion of a vaccinia virus gene encoding protein A35 is a deletion or functional deletion of gene WR158. Further provided herein is a composition in which the exogenous nucleic acid that causes activation or enhanced activation of MHC II presentation encodes a protein selected from apoptosis inhibitor protein; necrotic cell death activator protein; autophagy enhancer protein; asparaginyl endopeptidase; class II transactivator; interferon-gamma; Toll-like receptor activator; or dendritic cell maturation activator. Further provided herein is a composition in which the exogenous nucleic acid encodes an autophagy enhancer protein, and the autophagy enhancer protein is HMGB1 or a functional domain or variant thereof. Further provided herein is a composition in which the exogenous nucleic acid encodes a dendritic cell maturation activator, and the dendritic cell maturation activator comprises osteopontin, TNF-alpha, or a functional fragment or variant thereof. Further provided herein is a composition in which the protein encoded by the exogenous nucleic acid is fused to a secretory sequence, a cell-permeable domain, or a combination thereof.Further provided herein is a composition in which the oncolytic virus is a poxvirus, an adeno-associated virus, an adenovirus, a Newcastle disease virus (NDV), a reovirus (RV), a mengovirus, a myxoma virus (MYXV), a measles virus (MV), a herpes simplex virus (HSV), a vaccinia virus (VV), a vesicular stomatitis virus (VSV), and a poliovirus (PV). Further provided herein is a composition in which the poxvirus comprises a beta entomopoxvirus, a yatapoxvirus, a cervidopoxvirus, a gamma entomopoxvirus, a leporipoxvirus, a suipoxvirus, a molluscumpoxvirus, a crocodylidopoxvirus, an alpha entomopoxvirus, a capripoxvirus, an avipoxvirus, or a parapoxvirus. Further provided herein is a composition in which the poxvirus is a vaccinia virus. Further provided herein is a composition in which the MHC I inhibitor causes inhibition or partial inhibition of MHC I presentation. Further provided herein is a composition in which the exogenous nucleic acid encoding an MHC I inhibitor comprises a gene encoding one or more cowpox proteins. Further provided herein is a composition in which the exogenous nucleic acid encoding an MHC I inhibitor comprises a gene encoding cowpox protein CPXV012 or a functional fragment or variant thereof. Further provided herein is a composition in which the exogenous nucleic acid encoding an MHC I inhibitor comprises a gene encoding cowpox protein CPXV203 or a functional fragment or variant thereof.Further provided herein is a composition in which the exogenous nucleic acid encoding the MHC I inhibitor comprises a gene encoding at least one of the following: Epstein-Barr virus-encoded nuclear antigen 1 protein; Herpes simplex virus-encoded ICP47 protein; Herpes simplex virus-encoded UL49.5 protein; Cytomegalovirus-encoded US6, US2, US3, US11 or gp48 protein; Epstein-Barr virus-encoded BNLF2a protein; Adenovirus-encoded E3-19K protein; Human immunodeficiency virus-encoded Nef protein; Kaposi's sarcoma-associated herpes virus-encoded kK3, vIRF3 or kK5 protein; or a dominant negative form of IRF7 or IRF3. Further provided herein is a composition in which the MHC I inhibitor comprises a TAP inhibitor. Further provided herein is a composition in which the TAP inhibitor acts entirely or mainly within infected cells. Further provided herein is a composition in which the genomic modification reduces the immune response targeting the virus-infected tumor cells and increases the immune response targeting the cells surrounding the virus-infected tumor cells. Further provided herein is a composition further comprising a deletion of a thymidine kinase gene. Further provided herein is a composition further comprising an exogenous nucleic acid encoding a hyaluronidase. Further provided herein is a composition in which the hyaluronidase is PH-20 or HysA. Further provided herein is a composition in which the oncolytic virus is a vaccinia virus, and the vaccinia virus is a Western Reserve strain vaccinia virus (ATCC VR-1354), a Copenhagen strain, an IHD strain, a Wyeth strain (ATCC VR-325), a NYCBOH strain, a Tian Tan strain, a Lister strain, an Ankara strain (ATCC VR-1508 or ATTC VR1566), a USSR strain, or an ACAM2000 strain.

[0004] Further provided herein is a composition comprising an oncolytic virus, wherein the oncolytic virus comprises a genomic modification, wherein the genomic modification comprises a deletion or functional deletion of a vaccinia virus gene encoding the A35 protein, and an insertion of an exogenous gene encoding cowpox protein CPXV012 or cowpox protein CPXV203. Further provided herein is a composition wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, Newcastle disease virus (NDV), reovirus (RV), mengovirus, myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV), and poliovirus (PV). Further provided herein is a composition in which the poxvirus comprises a beta-entomopoxvirus, a yatapoxvirus, a cervidopoxvirus, a gamma-entomopoxvirus, a leporipoxvirus, a suipoxvirus, a molluscumpoxvirus, a crocodylidopoxvirus, an alpha-entomopoxvirus, a capripoxvirus, an avipoxvirus, or a parapoxvirus. Further provided herein is a composition in which the oncolytic virus is a vaccinia virus. Further provided herein is a composition in which an exogenous gene encoding cowpox protein CPXV012 is located at the locus of the gene encoding the A35 protein of the vaccinia virus. Further provided herein is a composition in which an exogenous gene encoding cowpox protein CPXV203 is located at the locus of the gene encoding the A35 protein of the vaccinia virus. Further provided herein are compositions wherein the genomic modification further comprises at least one of an exogenous nucleic acid encoding a chemokine receptor, or a functional domain or variant thereof; or an exogenous nucleic acid encoding a cytokine, or a functional domain or variant thereof.Further provided herein is a composition comprising an exogenous nucleic acid encoding a cytokine or a functional domain or variant thereof, wherein the cytokine comprises at least one of interleukin-2 (IL-2), interleukin-15 / interleukin-15Ra (IL15 / IL15Ra), interleukin-7 (IL-7), or a functional domain or variant thereof. Further provided herein is a composition wherein the genomic modification comprises an insertion of an exogenous nucleic acid encoding a fusion protein comprising a cytokine and a metabolic modulator regulating protein. Further provided herein is a composition comprising an exogenous nucleic acid encoding a chemokine receptor or a functional domain or variant thereof, wherein the chemokine receptor comprises at least one of CXCR4, CCR2, or a functional domain or variant thereof. Further provided herein is a composition wherein the chemokine receptor comprises CXCR4 or a functional domain or variant thereof. Further provided herein is a composition wherein the chemokine receptor comprises CCR2 or a functional domain or variant thereof, wherein the CCR2 comprises wild-type CCR2 or a mutant CCR2. Further provided herein is a composition in which the exogenous nucleic acid encoding a chemokine receptor or its functional domain or variant comprises a codon-optimized sequence. Further provided herein is a composition in which the exogenous nucleic acid encoding a chemokine receptor or its functional domain or variant comprises a sequence that is not codon-optimized. Further provided herein is a composition in which the genomic modification comprises a mutation or a complete or partial deletion of a viral gene comprising at least one of A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, or VH1 of vaccinia virus, or a functional domain or fragment or variant thereof, or any combination thereof. Further provided herein is a composition in which the thymidine kinase gene further comprises a deletion. Further provided herein is a composition in which the exogenous nucleic acid encoding a hyaluronidase further comprises a hyaluronidase PH-20 or HysA.Further provided herein is a composition wherein the oncolytic virus is a vaccinia virus, and the vaccinia virus is a Western Reserve strain vaccinia virus (ATCC VR-1354), a Copenhagen strain, an IHD strain, a Wyeth strain (ATCC VR-325), a NYCBOH strain, a Tian Tan strain, a Lister strain, an Ankara strain (ATCC VR-1508 or ATTC VR1566), a USSR strain, or an ACAM2000 strain.

[0005] Provided herein is a pharmaceutical composition comprising the composition described herein and a pharma- ceutically acceptable excipient. Further provided herein is a pharmaceutical composition, wherein the excipient comprises one or more of a buffer, a stabilizer, an antioxidant, a binder, a diluent, a dispersant, a rate control agent, a lubricant, a glidant, a disintegrant, a plasticizer, a preservative, or any combination thereof. Further provided herein is a pharmaceutical composition, wherein the excipient comprises dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, myo-inositol, sorbitol, or any combination thereof. Further provided herein is a pharmaceutical composition, wherein the pharmaceutical composition does not comprise a preservative. Further provided herein is a pharmaceutical composition, wherein the pharmaceutical composition further comprises one or more of a preservative, a diluent, and a carrier. Further provided herein is a pharmaceutical composition, wherein the pharmaceutical composition further comprises an additional active ingredient or a salt thereof. Further provided herein is a pharmaceutical composition, wherein the excipient is sterile water. Further provided herein is a pharmaceutical composition, wherein the pharmaceutical composition further comprises an additional active ingredient, wherein the additional active ingredient is an anticancer agent or an additional oncolytic virus.

[0006] Provided herein is a method of reducing the growth of cancer cells, comprising administering to the cancer cells a composition or pharmaceutical composition described herein.

[0007] Provided herein is a method of treating cancer, comprising administering a composition or pharmaceutical composition described herein to a subject with cancer.Further provided herein is a method, wherein the administration comprises intratumoral administration, intraperitoneal administration, oral administration, intravenous administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.Further provided herein is a method, wherein the administration comprises administering an additional therapy, wherein the additional therapy comprises chemotherapy, radiation, oncolytic virus therapy with additional viruses, treatment with immunomodulatory proteins, CAR T cell therapy, anti-cancer drugs, or any combination thereof.Further provided herein is a method, wherein the additional therapy comprises administering an immunomodulatory agent, comprising anti-CD33 antibody and its variable region, anti-CD11b antibody and its variable region, COX2 inhibitor, cytokine, chemokine, anti-CTLA4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PD-L1 antibody or its antigen-binding fragment, or TLR agonist.

[0008] Provided herein is a method of treatment comprising administering the composition or pharmaceutical composition described herein to a subject in need thereof.Further provided herein is a method wherein the administration comprises intratumoral administration.Further provided herein is a method wherein the administration comprises systemic administration.Further provided herein is a method wherein the systemic administration comprises at least one of intraperitoneal administration, oral administration, intravenous administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. Further provided herein is a method in which the subject has cancer, and the cancer is at least one of melanoma, hepatocellular carcinoma, breast cancer, lung cancer, non-small lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cell carcinoma, pancreatic cancer, epithelial cancer, gastric / GE junction adenocarcinoma, cervical cancer, colon cancer, colorectal cancer, duodenal cancer, pancreatic adenocarcinoma, adenoid cyst, sarcoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or any combination thereof. Further provided herein is a method in which the subject has cancer, and the cancer is at least one of melanoma, hepatocellular carcinoma, breast cancer, lung cancer, non-small lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, peritoneal cancer, prostate cancer, cyst, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or any combination thereof. Further provided herein is a method in which the subject has cancer, and the cancer is at least one of melanoma, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell 6 PFU / mL~about 10 10 Methods are provided for administering an oncolytic virus at a dosage of about 3×10 PFU / mL. 9A method is provided herein for administering an oncolytic virus at a dosage of PFU / mL. Further provided herein is a method in which the composition or pharmaceutical composition is administered in three doses, each of the three doses being administered at a dosage and duration independent of any other dose. Further provided herein is a method in which the three doses are administered in a first dose, a second dose, and a third dose, the first dose being lower than the second dose, and the second dose being lower than the third dose. Further provided herein is a method in which the three doses are administered in a first dose, a second dose, and a third dose, the first dose being higher than the second dose, and the second dose being higher than the third dose. Further provided herein are methods wherein the administration period for the three doses is, each independently, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year. Further provided herein are methods in which the composition or pharmaceutical composition independently comprises a liquid dosage form administered in a volume of about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL. Further provided herein is a method in which the composition or pharmaceutical composition is administered in a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof.Further provided herein is a method in which the composition or pharmaceutical composition is administered for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year.Further provided herein is a method in which the composition or pharmaceutical composition is administered once a day, twice a day, once a week, once every two weeks, or once every three weeks. Further provided herein is a method in which the composition or pharmaceutical composition is administered as a bolus injection or slow infusion. Further provided herein is a method in which administration of the composition or pharmaceutical composition results in a first peak viral load about 1 hour to about 3 days and a second peak viral load about 3 days to about 10 days after administration of the first dose. Further provided herein is a method comprising administering an additional therapy, the additional therapy being administered for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 12 weeks. Further provided herein is the additional therapy administered once a day, twice a day, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 12 weeks, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once a year.Further provided herein is the additional therapy administered in a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof. Further provided herein is a method in which the additional therapy is administered orally, intravenously, intratumorally, intraperitoneally, or by radiation.Further provided herein is a method in which the additional therapy comprises chemotherapy, radiation therapy, oncolytic virus therapy with additional viruses, treatment with immunomodulatory proteins, CAR T cell therapy, anticancer drugs, or any combination thereof.Further provided herein is a method, wherein the additional therapy comprises administering an immunomodulatory agent, including an anti-CD33 antibody and its variable region, an anti-CD11b antibody and its variable region, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, or a TLR agonist. Further provided herein is a method, wherein the additional therapy comprises administering an anti-cancer agent, and the anti-cancer agent is a chemotherapeutic agent. Further provided herein is a method, wherein the subject is a human.

[0009] 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 specific examples, while indicating preferred 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.

[0010] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the change in B16 tumor volume, shown in cubic millimeters on the y-axis, following treatment with vehicle formulated buffer (VFB) or recombinant vaccinia virus containing the TK deletion (HCCTKM); the TK and A52R deletion, and replacement of the gene encoding the A35 protein with the cowpox CPXV012 gene (WO0434N); or the TK and A52R deletion without the insertion of exogenous nucleic acid (WO416N).

[0012] [Diagram 2]Figures 2A and 2B show the change in Renca (Figure 2A) or EMT6 (Figure 2B) tumor volume, shown in cubic millimeters on the y-axis, following treatment with vehicle formulated buffer (VFB) or recombinant vaccinia virus containing: (1) a TK gene deletion and insertion of a nucleic acid encoding a cowpox virus V012 protein; (2) a WR158 gene deletion and insertion of a nucleic acid encoding a cowpox virus V012 protein; (3) a TK gene deletion and an insertion of a nucleic acid encoding a dominant negative interferon regulatory factor 7 (dnIRF7); or (4) a TK gene deletion and an insertion of a nucleic acid encoding a viral interferon regulatory factor 3 (vIRF3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Disclosure While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may now be made by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in carrying out the present disclosure.

[0014] The present disclosure provides, in some embodiments, recombinant oncolytic viruses and methods of using said oncolytic viruses to treat cancer. In some embodiments, the oncolytic viruses of the present disclosure include modifications in the viral genome that encode exogenous nucleic acids to enhance oncolytic immunotherapy by remodeling tumor microenvironment and enhancing systemic delivery. The present disclosure further relates to compositions of matter that include such oncolytic viruses, as well as methods and kits for use in cancer treatment.

[0015] Specific Definitions As used herein, the singular forms "a", "an" and "the" can refer to both the singular and the plural unless the context clearly indicates otherwise. As used herein, the term "comprise" can mean "include". Thus, "comprising one or more modifications in the viral genome" can mean "including at least one modification in the virus" without excluding other elements. In case of conflict, the present specification, including the explanation of terms, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting. The term "about" or "approximately" can 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, e.g., the limitations of the measurement system. For example, "about" can mean within or more than one standard deviation per implementation in a given value. When particular values ​​are described in the application and claims, unless otherwise indicated, the term "about" should be assumed to mean an acceptable range of error for the particular value, such as ±10% of the value modified by the term "about."

[0016] The term "recombinant oncolytic virus" as defined herein includes oncolytic virus strains that have been engineered to delete or functionally delete one or more endogenous nucleic acid sequences and / or to insert or partially insert one or more exogenous nucleic acid sequences. The term also includes the replacement of one or more endogenous nucleic acid sequences in the viral genome with one or more exogenous nucleic acids at the same or different loci.

[0017] The term "effective amount" as used herein can refer to an amount of an agent (such as a recombinant oncolytic virus disclosed herein, as well as other anti-cancer agents) sufficient to produce beneficial or desired results. The effective amount (also referred to as a therapeutically effective amount) can vary depending on one or more of the subject and disease state to be treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be easily determined by those skilled in the art. Beneficial therapeutic effects include, but are not limited to, making diagnostic determinations; improving a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or condition. overview

[0018] Provided herein are compositions and methods relating to (i) inhibiting major histocompatibility complex (MHC) class I presentation within virus-infected cells as a means of decreasing immune responses targeting the virus and virus-infected cells while increasing immune responses targeting the surrounding tumor. Additionally provided herein are compositions and methods of recombinant oncolytic viruses engineered to activate major histocompatibility complex (MHC) class II presentation throughout the tumor microenvironment while helping to overcome immune resistance to immune-oncology therapy (e.g., immune checkpoint inhibitor therapy) mediated by tumor-mediated downregulation of MHC II presentation.

[0019] In some embodiments, the recombinant oncolytic virus is a vaccinia virus, which can be modified such that the virus is (i) deleted for the natural MHC II inhibitor of vaccinia; and (ii) engineered to express the MHC I inhibitor from cowpox virus. Herein, in some aspects, it is shown that replacement of the vaccinia virus gene WR158 (NCBI accession number YP_233040), which encodes protein A35, with a DNA sequence encoding the viral promoter P7.5 (SEQ ID NO: 1), which drives expression of the cowpox protein CPXV012 (NCBI accession number NP_619801) (SEQ ID NO: 2), or with a DNA sequence encoding the cowpox protein CPXV203, can result in greater therapeutic activity than the same virus without this replacement.

[0020] In some embodiments, the MHC I inhibitor may be selected from one or more TAP inhibitors, such as UL49.5; ICP47; US6, BNLF2a.

[0021] In some embodiments, better activation of MHC II presentation can be achieved by other methods, including or excluding the deletion of vaccinia's natural MHC II inhibitor.These other methods include, but are not limited to, the modification in recombinant vaccinia virus, such that the viral genome is modified by inserting at least one of the following genes: apoptosis inhibitor protein or necrotic cell death activator protein; autophagy enhancer protein (e.g., HMGB1); asparaginyl endopeptidase; class II transactivator (CIITA); interferon-gamma; Toll-like receptor (TLR) activator; dendritic cell (DC) maturation activator (e.g., osteopontin or TNF-alpha) or Fas ligand.

[0022] In some embodiments, inhibition of major histocompatibility complex (MHC) class I presentation can be achieved by other methods, including or excluding the insertion of genes from cowpox virus expressing MHC I inhibitors. These other methods include, but are not limited to, the insertion of exogenous genes into the viral genome from one or more MHC I inhibitors from herpes viruses, such as Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) or BNLF2a proteins, herpes simplex virus-encoded ICP47 or UL49.5 proteins, herpes simplex virus-encoded proteins, human cytomegalovirus (HCMV)-encoded US6, US2, US3, US11 or gp48 proteins, Epstein-Barr virus-encoded BNLF2a proteins, Kaposi's sarcoma-associated herpes virus (KSHV)-encoded kK3, vIRF3 or kK5 proteins, or dominant negative forms of IRF7.

[0023] Other viral proteins that down-regulate MHC I include, for example, the adenovirus-encoded E3-19K protein, the human immunodeficiency virus 1-encoded Nef protein, the human immunodeficiency virus 2-encoded Nef protein, and the simian immunodeficiency virus 1-encoded Nef protein. Oncolytic viruses

[0024] Provided herein is a composition comprising a modified oncolytic virus. Modifications include the addition of exogenous nucleic acid as described herein. Further modifications include the addition of genomic modifications as described herein. Exemplary oncolytic viruses for inclusion in the compositions described herein include, but are not limited to, poxvirus, adeno-associated virus, adenovirus, Newcastle disease virus (NDV), reovirus (RV), mengovirus, myxoma virus (MYXV), measles virus (MV), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV), and poliovirus (PV). These oncolytic viruses have a tendency to specifically target cancer cells, and upon viral replication, cause significant cell death and tumor regression. In some embodiments, the oncolytic viruses described herein kill cancer or tumor cells through mechanisms such as direct lysis of the cells by stimulating immune responses against the cells, apoptosis, expression of toxic proteins, shutting down autophagy and protein synthesis, inducing anti-tumor immunity, or any combination thereof. In some embodiments, the poxvirus comprises a beta entomopoxvirus, a yatapoxvirus, a cervidopoxvirus, a gamma entomopoxvirus, a leporipoxvirus, a suipoxvirus, a molluscum poxvirus, a crocodylidopoxvirus, an alpha entomopoxvirus, a capripoxvirus, an avipoxvirus, or a parapoxvirus. In some embodiments, the poxvirus comprises a vaccinia virus. In some embodiments, the poxvirus is a vaccinia virus.

[0025] Also based on these approaches, compositions and methods are provided for tumor therapy such that immune responses targeting infected cells in the tumor are reduced, and non-infected tumor cells are better targeted instead (e.g., this approach can reduce anti-viral immunity in favor of anti-tumor immune responses).

[0026] Provided herein are recombinant viruses incorporating genomic modifications including insertions, mutations or deletions, as well as the insertion of exogenous genes described herein. In some embodiments, such modifications are generated by natural recombination with a transfer vector. For example, the vector, which may be a circular plasmid or a linear DNA fragment, can contain the desired DNA sequence to be added to the viral genome, followed by a gene encoding a floxed fluorescent reporter protein under a strong viral promoter. Such components can be flanked by DNA sequences that are 200-1000 bases in length that are homologous to the viral genomic DNA immediately before and after the desired integration site that directs site-specific integration of the vector payload plus reporter. Purified vector DNA is transfected into a virus-susceptible adherent cell line, e.g., 143B, seeded at approximately 1 million cells in a single well of a 6-well cell culture plate. The transfected cells are then infected with the virus into which the vector DNA is intended to be integrated. Recombination between the vector and the viral genome occurs naturally during viral replication. One to three days after infection, recombinant virus (along with parental virus) is recovered from the transfected cells by removing the medium and lysing the cell monolayer. Recombinant virus is purified from parental virus by plaque selection. Multiwell plates (e.g., 96-well, treated for adherent cell culture) are seeded with 1.2 x 10 cells per well on the day of plaque selection. 4 ~3.0×10 4The cells are seeded with recombinant plaques. The lysate containing the mixture of recombinant and parental viruses is sonicated, then diluted in cell medium and distributed among the top row of wells of the seeded 96-well plate. The medium from the infected row is mixed and transferred to the next adjacent row of cells in the plate. A process of serial dilution is performed for every row of the selection plate. The infected plate is kept in a cell culture incubator for 2-3 days to allow plaques to develop. The plaques formed by the recombinant viruses are fluorescent due to reporter gene expression and are identified using a fluorescent microscope. Recombinant plaques are manually picked using a single channel micropipette, typically 0.5-3.0 microliters of material is picked from each plaque. Preferred plaques are round, uniform in reporter intensity, and single within their wells. The picked plaques are frozen, thawed, diluted, and used to infect further 96-well selection plates. A pure population of recombinant plaques (fully GFP positive) is achieved after several rounds of selection. The reporter gene is then deleted from the viral genome by transfecting cells with a vector encoding the cre recombinase under the viral promoter. The transfected cells are infected with a reporter-positive virus, allowing the expressed cre to remove the floxed reporter. The reporter-free virus is purified by plaque selection, similar to the method described above, and targets GFP-deficient plaques that are present alone in those wells. Recombinant vaccine viruses

[0027] In some embodiments, the oncolytic virus is a vaccinia virus. When used interchangeably herein, the term "recombinant vaccinia virus" or "recombinant vaccinia virus" may refer to modified vaccinia virus. Exemplary modifications include, but are not limited to, the introduction of viral backbone mutations to express vaccinia virus genes / peptides, or the deletion of genes that specifically promote increased immune response. In some embodiments, introducing viral backbone mutations includes the complete or partial deletion of one or more toxic genes, or the replacement of one or more viral toxic genes (including, as non-limiting examples, genes known to inhibit Th1 immune response or cytokines involved in innate immune signaling, or inhibitors of other components of immune response, or vaccinia toxic genes replaced with more or less potent genes with equivalent functions from other poxviruses).

[0028] The modification in the genome of the virus can be at one or more positions in the genome.In some embodiments, the modification in the genome of the virus is located consecutively in the genome.In some embodiments, the modification in the genome of the virus is distributed throughout the genome.

[0029] Exemplary vaccinia viruses include, but are not limited to, the following strains for modification by including the fusion constructs described herein: Western Reserve Vaccinia Virus (ATCC VR-1354), Vaccinia Virus Ankara (ATCC VR-1508), Vaccinia Virus Ankara (ATCC VR-1566), Vaccinia Virus Strain Wyeth (ATCC VR-1536), or Vaccinia Virus Wyeth (ATCC VR-325). Furthermore, in some embodiments, the recombinant vaccinia virus is a modified version of a wild-type or attenuated vaccinia virus strain. Non-limiting examples of vaccinia virus strains include the Western Reserve, Copenhagen, Wyeth (NYCBOH), Tian Tan, Lister, USSR, Ankara, NYVAC strains, and recombinant vaccinia virus Ankara (MVA). Further exemplary strains for inclusion in the compositions described herein are, but are not limited to, Western Reserve strain vaccinia virus, Copenhagen strain, IHD strain, Wyeth strain, NYCBOH strain, Tian Tan strain, Lister strain, Ankara strain, USSR strain, or ACAM2000 strain. The modified basic vaccinia virus strain described herein can itself comprise one or more mutations relative to its parent strain, such as, but not limited to, one or more of the following: deletion in TK (also referred to herein as "TK-"); deletion in A52 (also referred to herein as "A52-"). An exemplary vaccinia virus is Western Reserve vaccinia virus. Any known vaccinia virus or modifications thereof that correspond to those provided herein or known to those skilled in the art are also included in the scope of this application. Modifications that inhibit MHC I presentation

[0030] Peptide presentation on major histocompatibility complex I or MHC I is a pathway to present peptides on cells and alert the immune system to virus-infected cells. For example, infected cells can present viral peptides on MHC I, thereby alerting cytotoxic T lymphocytes to destroy these cells. Infection of the host with a recombinant oncolytic virus inhibits peptide presentation on MHC I.

[0031] In some embodiments of the present disclosure, a modification of a recombinant oncolytic virus is provided herein that provides inhibition or partial inhibition of MHC I presentation. The modification can include insertion or partial insertion of an exogenous MHC I inhibitor. The insertion of an MHC I inhibitor can be provided by an exogenous nucleic acid encoding said MHC I inhibitor. The exogenous nucleic acid encoding said MHC I inhibitor can be inserted into a nucleic acid sequence of an oncolytic virus genome, such as a cowpox virus. In certain cases, the MHC I inhibitor is inserted into a non-coding region. In other examples, the inhibitor is inserted into a nucleic acid sequence encoding a viral protein of an oncolytic virus. In certain cases, the MHC I inhibitor is inserted into a region of an oncolytic virus genome that allows the virus to replicate in tumor cells. In certain cases, with respect to a cowpox virus, the MHC I inhibitor can be inserted into at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes. The gene encoding the cowpox virus protein can be CPXV012, CPXV203, or any combination thereof. In some embodiments, the cowpox virus protein containing any combination of substitutions, insertions, and deletions can result in a sequence having less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less sequence homology with the wild-type sequence of the viral gene or viral protein encoded by the gene. MHC I presentation can be reduced by at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 100%.

[0032] In some embodiments, exogenous nucleic acid can code at least one TAP inhibitor.In some embodiments, exogenous nucleic acid can code at least one gene selected from the group consisting of Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) protein, Herpes simplex virus-encoded ICP47 protein, Herpes simplex virus-encoded UL49.5 protein, Human cytomegalovirus (HCMV)-encoded US6, US2, US3 or US11 protein, Epstein-Barr virus-encoded BNLF2a protein, Adenovirus-encoded E3-19K protein, Cytomegalovirus-encoded gp48 protein, Human immunodeficiency virus-encoded Nef protein, Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded kK3, vIRF3 or kK5 protein, or dominant negative form of IRF7. Modifications that activate MHC class II presentation

[0033] In some embodiments of the present disclosure, a modification of recombinant oncolytic virus is provided that results in activation of MHC II presentation, and the modification can include at least one of the following: i) deletion or partial deletion of one or more MHC II inhibitors (e.g., natural MHC II inhibitors); ii) insertion of apoptosis inhibitor protein or necrotic cell death activator protein; or any combination thereof. The modification can be deletion of oncolytic virus gene. The modification can be deletion (complete or partial deletion) of gene encoding A35. The modification of deletion of A35 coding can be deletion or functional deletion of viral gene WR158. In some embodiments, MHC II inhibitors comprising any combination of substitutions, insertions, and deletions can result in sequences having less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less sequence identity with the wild-type sequence of the viral gene or the viral protein encoded by the gene, i.e., the native MHC II inhibitor of the virus. Compared to an otherwise identical virus that does not contain a modification that activates or enhances MHC II presentation, MHC II presentation can be activated or enhanced by at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 100%.

[0034] Modifications that cause activation or enhancement of MHC II presentation can include the insertion of one or more genes selected from the group consisting of apoptosis inhibitor protein or necrotic cell death activator protein; autophagy enhancer protein; asparaginyl endopeptidase; class II transactivator (CIITA); interferon-gamma; Toll-like receptor (TLR) activator; dendritic cell (DC) maturation activator (e.g., osteopontin or TNF alpha). In certain cases, the MHC II enhancer can be modified to include a secretory sequence and a cell-permeable domain to provide an immunological bystander effect. In some cases, the autophagy enhancer protein includes HMGB1, PIAS3, LIGHT, ITAC, fractalkine, functional domain or fragment or variant thereof, or any combination thereof. In some cases, the autophagy enhancer protein includes HMGB1. In some cases, the HMGB1 includes the nucleic acid sequence described in SEQ ID NO:4 or the amino acid sequence shown in SEQ ID NO:5. In some cases, the secretory sequence comprises an IgE-derived signal sequence. In some cases, the IgE-derived signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 6 or the amino acid sequence set forth in SEQ ID NO: 7. In some cases, HMGB1 is modified with an IgE-derived signal sequence, and the modified HMGB1 comprises the nucleic acid sequence set forth in SEQ ID NO: 8 or the amino acid sequence set forth in SEQ ID NO: 9.

[0035] Concomitant upregulation of MHC II (ideally in uninfected cells, but not a requirement) results in more efficient targeting of tumor antigens (e.g., downregulation of MHC II is one of the common tumor resistance mechanisms to anti-PD1 / PDL1 antibodies).

[0036] Provided herein is a recombinant oncolytic virus modification that provides inhibition or partial inhibition of MHC I presentation as described herein, and further provides activation of MHC II presentation as described herein.In some cases, the modifications are at separate positions in the genome.In some cases, the modifications are at the same position in the genome.In some cases, the modifications are at consecutive positions in the genome. Several other modifications

[0037] In some embodiments, the present application provides a vaccine comprising recombinant oncolytic viruses with insertion, mutation or deletion in viral genome (also referred to herein as viral backbone).For oncolytic vaccinia viruses, they are preferably recombinant or selected to have low toxicity and accumulate in target tissue.In some embodiments, the modification in viral backbone / viral genome is such that vaccinia virus does not replicate or contains poor replication ability. Non-limiting examples of such modifications can include mutations in the following viral genes: TK, A1, A2, VH1, A33, I7, F13L, A36R, A34R, A46R, A49R, B8R, B14R, B15R, B18R, C12L, C4, C16, SPI-1, SPI-2, B15R, VGF, E3L, K1L, K3L, K7R, A41L, M2L, N1L, A52R, functional domains or fragments or variants thereof, or any combination thereof.

[0038] In some embodiments, with respect to vaccinia virus, the viral backbone mutations include a complete or partial deletion of the A52R gene; a complete or partial deletion of the TK gene; a complete or partial deletion of the F13L gene; a complete or partial deletion of the A36R gene; a complete or partial deletion of the A34R gene; a complete or partial deletion of the B8R gene; a complete or partial deletion of the B18R gene; a complete or partial deletion of the C12L gene; a complete or partial deletion of the C4 gene; a complete or partial deletion of the C16 gene; a complete or partial deletion of the SPI-1 gene; a complete or partial deletion of the SPI-2 gene; a complete or partial deletion of the VGF gene; a complete or partial deletion of the E3L gene; a complete or partial deletion of the K3L gene; The viral gene may be selected from the group consisting of a complete or partial deletion of a gene, a complete or partial deletion of an A41L gene, a complete or partial deletion of an A52R gene, a complete or partial deletion of a B15R gene, a complete or partial deletion of a K7R gene, a complete or partial deletion of a B14R gene, a complete or partial deletion of an N1L gene, a complete or partial deletion of a K1L gene, a complete or partial deletion of a M2L gene, a complete or partial deletion of an A49R gene, a complete or partial deletion of a VH1 gene, a complete or partial deletion of an A33 gene, a complete or partial deletion of an A1 gene, a complete or partial deletion of an A2 gene, a complete or partial deletion of an I7 gene, and a complete or partial deletion of an A46R gene. As used herein, reference to a viral gene may be made by reference to the protein encoded by the gene (e.g., the A33 gene may refer to the gene encoding the A33 protein).

[0039] In some embodiments, the viral backbone mutations of the oncolytic virus, including any combination of substitutions, insertions, and deletions, can result in sequences with less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less sequence homology with the wild-type sequence of the viral gene or viral protein encoded by the gene. In some embodiments, the viral genes and proteins encoded by the same are selected from the group consisting of TK, A1, A2, VH1, A33, I7, F13L, A36R, A34R, A46R, A49R, B8R, B14R, B15R, B18R, C12L, C4, C16, SPI-1, SPI-2, B15R, VGF, E3L, K1L, K3L, K7R, A41L, M2L, N1L, and A52R.

[0040] In some embodiments, the viral backbone can include one, two, three, four, five or more mutations in the amino acid sequence of a viral protein (e.g., a viral antigen). The viral antigen, in some examples, is selected from the group consisting of TK, A1, A2, VH1, A33, I7, F13L, A36R, A34R, A46R, A49R, B8R, B14R, B15R, B18R, C12L, C4, C16, SPI-1, SPI-2, B15R, VGF, E3L, K1L, K3L, K7R, A41L, M2L, N1L, and A52R.

[0041] The present disclosure provides, in some embodiments, a recombinant oncolytic virus comprising one or more mutations in the viral genome (viral backbone), such that the mutations increase the T cell arm of the immune response. The mutations can be additions, deletions or substitutions of one or more nucleic acids in the viral genome (wild type or attenuated native strain of the oncolytic virus).

[0042] In a non-limiting example, the mutation can be a complete or partial deletion of a gene known to inhibit cytokines involved in Th1 immune responses. As a non-limiting example, the mutation can be a deletion of a nucleic acid encoding B8R (interferon gamma (IFN-g) binding protein); C12L (interleukin-18 (IL-18) binding protein).

[0043] In another non-limiting example, the mutation can be the complete or partial deletion of the gene in innate immune signal transduction.As a non-limiting example, the mutation can be the deletion of the nucleic acid encoding B18R (type I interferon (IFN) binding protein); A52R (nuclear factor kappa B (NF-κB) inhibitor protein); E3L (protein kinase (PKR) inhibitor); C4, C16 (STING pathway inhibitor).

[0044] In a further non-limiting example, the mutation can be a complete or partial deletion of a gene that encodes a protein for inhibiting other components of the immune response.As a non-limiting example, the mutation can be a complete or partial deletion of the nucleic acid that encodes B15, K7, B14, N1, K1, M2, A49, VH1, A46, or a combination thereof.Virus backbone mutations can also include replacing oncolytic toxic genes with the most potent genes with equivalent functions from other poxviruses.

[0045] The amino acid sequence variants of the polypeptides of the present disclosure can be substitution, insertion or deletion variants. Mutations in genes encoding viral polypeptides can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more non-contiguous or contiguous amino acids of the polypeptide may be affected.

[0046] Deletion variants can lack one or more residues of the native or wild-type protein. Individual residues can be deleted, or whole or part of a domain (such as catalytic or binding domain) can be deleted. Stop codons can be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate truncated proteins. Insertion mutants typically involve the addition of material at a non-terminal point of a polypeptide. This can include the insertion of an immunoreactive epitope, or simply the insertion of one or more residues. Terminal additions, called fusion proteins, can also occur.

[0047] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid can be replaced with one of similar shape and charge. Conservative substitutions may include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative, such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically different amino acid, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, or vice versa.

[0048] The oncolytic virus provided herein comprises additional insertions, mutations, deletions or substitutions in the viral genome. The oncolytic virus may comprise one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more of chemokine receptors, TRIF protein or its functional domains, or leptin, interleukin-2 (IL2), interleukin-15 / interleukin-15Ra (IL15 / IL15Ra), interleukin-7 (IL-7), leptin-interleukin fusion protein (e.g., leptin-IL2 fusion protein shown in Example 1 as L2). In some cases, the nucleic acid encodes the IL15 amino acid sequence described in SEQ ID NO: 10. In some cases, the nucleic acid encodes the IL-7 amino acid sequence described in either SEQ ID NO: 13 or 14. The modification such as the insertion of chemokine receptors is the insertion of wild type and / or mutant CCL5, CXCR4, CCR2, CCL2. In some cases, the nucleic acid encodes a CXCR4 amino acid sequence set forth in any one of SEQ ID NOs: 18-20. In some cases, the nucleic acid encodes a CXCR4 amino acid sequence set forth in SEQ ID NO: 18. In some cases, the nucleic acid encodes a CCR2 amino acid sequence set forth in any one of SEQ ID NOs: 23-24. The oncolytic virus may further comprise one or more additional deletions or partial deletions of one or more genes from TK, A1, A2, VH1, A33, I7, F13L, A36R, A34R, A46R, A49R, B8R, B14R, B15R, B18R, C12L, C4, C16, SPI-1, SPI-2, B15R, VGF, E3L, K1L, K3L, K7R, A41L, M2L, N1L, A52R, functional domains or fragments or variants thereof, or any combination thereof. In some cases, the oncolytic viruses provided herein can include a full or partial deletion of the A52R gene and an insertion of a chemokine receptor, such as CCR2. In some cases, the oncolytic viruses provided herein can include a full or partial deletion of the A52R gene and an insertion of a chemokine receptor, such as CCR2.In some cases, the oncolytic viruses provided herein can include a complete or partial deletion of at least one of the A52R or TK viral genes and the insertion of an exogenous nucleic acid encoding a fusion protein (e.g., a metabolic modulator protein fused to a cytokine, such as a leptin-IL2 fusion protein).

[0049] Hyaluronic acid (HA) is a structural element of the ECM and is a high molecular weight linear glycosaminoglycan composed of repeating disaccharide units. It can be widely distributed throughout connective, epithelial, and neural tissues, and its expression level is significantly elevated in many types of tumors. Hyaluronidase is a family of enzymes that catalyze the degradation of HA. At least five functional hyaluronidases have been identified in humans to date: HYAL1, HYAL2, HYAL3, HYAL4, HYAL5 (also known as PH-20 or SPAM1), of which PH-20 is the only one known to date to function at a relatively neutral pH. In some cases of the present disclosure, combining hyaluronidase with other tumor-targeting therapeutic agents (e.g., transgenes, also referred to herein as exogenous nucleic acids) can promote the therapeutic effect of modified oncolytic viruses by at least reducing ECM and enhancing transport of therapeutic agents within and between tumors.

[0050] Some embodiments herein disclose modified oncolytic viruses that can include exogenous nucleic acids encoding membrane-bound proteins capable of degrading hyaluronic acid, such as hyaluronidase. Note that the term "hyaluronidase" as used herein can refer to any enzyme or fragment thereof that catalyzes the degradation of HA in tumors, including, but not limited to, PH-20 and its homologues from other species, as well as other engineered / designed proteins with similar enzymatic functions. As used herein, hyaluronidase can refer to a class of hyaluronic acid degrading enzymes. In some cases, PH-20 comprises the amino acid sequence set forth by SEQ ID NO:26. In some cases, hyaluronidase comprises the amino acid sequence set forth by SEQ ID NO:28. In some cases, hyaluronidase further comprises a secretory sequence. In some cases, the secretory sequence comprises an IgE-derived signal sequence. In some cases, the IgE-derived signal sequence comprises the amino acid sequence set forth by SEQ ID NO:7. In some cases, the hyaluronidase having an IgE-derived signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO:29 or the amino acid sequence set forth in SEQ ID NO:30. Vaccines, pharmaceutical compositions, and delivery of recombinant vaccine viruses

[0051] The present disclosure further provides a pharmaceutical composition or immunogenic composition for treating cancer. In addition, the present disclosure further provides a pharmaceutical composition or immunogenic composition that can include a vaccine comprising the recombinant vaccinia virus described above and a suitable carrier in some embodiments. The vaccine can be provided as a kit that includes the recombinant vaccinia virus-based vaccine described above or a pharmaceutical composition of the recombinant vaccinia virus-based vaccine described above. The pharmaceutical composition for vaccine delivery can be for parenteral or oral delivery or nasal delivery. The pharmaceutical composition can be administered to a human at least once. The pharmaceutical composition can be administered to a human one or more times.

[0052] Also provided herein are recombinant vaccinia viruses for use as medicaments or vaccines, compositions comprising the recombinant vaccinia viruses, and / or vaccines.

[0053] In some embodiments, a pharmaceutical composition comprising a vaccine comprising a recombinant vaccinia virus provided herein is administered in a dose of 10 6 ~10 9 Dose of PFU, 10 6 ~5×10 8 Dose of PFU, or 10 7 ~10 8 The recombinant vaccinia viruses provided herein can also be administered to a subject at 10 PFU. 6 , 10 7 PFU, 10 8 , or 5 × 10 8 The recombinant vaccinia viruses provided herein can be administered to a subject at a dose of 10 PFU. 7 PFU, 10 8 PFU, or 5 × 10 8 It can be administered to a human subject in a dose of PFU.

[0054] The recombinant vaccinia virus, vaccine composition, or pharmaceutical composition described herein may be 4 ~10 9 PFU / mL, 10 5 ~5×10 8 PFU / mL, 10 6 ~10 8 PFU / mL, or 10 7 ~10 8 The vaccine can be formulated in a range of concentrations in solution per mL of PFU / mL. In some embodiments, the vaccination dose for a human is about 10 6 ~10 9 PFU, e.g., about 10 6 PFU, approx. 10 7 PFU, or about 10 8 In some embodiments, a dose for a human may include a dose of at least about 2×10 PFU in a volume of 0.1-0.5 ml. 7 PFU, at least about 3 × 107 PFU, at least about 5 × 10 7 PFU, at least about 1 × 10 8 PFU, at least about 2 × 10 8 It may contain PFUs.

[0055] The pharmaceutical / immunogenic compositions provided herein can generally include one or more pharma- ceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, and the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0056] The corresponding prime-boost regimen can be used, and the recombinant vaccinia virus defined herein can be administered in the first dosage.One or more subsequent administrations of the recombinant vaccinia virus defined herein can be given to boost the immune response provided in the first administration.In some embodiments, the one or more antigens delivered by the recombinant vaccinia virus can be the same or similar to those of the first administration.

[0057] The pharmaceutical compositions comprising the recombinant vaccinia virus provided herein can be administered to a subject in a single dose or multiple doses (e.g., 2, 3, 4, etc.). The recombinant vaccinia virus can be administered in a first dose (priming) and a second dose (booster). The first dose is about 10 6 ~about 10 9 PFU / mL of recombinant vaccinia virus, and the second dose may contain about 10 6 ~about 10 9 PFU / mL of the recombinant vaccinia virus. The first and second doses may contain at least 1000 PFU / mL of the modified vaccine virus.

[0058] The second dose of the vaccine or pharmaceutical / immunogenic composition can be administered 24 hours to about 3 months, for example, about 7 days to about 2 months, after administration of the first dose. The second dose can be a booster dose.

[0059] The recombinant vaccinia virus or a pharmaceutical / immunogenic composition comprising the recombinant vaccinia virus can be administered intraperitoneally, systemically, topically, parenterally, subcutaneously, intravenously, intramuscularly, or intranasally. The vaccine can be administered with an adjuvant, such as the adjuvants described herein.

[0060] Another aspect of the present disclosure relates to a method of affecting an immune response in a subject comprising administering to a subject a recombinant vaccinia virus described herein, a vaccine comprising same, or a pharmaceutical / immunogenic composition comprising a recombinant vaccinia virus or vaccine composition described herein.

[0061] The immunization protocol may include immunization with more than one antigen in a single dose of the vaccine, multiple doses, and multiple doses with different antigens in each dose. In some embodiments, the immunization protocol may include immunization with an antigen and an adjuvant. Adjuvants, in this context, may include cytokines and other immunomodulatory molecules, such as TLR (toll-like receptor) agonists and their derivatives, that stimulate the immune response.

[0062] Also provided herein are methods of treating a disease, disorder, or condition by administering a recombinant vaccinia virus described herein. In some embodiments, the amount of recombinant vaccinia virus of the present disclosure administered to a subject is about 10 3 ~10 12 of infectious viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8PFU, or about 10 8 ~10 10 In some embodiments, the amount of recombinant vaccinia virus of the present disclosure administered to a subject is about 10 3 ~10 12 of virus particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or about 10 5 ~10 8 PFU, or about 10 8 ~10 10 In some embodiments, the recombinant vaccine virus of the present disclosure can be about 10 PFU. 3 PFU / dose~about 10 4 PFU / dose, approximately 10 4 PFU / dose~about 10 5 PFU / dose, approximately 10 5 PFU / dose~about 10 6 PFU / dose, approximately 10 7 PFU / dose~about 10 8 PFU / dose, approximately 10 9 PFU / dose~about 10 10 PFU / dose, approximately 10 10 PFU / dose~about 10 11 PFU / dose, approximately 10 11 PFU / dose~about 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered in a dose that may include about 2×10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, approximately 104 PFU / dosage, approximately 2×10 4 PFU / dosage, approximately 3×10 4 PFU / dosage, approximately 4×10 4 PFU / dosage, approximately 5×10 4 PFU / dosage, approximately 6×10 4 PFU / dosage, approximately 7×10 4 PFU / dosage, approximately 8×10 4 PFU / dosage, approximately 9×10 4 PFU / dosage, approximately 10 5 PFU / dosage, 2×10 5 PFU / dosage, 3×10 5 PFU / dosage, 4×10 5 PFU / dosage, 5×10 5 PFU / dosage, 6×10 5 PFU / dosage, 7×10 5 PFU / dosage, 8×10 5 PFU / dosage, 9×10 5 PFU / dosage, approximately 10 6 PFU / dosage, approximately 2×10 6 PFU / dosage, approximately 3×10 6 PFU / dosage, approximately 4×10 6 PFU / dosage, approximately 5×10 6 PFU / dosage, approximately 6×10 6 PFU / dosage, approximately 7×10 6 PFU / dosage, approximately 8×10 6 PFU / dosage, approximately 9×10 6 PFU / dosage, approximately 10 7 PFU / dosage, approximately 2×10 7 PFU / dosage, approximately 3×10 7 PFU / dosage, approximately 4×10 7 PFU / dosage, approximately 5×10 7 PFU / dosage, approximately 6×10 7 PFU / dosage, approximately 7×10 7 PFU / dosage, approximately 8×10 7 PFU / dosage, approximately 9×10 7 PFU / dosage, approximately 10 8 PFU / dosage, approximately 2×10 8 PFU / dosage, approximately 3×10 8 PFU / dosage, approximately 4×10 8 PFU / dosage, approximately 5×10 8PFU / dosage, approximately 6×10 8 PFU / dosage, approximately 7×10 8 PFU / dosage, approximately 8×10 8 PFU / dosage, approximately 9×10 8 PFU / dosage, approximately 10 9 PFU / dosage, approximately 2×10 9 PFU / dosage, approximately 3×10 9 PFU / dosage, approximately 4×10 9 PFU / dosage, approximately 5×10 9 PFU / dosage, approximately 6×10 9 PFU / dosage, approximately 7×10 9 PFU / dosage, approximately 8×10 9 PFU / dosage, approximately 9×10 9 PFU / dosage, approximately 10 10 PFU / dosage, approximately 2×10 10 PFU / dosage, approximately 3×10 10 PFU / dosage, approximately 4×10 10 PFU / dosage, approximately 5×10 10 PFU / dosage, approximately 6×10 10 PFU / dosage, approximately 7×10 10 PFU / dosage, approximately 8×10 10 PFU / dosage, approximately 9×10 10 PFU / dosage, approximately 10 10 PFU / dosage, approximately 2×10 10 PFU / dosage, approximately 3×10 10 PFU / dosage, approximately 4×10 10 PFU / dosage, approximately 5×10 10 PFU / dosage, approximately 6×10 10 PFU / dosage, approximately 7×10 10 PFU / dosage, approximately 8×10 10 PFU / dosage, approximately 9×10 10 PFU / dosage, approximately 10 11 PFU / dosage, approximately 2×10 11 PFU / dosage, approximately 3×10 11 PFU / dosage, approximately 4×10 11 PFU / dosage, approximately 5×10 11 PFU / dosage, approximately 6×10 11 PFU / dosage, approximately 7×10 11 PFU / dosage, approximately 8×10 11 PFU / dosage, approximately 9×10 11PFU / dose, or approximately 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered at a dose that may include 3×10 PFU / dose. 9 In some embodiments, the modified oncolytic vaccinia virus of the present disclosure can be administered in doses that can include up to 5×10 PFU / dose. 9 It can be administered in doses that can include PFU / dose.

[0063] In some embodiments, the recombinant vaccinia virus of the present disclosure is 3 Virus particles / dose ~ approx. 10 4 Viral particles / dose, approximately 10 4 Virus particles / dose ~ approx. 10 5 Viral particles / dose, approximately 10 5 Virus particles / dose ~ approx. 10 6 Viral particles / dose, approximately 10 7 Virus particles / dose ~ approx. 10 8 Viral particles / dose, approximately 10 9 Virus particles / dose ~ approx. 10 10 Viral particles / dose, approximately 10 10 Virus particles / dose ~ approx. 10 11 Viral particles / dose, approximately 10 11 Virus particles / dose ~ approx. 10 12 Viral particles / dose, approximately 10 12 Virus particles / dose ~ approx. 10 13 Viral particles / dose, approximately 10 13 Virus particles / dose ~ approx. 10 14 viral particles / dose, or approximately 10 14 Virus particles / dose ~ approx. 10 15 It may be administered in a dose that can include a viral particle / dose.

[0064] In some embodiments, the recombinant vaccine virus of the present disclosure is about 10 3 PFU / kg ~ approx. 10 4 PFU / kg, approximately 10 4 PFU / kg ~ approx. 10 5 PFU / kg, approximately 10 5 PFU / kg ~ approx. 10 6 PFU / kg, approximately 10 7 PFU / kg ~ approx. 10 8 PFU / kg, approximately 10 9 PFU / kg ~ approx. 10 10 PFU / kg, approximately 10 10 PFU / kg ~ approx. 10 11 PFU / kg, approximately 10 11 PFU / kg ~ approx. 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic vaccinia virus of the present disclosure may be administered at a dose that may include about 2×10 PFU / kg. 3 PFU / kg, 3×10 3 PFU / kg, 4×10 3 PFU / kg, 5×10 3 PFU / kg, 6×10 3 PFU / kg, 7×10 3 PFU / kg, 8×10 3 PFU / kg, 9×10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2×10 5PFU / kg, 3×10 5 PFU / kg, 4×10 5 PFU / kg, 5×10 5 PFU / kg, 6×10 5 PFU / kg, 7×10 5 PFU / kg, 8×10 5 PFU / kg, 9×10 5 PFU / kg, approximately 10 6 PFU / kg, approximately 2×10 6 PFU / kg, approximately 3×10 6 PFU / kg, approximately 4×10 6 PFU / kg, approximately 5×10 6 PFU / kg, approximately 6×10 6 PFU / kg, approximately 7×10 6 PFU / kg, approximately 8×10 6 PFU / kg, approximately 9×10 6 PFU / kg, approximately 10 7 PFU / kg, approximately 2×10 7 PFU / kg, approximately 3×10 7 PFU / kg, approximately 4×10 7 PFU / kg, approximately 5×10 7 PFU / kg, approximately 6×10 7 PFU / kg, approximately 7×10 7 PFU / kg, approximately 8×10 7 PFU / kg, approximately 9×10 7 PFU / kg, approximately 10 8 PFU / kg, approximately 2×10 8 PFU / kg, approximately 3×10 8 PFU / kg, approximately 4×10 8 PFU / kg, approximately 5×10 8 PFU / kg, approximately 6×10 8 PFU / kg, approximately 7×10 8 PFU / kg, approximately 8×10 8 PFU / kg, approximately 9×10 8 PFU / kg, approximately 10 9 PFU / kg, approximately 2×10 9 PFU / kg, approximately 3×10 9 PFU / kg, approximately 4×10 9 PFU / kg, approximately 5×10 9 PFU / kg, approximately 6×10 9 PFU / kg, approximately 7×109 PFU / kg, approximately 8×10 9 PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered at a dose that may include 3×10 PFU / kg. 9In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered at a dose that may include up to 5×10 PFU / kg. 9 It may be administered in a dose that may contain PFU / kg.

[0065] In some embodiments, the recombinant vaccine virus of the present disclosure is about 10 3 Virus particles / kg ~ approx. 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg ~ approx. 10 5 Virus particles / kg, approximately 10 5 Virus particles / kg ~ approx. 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg ~ approx. 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg ~ approx. 10 10 Virus particles / kg, approximately 10 10 Virus particles / kg ~ approx. 10 11 Virus particles / kg, approximately 10 11 Virus particles / kg ~ approx. 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg ~ approx. 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg ~ approx. 10 14 Virus particles / kg, or approximately 10 14 Virus particles / kg ~ approx. 10 15 It may be administered in a dose that may contain viral particles / kg.

[0066] The liquid dosage forms of the recombinant vaccinia viruses described herein may, in certain embodiments, be administered in a volume of about 10 3 PFU / mL~about 10 4 PFU / mL, approximately 10 4 PFU / mL~about 10 5 PFU / mL, approximately 10 5 PFU / mL~about 10 6 PFU / mL, approximately 10 7 PFU / mL~about 10 8 PFU / mL, approximately 10 9 PFU / mL~about 10 10PFU / mL, approximately 10 10 PFU / mL~about 10 11 PFU / mL, approximately 10 11 PFU / mL~about 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the recombinant vaccinia virus of the present disclosure may comprise a viral dose of about 2×10 PFU / mL. 3 PFU / mL, 3×10 3 PFU / mL, 4×10 3 PFU / mL, 5×10 3 PFU / mL, 6×10 3 PFU / mL, 7×10 3 PFU / mL, 8×10 3 PFU / mL, 9×10 3 PFU / mL, approximately 10 4 PFU / mL, approximately 2×10 4 PFU / mL, approximately 3×10 4 PFU / mL, approximately 4×10 4 PFU / mL, approximately 5×10 4 PFU / mL, approximately 6×10 4 PFU / mL, approximately 7×10 4 PFU / mL, approximately 8×10 4 PFU / mL, approximately 9×10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6 PFU / mL, approximately 2×10 6 PFU / mL, approximately 3×10 6 PFU / mL, approximately 4×10 6PFU / mL, approximately 5×10 6 PFU / mL, approximately 6×10 6 PFU / mL, approximately 7×10 6 PFU / mL, approximately 8×10 6 PFU / mL, approximately 9×10 6 PFU / mL, approximately 10 7 PFU / mL, approximately 2×10 7 PFU / mL, approximately 3×10 7 PFU / mL, approximately 4×10 7 PFU / mL, approximately 5×10 7 PFU / mL, approximately 6×10 7 PFU / mL, approximately 7×10 7 PFU / mL, approximately 8×10 7 PFU / mL, approximately 9×10 7 PFU / mL, approximately 10 8 PFU / mL, approximately 2×10 8 PFU / mL, approximately 3×10 8 PFU / mL, approximately 4×10 8 PFU / mL, approximately 5×10 8 PFU / mL, approximately 6×10 8 PFU / mL, approximately 7×10 8 PFU / mL, approximately 8×10 8 PFU / mL, approximately 9×10 8 PFU / mL, approximately 10 9 PFU / mL, approximately 2×10 9 PFU / mL, approximately 3×10 9 PFU / mL, approximately 4×10 9 PFU / mL, approximately 5×10 9 PFU / mL, approximately 6×10 9 PFU / mL, approximately 7×10 9 PFU / mL, approximately 8×10 9 PFU / mL, approximately 9×10 9 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 11 PFU / mL, approximately 2×10 11 PFU / mL, approximately 3×10 11 PFU / mL, approximately 4×10 11 PFU / mL, approximately 5×10 11 PFU / mL, approximately 6×10 11 PFU / mL, approximately 7×10 11 PFU / mL, approximately 8×10 11 PFU / mL, approximately 9×10 11 PFU / mL, or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered at a dose that may contain 5×10 PFU / mL. 9 In some embodiments, the recombinant vaccinia virus of the present disclosure may be administered in a dose that may contain up to 5×10 PFU / mL. 9 It may be administered in a dose that may contain PFU / mL.

[0067] In some cases, the recombinant vaccinia virus can be administered by injection, with the dosage being about 10 3 Viral particles, 10 per injection 4 Viral particles, 10 per injection 5 Viral particles, 10 per injection 6 Viral particles, 10 per injection7 Viral particles, 10 per injection 8 Viral particles, 10 per injection 9 Viral particles, 10 per injection 10 Viral particles, 10 per injection 11 Viral particles, 10 per injection 12 Viral particles, 2 x 10 per injection 12 Viral particles, 10 per injection 13 Viral particles, 10 per injection 14 viral particles, or 10 per injection 15 In a further embodiment, the recombinant vaccinia virus is administered by injection and the dosage is about 10 3 Infectious viral particles, 10 per injection 4 Infectious viral particles, 10 per injection 5 Infectious viral particles, 10 per injection 6 Infectious viral particles, 10 per injection 7 Infectious viral particles, 10 per injection 8 Infectious viral particles, 10 per injection 9 Infectious viral particles, 10 per injection 10 Infectious viral particles, 10 per injection 11 Infectious viral particles, 10 per injection 12 Infectious viral particles, 2 x 10 per injection 12 Infectious viral particles, 10 per injection 13 Infectious viral particles, 10 per injection 14 infectious viral particles, or 10 per injection 15 In a further embodiment, the recombinant vaccinia virus of the present disclosure can comprise an infectious viral particle. 3 Tissue culture inhibitor dose 50% (PFU) / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 104 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 10 4 PFU / kg, 3×10 8 PFU / kg, 4×10 8 PFU / kg, 5×10 8 PFU / kg, 3×10 9 PFU / kg, 4×10 9 PFU / kg, 5×10 9 PFU / kg, 3×10 10 PFU / kg, 4×10 10 PFU / kg, or 4 × 10 10 The dose may be administered at a level that may be 10 PFU / kg. X Note that is alternatively expressed as 1eX. In certain embodiments, the recombinant vaccinia virus can be administered in one or more doses. In certain embodiments, the virus can be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor.

[0068] In certain embodiments, a single dose of recombinant virus can refer to the amount administered to a subject or tumor over 1, 2, 5, 10, 15, 20 or 24 hours.In certain embodiments, the dose can be spread over time or by separate injections.In certain embodiments, repeated doses (e.g., 2, 3, 4, 5, 6 or more doses) of vaccinia virus can be administered to a subject, for example, where a second treatment can occur within 1, 2, 3, 4, 5, 6, 7 days or weeks of a first treatment.In certain embodiments, multiple doses of modified oncolytic virus can be administered to a subject over 1, 2, 3, 4, 5, 6, 7 days or weeks or longer. In certain embodiments, the recombinant vaccinia virus or pharmaceutical composition disclosed herein may be administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or longer. The frequency of administration of the recombinant virus or pharmaceutical composition described herein may be, in certain cases, once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months). In some embodiments of the method disclosed herein, the recombinant vaccinia virus or pharmaceutical composition can be administered at an initial dose in a first period, an intermediate dose in a second period, and a high dose in a third period, independently. In some embodiments, the initial dose can be lower than the intermediate dose, and the intermediate dose can be lower than the high dose. In some embodiments of the method disclosed herein, the recombinant vaccinia virus or pharmaceutical composition can be administered at a high dose in a first period, an intermediate dose in a second period, and a low dose in a third period, independently. In some embodiments, the initial dose can be higher than the intermediate dose, and the intermediate dose can be higher than the low dose.In some embodiments, the first, second, and third periods can be, independently, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer. In some embodiments, the recombinant oncolytic vaccinia viruses described herein can be administered using a prime-boost regimen.

[0069] In some examples, a reduced carbohydrate diet, e.g., a ketogenic diet, can be followed before, during, and after administration of a modified oncolytic vaccinia virus or a pharmaceutical composition comprising the same, as described herein, in accordance with any of the treatment methods described herein. In certain embodiments, a subject may be on a diet consuming less than 500 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 400 grams of carbohydrates per day, less than 350 grams of carbohydrates per day, less than 300 grams of carbohydrates per day, less than 250 grams of carbohydrates per day, less than 200 grams of carbohydrates per day, less than 150 grams of carbohydrates per day, less than 100 grams of carbohydrates per day, less than 90 grams of carbohydrates per day, less than 80 grams of carbohydrates per day, less than 70 grams of carbohydrates per day, less than 60 grams of carbohydrates per day, less than 50 grams of carbohydrates per day, less than 40 grams of carbohydrates per day, less than 30 grams of carbohydrates per day, less than 20 grams of carbohydrates per day, or less than 10 grams of carbohydrates per day. An exemplary method for delivering the recombinant vaccinia virus of the present disclosure or pharmaceutical composition comprising the same to cancer or tumor cells can be via intratumoral injection. However, alternative methods of administration, such as intravenous, by injection, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, ​​intravaginal, intranasal, intrathecal, or intraperitoneal, can also be used. The route of administration can vary depending on the location and nature of the tumor. In certain embodiments, the route of administration can be intradental, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, topical (e.g., in the vicinity of the tumor, particularly with the tumor vasculature or adjacent vasculature), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, or oral. The injectable dose of recombinant vaccinia virus can be administered as a bolus injection or slow infusion. In certain embodiments, modified oncolytic vaccinia virus can be administered to patient from the source implanted in patient.In certain embodiments, modified oncolytic vaccinia virus can be administered by continuous infusion over a selected period of time.In some cases, recombinant vaccinia virus as described herein or pharmaceutical composition comprising it can be administered at a therapeutically effective dose by infusion over about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or longer. The recombinant vaccinia virus or pharmaceutical composition of the present disclosure may be administered as a liquid dosage having a total administration volume of about 1 mL to about 5 mL, about 5 mL to about 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to about 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to about 500 mL, about 500 mL to about 750 mL, or about 750 mL to about 1000 mL. Methods of Using Recombinant Oncolytic Viruses

[0070] The recombinant oncolytic virus described herein or pharmaceutical composition or vaccine comprising it can be used for cancer, as described above, in cancer immunotherapy and tumor treatment.Tumor can be solid tumor and liquid tumor, including but not limited to melanoma, hepatocellular carcinoma, breast cancer, lung cancer, non-small cell lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cell carcinoma, pancreatic cancer, epithelial cancer, gastric / GE junction adenocarcinoma, cervical cancer, colon cancer, colorectal cancer, duodenal cancer, pancreatic adenocarcinoma, adenoid cyst, sarcoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma or myeloproliferative neoplasm. Thus, some embodiments of the present disclosure provide methods of treating cancer, tumors, cancer immunotherapy by administering a recombinant oncolytic virus described herein, or a pharmaceutical or immunogenic composition comprising same.

[0071] The recombinant vaccinia virus described herein or pharmaceutical composition or vaccine comprising it can be used to treat cancer, in cancer immunotherapy, and in tumor treatment, as described above.Tumor can be solid tumor and liquid tumor, including but not limited to melanoma, hepatocellular carcinoma, breast cancer, lung cancer, non-small cell lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cell carcinoma, pancreatic cancer, epithelial cancer, gastric / GE junction adenocarcinoma, cervical cancer, colon cancer, colorectal cancer, duodenal cancer, pancreatic adenocarcinoma, adenoid cyst, sarcoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma or myeloproliferative neoplasm. Thus, some embodiments of the present disclosure provide methods for treating cancer, tumors, cancer immunotherapy by administering a recombinant vaccinia virus described herein, or a pharmaceutical or immunogenic composition comprising same.

[0072] Cancer cells that can be treated by the methods of the present disclosure include cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. Further, cancer may include, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; hairy cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastric cancer, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; branchial alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; acidophilic adenocarcinoma; basophilic adenocarcinoma. Glomerular carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Non-encapsulating sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Cutaneous adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous adenocarcinoma;Seleminal adenocarcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Sheath tumor, malignant;Granulosa cell tumor, malignant;Androblastoma, malignant mammary;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomerular sarcoma;malignant melanoma;amelanomatous melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymal tumor , malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Germinal dysplasia;Embryonal carcinoma;Teratoma, malignant;Ovarian ventricle, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Juxtamebocyte osteosarcoma;Chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neuroblastoma;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's;Paragranuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse; The tumor may be malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some cases, solid tumors that are metastatic can be treated with the recombinant oncolytic virus of the present disclosure, such as recombinant oncolytic vaccinia virus that is favorable for systemic delivery. In some cases, solid tumors that are inaccessible or difficult to access, such as for the purpose of intratumoral delivery of therapeutic agents, can be treated with the recombinant oncolytic virus of the present disclosure, such as recombinant oncolytic virus that is favorable for systemic delivery. Cancers associated with increased expression of free fatty acids can, in some instances, be treated with the recombinant oncolytic viruses of the present disclosure, such as recombinant oncolytic vaccinia viruses that are advantageous for systemic delivery and form increased amounts of EEV; Combination therapy

[0073] The method of the present disclosure, in some aspects, includes administering a recombinant oncolytic virus disclosed herein, or a pharmaceutical or immunogenic composition comprising the same, followed by administration prior to or in combination with one or more additional therapies. Examples of additional therapies can include, but are not limited to, chemotherapy, radiation, oncolytic virus therapy with additional viruses, treatment with immunomodulatory proteins, anti-cancer drugs, or any combination thereof. The additional therapies can be administered simultaneously or sequentially with respect to the administration of the modified virus, such as an oncolytic vaccinia virus. In certain embodiments, the method of the present disclosure can include administering a modified oncolytic virus disclosed herein, followed by administration prior to or in combination with one or more anti-cancer drugs or cancer therapies. Anti-cancer drugs can include, but are not limited to, chemotherapeutic drugs, radiotherapeutic drugs, cytokines, immune checkpoint inhibitors, anti-angiogenic drugs, apoptosis inducers, anti-cancer antibodies, and / or anti-cyclin-dependent kinase agents. In certain embodiments, the cancer therapy can include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, antivascular therapy, cryotherapy, toxin therapy, and / or surgery, or a combination thereof. In certain embodiments, the methods of the present disclosure can include administering a recombinant virus disclosed herein, followed by administration prior to or in combination with a modified oncolytic virus of the present disclosure.

[0074] In certain embodiments, treatment with recombinant oncolytic viruses can be used alone or in combination with one or more immunomodulatory agents. Immunomodulatory agents can include any compound, molecule, or substance that can suppress antiviral immunity associated with tumors or cancer. In certain embodiments, immunomodulatory agents can suppress innate or adaptive immunity against the modified virus. Non-limiting examples of immunomodulatory agents include anti-CD33 antibodies or variable regions thereof, anti-CD11b antibodies or variable regions thereof, COX2 inhibitors, such as celecoxib, cytokines, such as IL-12, GM-CSF, IL-2 (having an amino acid sequence set forth in either SEQ ID NO: 33 or 34), IFN3 and IFN-g, and chemokines, such as MIP-1, MCP-1, and IL-8. In certain embodiments, immunomodulatory agents can include immune checkpoint modulators, such as, but not limited to, anti-CTLA4, anti-PD-1, and anti-PD-L1 and TLR agonists (e.g., poly I:C). In some examples, the immunomodulatory agent is an immune checkpoint inhibitor, e.g., an antagonist of PD-1 (e.g., an antagonist antibody that binds to PD-1), an antagonist of PD-L1 (e.g., an antagonist antibody that binds to PD-L1), an antagonist of CTLA-4 (e.g., an antagonist antibody that binds to CTLA-4), an antagonist of A2AR (e.g., an antagonist antibody that binds to A2AR), an antagonist of B7-H3 (e.g., an antagonist antibody that binds to B7-H3 ... The additional therapy can include an antagonist of 7-H4 (e.g., an antagonist antibody that binds to B7-H4), an antagonist of BTLA (e.g., an antagonist antibody that binds to BTLA), an antagonist of IDO (e.g., an antagonist antibody that binds to IDO), an antagonist of KIR (e.g., an antagonist antibody that binds to KIR), an antagonist of LAG3 (e.g., an antagonist antibody that binds to LAG3), an antagonist of TIM-3 (e.g., an antagonist antibody that binds to TIM3). In some embodiments, the additional therapy can include administering an immune checkpoint modulator.In one example, the immune checkpoint modulator can be TGN1412. In one example, the immune checkpoint modulator can be NKTR-214. In one example, the immune checkpoint modulator can be MEDI0562. In one example, the immune checkpoint modulator can be MEDI6469. In one example, the immune checkpoint modulator can be MEDI6383. In one example, the immune checkpoint modulator can be JTX-2011. In one example, the immune checkpoint modulator can be pembrolizumab. In one example, the immune checkpoint modulator can be nivolumab. In one example, the immune checkpoint modulator can be ipilimumab. In one example, the immune checkpoint modulator can be tremelimumab. In one example, the immune checkpoint modulator can be atezolizumab. In one example, the immune checkpoint modulator can be MGA271. In one example, the immune checkpoint modulator can be indoximod. In one example, the immune checkpoint modulator can be epacadostat. In one example, the immune checkpoint modulator can be lirilumab. In one example, the immune checkpoint modulator can be BMS-986016. In one example, the immune checkpoint modulator can be MPDL3280A. In one example, the immune checkpoint modulator can be avelumab. In one example, the immune checkpoint modulator can be durvalumab. In one example, the immune checkpoint modulator can be MEDI4736. In one example, the immune checkpoint modulator can be MEDI4737. In one example, the immune checkpoint modulator can be TRX518. In one example, the immune checkpoint modulator can be MK-4166. In one example, the immune checkpoint modulator can be urelumab (BMS-663513). In one example, the immune checkpoint modulator can be PF-05082566 (PF-2566).

[0075] In certain instances, when the additional therapy is radiation, an exemplary dose can be 5,000 Rad (50 Gy) to 100,000 Rad (1000 Gy), or 50,000 Rad (500 Gy), or other suitable dose within the recited range. Alternatively, the radiation dose can be about 30 to 60 Gy, about 40 to about 50 Gy, about 40 to 48 Gy, or about 44 Gy, or other suitable dose within the recited range, the dose being determined, for example, by dosimetry studies as described above. As used herein, "Gy" can refer to a unit for a specific absorbed dose of radiation equal to 100 Rad. Gy is an abbreviation for "Gray."

[0076] In certain examples where the additional therapy is chemotherapy, exemplary chemotherapeutic agents may include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethylenimines, alkylsulfonates, hydrazines and triazines, nitrosureas, and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins, and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, and the like), antimetabolites (e.g., folate antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and various antitumor agents (e.g., ribonucleotide reductase inhibitors, corticosteroid inhibitors, enzymes, antimicrotubule agents, and retinoids). Exemplary chemotherapeutic agents include, but are not limited to, anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5-fluorouracil, flutamide, tezamido. These may include thibavirin, gemcitabine (difluorodeoxycytidine), hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, leucovorin calcium, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, mylotarg, paclitaxel, phoenix, pentostatin, polypheprosan 20 with carmustine implant, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine, ibrutinib, idelalisib, and brentuximab vedotin.

[0077] Exemplary alkylating agents may include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas and triazenes, such as uracil mustard chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, temozolomide, thiotepa, busulfan, and dacarbazine. Additional exemplary alkylating agents include, but are not limited to, oxaliplatin, temozolomide, dactinomycin, L-PAM, L-sarcolysin, hexamethylmelamine, carmustine, bendamustine, busulfan; carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, altretamine, ifofamide, prednumstine, procarbazine, mechlorethamine, streptozocin, thiotepa, cyclophosphamide; and bendamustine HCl.

[0078] Exemplary anthracyclines can include, but are not limited to, for example, doxorubicin, bleomycin, daunorubicin, daunomycin, rubidomycin hydrochloride, mitoxantrone, epirubicin, idarubicin, mitomycin C, geldanamycin, herbimycin, rabidomycin, and desacetylrabidomycin.

[0079] Exemplary vinca alkaloids can include, but are not limited to, vinorelbine tartrate, vincristine, vindesine, vinblastine, and vinorelbine.

[0080] Exemplary proteasome inhibitors include, but are not limited to, bortezomib; carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamide). -4-phenylbutanamide)-pentanamide), marizomib (NPI-0052), ixazomib citrate, delanzomib, and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide.

[0081] "In combination" as used herein can mean that a recombinant virus, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising it, and an additional therapy, such as an additional therapy comprising one or more drugs, are administered to a subject as part of a treatment regimen or plan.In certain embodiments, using in combination may not require that the recombinant virus and the one or more drugs are physically combined before administration, or that they are administered over the same time frame.For example, but not limited to, the recombinant virus and the one or more drugs can be administered to the subject to be treated at the same time, or can be administered at the same time or in any order, or sequentially at different times.

[0082] The additional therapy may be administered in various embodiments in a liquid dosage form, a solid dosage form, a suppository, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof. In certain embodiments, the additional therapy is administered for a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer. The frequency of administration of the additional therapy may be once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months) in certain cases. In certain embodiments, the method of treating a subject with cancer may include administering to the subject an effective amount of a recombinant virus of the present disclosure, such as a recombinant vaccinia virus. In certain embodiments, the method of the present disclosure may further include administering to the subject an effective amount of one or more agents. For example, but not limited to, the agent may be an anti-cancer agent, an immunomodulatory agent, or any combination thereof, as described above. As used herein, an "anti-cancer agent" may be any molecule, compound, chemical, or composition that has an anti-cancer effect. The anti-cancer agent may include, but is not limited to, a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an immune checkpoint inhibitor, an anti-angiogenic agent, an apoptosis inducer, an anti-cancer antibody, and / or an anti-cyclin-dependent kinase agent. Additional Embodiments

[0083] Provided herein is a recombinant oncolytic virus comprising a modification in the viral genome, said modification comprising at least one of the following: deletion or functional deletion of an endogenous nucleic acid encoding an MHC class II inhibitor; insertion of an exogenous nucleic acid resulting in activation or enhanced activation of MHC class II presentation; and insertion of an exogenous nucleic acid encoding an MHC class I inhibitor acting entirely or mainly within infected cells.Further provided herein is a recombinant oncolytic virus comprising a deletion or functional deletion of an endogenous nucleic acid encoding an MHC class II inhibitor, said deletion or functional deletion resulting in increased MHC class II presentation, said oncolytic virus being a vaccinia virus.Further provided herein is a recombinant oncolytic virus comprising a deletion or functional deletion of an endogenous nucleic acid encoding an MHC class II inhibitor, said deletion or functional deletion of said endogenous nucleic acid encoding an MHC class II inhibitor comprising deletion of a gene encoding protein A35 of vaccinia virus. Further provided herein is a recombinant oncolytic virus, wherein the deletion or functional deletion of the gene encoding vaccinia virus protein A35 is a deletion or functional deletion of gene WR158.Further provided herein is a recombinant oncolytic virus, wherein the modification comprises the insertion of an exogenous nucleic acid that results in activation or enhanced activation of MHC class II presentation, and wherein the exogenous nucleic acid encodes an apoptosis inhibitor protein or a necrotic cell death activator protein; an autophagy enhancer protein; an asparaginyl endopeptidase; a class II transactivator; an interferon-gamma; a Toll-like receptor activator; or a dendritic cell maturation activator.Further provided herein is a recombinant oncolytic virus comprising an autophagy enhancer protein, wherein the autophagy enhancer protein is HMGB1 or a functional domain or variant thereof. Further provided herein is a recombinant oncolytic virus comprising a dendritic cell maturation activator, wherein the dendritic cell maturation activator comprises osteopontin, or TNF-alpha, or a functional fragment or variant thereof.Further provided herein is a recombinant oncolytic virus in which the encoded MHC II upregulatory protein is fused to a secretory sequence, a cell-penetrating domain, or a combination thereof, to achieve MHC II upregulation throughout the tumor. Further provided herein is a recombinant oncolytic virus in which the modification comprises the insertion of an exogenous nucleic acid encoding an MHC class I inhibitor, the insertion resulting in inhibition or partial inhibition of MHC class I presentation. Further provided herein is a recombinant oncolytic virus in which the insertion of an exogenous nucleic acid encoding an MHC class I inhibitor comprises the insertion of a gene encoding one or more cowpox virus proteins. Further provided herein is a recombinant oncolytic virus in which the insertion of an exogenous nucleic acid encoding an MHC class I inhibitor comprises the insertion of a gene encoding cowpox protein CPXV012 or a functional fragment or variant thereof. Further provided herein is a recombinant oncolytic virus in which the insertion of an exogenous nucleic acid encoding an MHC class I inhibitor comprises the insertion of a gene encoding cowpox protein CPXV203 or a functional fragment or variant thereof. Further provided herein is a recombinant oncolytic virus, wherein the exogenous nucleic acid encoding MHC class I inhibitor comprises the insertion of a gene encoding at least one of the following: Epstein-Barr virus-encoded nuclear antigen 1 protein; Herpes simplex virus-encoded ICP47 protein; Herpes simplex virus-encoded UL49.5 protein; Cytomegalovirus-encoded US6, US2, US3, US11 or gp48 protein; Epstein-Barr virus-encoded BNLF2a protein; Adenovirus-encoded E3-19K protein; Human immunodeficiency virus-encoded Nef protein; Kaposi's sarcoma-associated herpes virus-encoded kK3, vIRF3 or kK5 protein; or IRF7 or IRF3 dominant negative form.Further provided herein is a recombinant oncolytic virus, wherein the MHC class I inhibitor comprises a TAP inhibitor. Further provided herein are recombinant oncolytic viruses in which the TAP inhibitor acts entirely or primarily within the infected cell.Further provided herein is a recombinant oncolytic virus in which the modification in the viral genome reduces the immune response targeting the virus-infected tumor cells and increases the immune response targeting the cells surrounding the virus-infected tumor cells.Further provided herein is a recombinant oncolytic virus in which the thymidine kinase gene is deleted from the viral genome.Further provided herein is a recombinant oncolytic virus further comprising an exogenous nucleic acid encoding a hyaluronidase.Further provided herein is a recombinant oncolytic virus in which the hyaluronidase is PH-20 or HysA. Further provided herein is a recombinant oncolytic virus, wherein the oncolytic virus is a vaccinia virus, wherein the vaccinia virus is a Western Reserve strain vaccinia virus (ATCC VR-1354), a Copenhagen strain, an IHD strain, a Wyeth strain (ATCC VR-325), a NYCBOH strain, a Tian Tan strain, a Lister strain, an Ankara strain (ATCC VR-1508 or ATTC VR1566), a USSR strain, or an ACAM2000 strain.

[0084] Provided herein is a recombinant oncolytic virus comprising a modification in the viral genome, the modification comprising a deletion or functional deletion of the vaccinia virus gene encoding the A35 protein, and an insertion of an exogenous gene encoding the cowpox protein CPXV012 or the cowpox protein CPXV203, the modification being such that the insertion of the exogenous gene encoding the cowpox protein CPXV012 is at the locus of the gene encoding the A35 protein of the vaccinia virus. Provided herein is a recombinant oncolytic virus further comprising an additional modification in the viral genome. Provided herein is a recombinant oncolytic virus, the additional modification comprising at least one of the following: an insertion of an exogenous nucleic acid encoding a chemokine receptor or a functional domain or variant thereof; or an insertion of an exogenous nucleic acid encoding a cytokine or a functional domain or variant thereof. Further provided herein is a recombinant oncolytic virus comprising an exogenous nucleic acid encoding a cytokine or a functional domain or variant thereof, wherein the cytokine comprises at least one of interleukin-2 (IL-2), interleukin-15 / interleukin-15Ra (IL15 / IL15Ra), interleukin-7 (IL-7), or a functional domain or variant thereof. Further provided herein is a recombinant oncolytic virus, wherein the additional modification comprises the insertion of an exogenous nucleic acid encoding a fusion protein comprising a cytokine and a metabolic modulator protein. Further provided herein is a recombinant oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor or a functional domain or variant thereof, wherein the chemokine receptor comprises at least one of CXCR4, CCR2, or a functional domain or variant thereof. Further provided herein is a recombinant oncolytic virus, wherein the chemokine receptor comprises CXCR4 or a functional domain or variant thereof.Further provided herein is a recombinant oncolytic virus, wherein the chemokine receptor comprises CCR2 or a functional domain or variant thereof, and wherein the CCR2 comprises wild-type CCR2 or mutant CCR2. Further provided herein is a recombinant oncolytic virus, wherein the exogenous nucleic acid encoding the chemokine receptor or a functional domain or variant thereof comprises a codon-optimized sequence. Further provided herein is a recombinant oncolytic virus, wherein the exogenous nucleic acid encoding the chemokine receptor or a functional domain or variant thereof comprises a sequence that is not codon-optimized. Further provided herein is a recombinant oncolytic virus, wherein the additional modification comprises a mutation or a complete or partial deletion of a viral gene, comprising at least one of A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, VH1, or a functional domain or fragment or variant thereof, or any combination thereof, of vaccinia virus. Further provided herein is a recombinant oncolytic virus in which the thymidine kinase gene is deleted from the viral genome. Further provided herein is a recombinant oncolytic virus further comprising an exogenous nucleic acid encoding a hyaluronidase. Further provided herein is a recombinant oncolytic virus in which the hyaluronidase is PH-20 or HysA. Further provided herein is a recombinant oncolytic virus in which the oncolytic virus is a vaccinia virus, and the vaccinia virus is a Western Reserve strain vaccinia virus (ATCC VR-1354), a Copenhagen strain, an IHD strain, a Wyeth strain (ATCC VR-325), a NYCBOH strain, a Tian Tan strain, a Lister strain, an Ankara strain (ATCC VR-1508 or ATTC VR1566), a USSR strain, or an ACAM2000 strain.

[0085] Provided herein is an immunogenic composition comprising a recombinant oncolytic virus according to any embodiment described herein.

[0086] Provided herein is a pharmaceutical composition comprising a recombinant oncolytic virus or immunogenic composition according to any embodiment described herein and at least one of a solubilizing agent, an excipient, or a pharma- ceutically acceptable carrier. Further provided herein is a pharmaceutical composition wherein the excipient comprises one or more of a buffer, a stabilizer, an antioxidant, a binder, a diluent, a dispersant, a rate control agent, a lubricant, a glidant, a disintegrant, a plasticizer, a preservative, or any combination thereof. Further provided herein is a pharmaceutical composition wherein the excipient comprises disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, myo-inositol, sorbitol, or any combination thereof. Further provided herein is a pharmaceutical composition that does not contain a preservative. Further provided herein is a pharmaceutical composition further comprising one or more of a preservative, a diluent, and a carrier. Further provided herein is a pharmaceutical composition further comprising an additional active ingredient or a salt thereof. Further provided herein is a pharmaceutical composition wherein the solubilizing agent is sterile water. Further provided herein are pharmaceutical compositions further comprising an additional active ingredient which is an anti-cancer agent or an additional oncolytic virus.

[0087] Provided herein are methods of reducing the growth of cancer cells, exemplary methods include administering to the cancer cells an effective amount of a recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition according to any embodiment described herein.

[0088] Provided herein is a method of regressing tumor growth, an exemplary method comprising administering to the tumor an effective amount of a recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition according to any embodiment described herein. Further provided herein is a method in which the tumor is in a subject, and the administering comprises administering to the subject. Further provided herein is a method comprising administering an additional therapy, the additional therapy comprising chemotherapy, radiation therapy, oncolytic virus therapy with an additional virus, treatment with an immunomodulatory protein, CAR T cell therapy, an anti-cancer drug, or any combination thereof. Further provided herein is a method in which the additional therapy comprises administering an immunomodulatory agent comprising an anti-CD33 antibody and its variable region, an anti-CD11b antibody and its variable region, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, or a TLR agonist.

[0089] A method of treatment comprising administering an effective amount of a recombinant oncolytic virus, immunogenic composition or pharmaceutical composition according to any embodiment described herein to a subject in need thereof. Further provided herein is a method, wherein the administration comprises intratumoral administration. Further provided herein is a method, wherein the administration comprises systemic administration. Further provided herein is a method, wherein the systemic administration comprises at least one of intraperitoneal administration, oral administration, intravenous administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. Further provided herein is a method in which the subject has cancer, and the cancer is at least one of melanoma, hepatocellular carcinoma, breast cancer, lung cancer, non-small lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, renal cell carcinoma, pancreatic cancer, epithelial cancer, gastric / GE junction adenocarcinoma, cervical cancer, colon cancer, colorectal cancer, duodenal cancer, pancreatic adenocarcinoma, adenoid cyst, sarcoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or any combination thereof. Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered to a subject in need thereof at least about 10 6 PFU / mL~about 10 10 Further provided herein is a method in which the recombinant oncolytic virus or pharmaceutical composition is administered in a dosage containing about 3×10 PFU / mL of recombinant vaccinia virus. 9A method is provided herein, in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered in a dosage comprising PFU / mL of recombinant vaccinia virus. Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered independently at an initial dose for a first period of time, an intermediate dose for a second period of time, and a high dose for a third period of time. Further provided herein is a method comprising independently administering the initial, intermediate, and high doses, wherein the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose. Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered independently at a high dose for a first period of time, an intermediate dose for a second period of time, and a low dose for a third period of time. Further provided herein is a method comprising independently administering the initial, intermediate, and low doses, wherein the initial dose is higher than the intermediate dose, and the intermediate dose is higher than the low dose. Further provided herein are methods wherein the first, second, and third periods of time are, respectively, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year. Further provided herein are methods in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition independently comprises a liquid dosage form administered in a volume of about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL. Further provided herein are methods wherein the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered in a liquid dosage form, 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.Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year. Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered once a day, twice a day, once a week, once every two weeks, or once every three weeks. Further provided herein is a method in which the recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition is administered as a bolus injection or slow infusion. Further provided herein is a method, wherein administration of a recombinant oncolytic virus, immunogenic composition, or pharmaceutical composition results in a first peak viral load about 1 hour to about 3 days and a second peak viral load about 3 to about 10 days after administration of a first dose. Further provided herein is a method, comprising administering an additional therapy, wherein the additional therapy is administered for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 12 weeks. Further provided herein is a method in which the additional therapy is administered once a day, twice a day, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 12 weeks, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once a year.Further provided herein is a method in which the additional therapy is administered in a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof.Further provided herein is a method in which the additional therapy is administered in a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof. Further provided herein is a method in which the additional therapy is administered orally, intravenously, intratumorally, intraperitoneally, or by radiation. Further provided herein is a method in which the additional therapy comprises chemotherapy, radiation therapy, oncolytic virus therapy with additional viruses, treatment with immunomodulatory proteins, CAR T cell therapy, anticancer drugs, or any combination thereof. Further provided herein is a method in which the additional therapy comprises administering an immunomodulatory agent comprising an anti-CD33 antibody and its variable region, an anti-CD11b antibody and its variable region, a COX2 inhibitor, a cytokine, a chemokine, an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, or a TLR agonist. Further provided herein is a method, wherein the additional therapy comprises administration of an anti-cancer agent, and the anti-cancer agent is a chemotherapeutic agent. Further provided herein is a method, wherein the subject is a human. EXAMPLES

[0090] The following examples further illustrate the described embodiments without limiting the scope of the disclosure. Example 1 Tumor models

[0091] The recombinant vaccinia viruses were assayed in tumor model systems in comparison to the same virus strains without the modifications described herein. The table below lists the viruses used in the study and the modifications in their viral genomes. Table 1 [Table 1]

[0092] Table 1 above shows that the viruses used in this study were recombinant vaccinia virus WO0434N (designated as A52RmutCXCR4 TK-158-cpx012+) and reference vaccinia virus WO0416N (A52RmutCXCR4 TK-). Modifications to WO0434N were by deletion of the TK gene and replacement of the A52 gene with the P7.5-inducible mutant CXCR4, and replacement of the A35 gene (WR158) with the cowpox virus gene CPXV012. Reference vaccinia virus WO0416N had deletion of the TK gene and replacement of the A52 gene with the P7.5-inducible mutant CXCR4. Controls were the Western Reserve thymidine kinase negative (WR.TK-) strain of vaccinia virus (HCCTKM) and vehicle formulation buffer (VFB). The nucleic acid sequence of the P7.5 promoter is defined by SEQ ID NO: 1. The nucleic acid sequence of the mutant CXCR4 gene is defined by SEQ ID NO: 15. The nucleic acid sequence of CPXV012 is defined by SEQ ID NO: 2. The study was carried out in a B16 tumor model. Tumor volumes were measured 17 days after virus administration for each group and control. The results shown in Figure 1 show enhanced therapeutic activity with WO0434N virus compared to the reference virus WO0416N or the controls HCCTKM and VFB. Example 2 Strain characterization

[0093] To assess whether a given virus is replication-competent or replication-deficient in a cell type, the replication capacity of recombinant vaccinia viruses is assessed using serum from mammals or rodents infected with the recombinant virus, via PCR assays, viral plaque assays, or any combination thereof. In viral plaque assays, confluent monolayers of susceptible cells in tissue culture flasks are infected with vaccinia virus. After incubation, cytopathic effect (CPE) is observed, visualized through the formation of halos or circles, and the cell monolayer is removed. The cell medium is replaced with a solution that increases viscosity. The replaced solution includes gelatin or carboxymethylcellulose. Viral plaque assays are visualized by staining with an agent that increases cell contrast by eye or microscope. Post-infection incubation can be 4-48 hours, during which plaques can be observed. The staining agent is crystal violet. In PCR-based assays, vaccinia virus content is quantified using a qPCR-based approach. Example 3 Tumor growth inhibition

[0094] Animal assays were performed in mouse models of cancer to evaluate the tumor growth impact of the compositions described herein. Briefly, modified vaccinia viruses were intratumorally injected (IT) into Renca and EMT6 tumor-bearing mice.

[0095] The modified vaccinia viruses were evaluated in comparison to the vehicle formulation buffer (VFB). Group 1 was treated with a modified vaccinia virus containing a TK gene deletion, an insertion of a nucleic acid (SEQ ID NO:2) encoding the cowpox virus V012 protein (CPXV012), a P7.5 promoter (SEQ ID NO:1) and a loxP sequence (SEQ ID NO:35). Group 2 was treated with a modified vaccinia virus containing a WR158 gene deletion, an insertion of a nucleic acid (SEQ ID NO:2) encoding the CPXV012 protein, a P7.5 promoter (SEQ ID NO:1), and a loxP sequence (SEQ ID NO:35). Group 3 was treated with a vaccinia virus containing a TK gene deletion and an insertion of a nucleic acid (dnIRF7, SEQ ID NO:36) encoding a dominant negative interferon regulatory factor 7. Group 4 was treated with a modified vaccinia virus containing a TK gene deletion and an insertion of a nucleic acid (vIRF3, SEQ ID NO:38) encoding a viral interferon regulatory factor 3.

[0096] Balb / c mice were subcutaneously implanted with RENCA or EMT6 cell tumors. Mice were divided into 10 groups. Tumors were 1×10 7 pfu of modified vaccinia virus or vehicle control were injected at a single dose.

[0097] Tumor volumes in Renca tumor-bearing mice were measured after 23 days, as shown in Figure 2 A. Groups treated with cowpox virus V012 and virus expressing vIRF3 showed the most effective reduction in Renca tumor volume.

[0098] As shown in Figure 2B, tumor volumes in EMT6 tumor-bearing mice were measured after 27 days. The groups treated with cowpox virus V012 and the virus expressing dnIRF7 showed the most effective reduction in EMT6 tumor volume.

[0099] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions may now be made by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. Table 2: Sequences [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12]

Claims

1. A composition comprising an oncolytic virus, wherein the oncolytic virus comprises a genomic modification, the genomic modification comprises an exogenous nucleic acid encoding an MHC I inhibitor, and the MHC I inhibitor comprises vIRF3.

2. The genome modification a) a deletion or functional deletion of an endogenous nucleic acid encoding an MHC II inhibitor; or b) Exogenous nucleic acids that result in activation or enhanced activation of MHC II presentation The composition of claim 1 further comprising:

3. The composition of claim 2, wherein the deletion or functional deletion of the endogenous nucleic acid encoding the MHC II inhibitor comprises a deletion or functional deletion of a vaccinia virus gene encoding protein A35.

4. The composition described in claim 3, wherein the deletion or functional deletion of the vaccinia virus gene encoding protein A35 is a deletion or functional deletion of gene WR158.

5. The exogenous nucleic acid that results in activation or enhanced activation of MHC II presentation, a) apoptosis inhibitor proteins; b) necrotic cell death activator protein; c) autophagy enhancer proteins; d) asparaginyl endopeptidase; e) class II transactivators; f) interferon gamma; g) a Toll-like receptor activator; or h) Dendritic cell maturation activator The composition of claim 2 , which encodes a protein selected from:

6. The composition described in claim 5, wherein the exogenous nucleic acid that brings about activation or enhanced activation of the MHC II presentation encodes the autophagy enhancer protein, and the autophagy enhancer protein is HMGB1 or a functional domain or variant thereof.

7. The composition of claim 5, wherein the exogenous nucleic acid that effects activation or enhanced activation of MHC II presentation encodes a dendritic cell maturation activator, the dendritic cell maturation activator comprising osteopontin, TNF-alpha, or a functional fragment or variant thereof.

8. The composition described in claim 5, wherein the protein encoded by the exogenous nucleic acid is fused to a secretory sequence, a cell-permeable domain, or a combination thereof.

9. The composition of claim 1, wherein the oncolytic virus comprises a poxvirus, an adeno-associated virus, an adenovirus, a Newcastle disease virus (NDV), a reovirus (RV), a mengovirus, a myxoma virus (MYXV), a measles virus (MV), a herpes simplex virus (HSV), a vaccinia virus (VV), a vesicular stomatitis virus (VSV), and a poliovirus (PV).

10. The composition of claim 9, wherein the poxvirus comprises a betaentomopoxvirus, a yatapoxvirus, a cerbidopoxvirus, a gammaentomopoxvirus, a leporipoxvirus, a suipoxvirus, a molluscuspoxvirus, a crocodylidopoxvirus, an alphaentomopoxvirus, a capripoxvirus, an avipoxvirus, or a parapoxvirus.

11. The composition described in claim 1, wherein the oncolytic virus is a vaccinia virus.

12. The composition of claim 1, wherein the MHC I inhibitor causes inhibition or partial inhibition of MHC I presentation.

13. The composition described in claim 1, wherein the genomic modification reduces an immune response targeted to virally-infected tumor cells and increases an immune response targeted to cells surrounding the virally-infected tumor cells.

14. The composition of claim 1, wherein the genomic modification further comprises a mutation or complete or partial deletion of a viral gene comprising at least one of A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, or VH1 of vaccinia virus, or a functional domain or fragment or variant thereof, or any combination thereof.

15. The composition described in claim 1, wherein the genomic modification further comprises a deletion of a thymidine kinase gene.

16. The composition of claim 1, wherein the oncolytic virus is a vaccinia virus, the vaccinia virus being Western Reserve strain vaccinia virus (ATCC VR-1354), Copenhagen strain, IHD strain, Wyeth strain (ATCC VR-325), NYCBOH strain, Tian Tan strain, Lister strain, Ankara strain (ATCC VR-1508 or ATCC VR1566), USSR strain, or ACAM2000 strain.

17. A pharmaceutical composition comprising the composition of claim 1 and a pharma- ceutically acceptable excipient.

18. The pharmaceutical composition of claim 17, wherein the excipient comprises one or more of a buffer, stabilizer, antioxidant, binder, diluent, dispersant, rate control agent, lubricant, glidant, disintegrant, plasticizer, preservative, or any combination thereof.

19. The pharmaceutical composition of claim 17, wherein the excipient comprises disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, myo-inositol, sorbitol, or any combination thereof.

20. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition does not contain a preservative.

21. The pharmaceutical composition of claim 17, further comprising one or more of a preservative, a diluent, and a carrier.

22. The pharmaceutical composition of claim 17, further comprising an additional active ingredient or salt thereof.

23. The pharmaceutical composition of claim 17, wherein the excipient is sterile water.

24. The pharmaceutical composition of claim 17, further comprising an additional active ingredient, wherein the additional active ingredient is an anticancer agent or a further oncolytic virus.

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