Oncolytic vaccinia viruses, recombinant viruses and methods of use thereof

Abstract

This invention relates to oncolytic vaccinia viruses, recombinant viruses thereof, and methods of use. Specifically, the invention provides vaccinia virus clone lines and recombinant vaccinia viruses derived therefrom, said vaccinia virus clone lines exhibiting enhanced antitumor properties and / or reduced immunogenicity. The invention also provides recombinant oncolytic virus lines comprising inactivating mutations in one or more viral genes, and / or one or more heterologous nucleic acids each encoding one or more heterologous gene products. The viruses provided by this invention (such as vaccinia viruses, including recombinant vaccinia viruses) can be used as oncolytic virus therapies (such as oncolytic vaccinia virus therapy) for the treatment of cancer. The invention also provides pharmaceutical compositions of viruses (such as vaccinia viruses), methods and uses of viruses (such as vaccinia viruses) for the treatment of cancer, and nucleic acids encoding said viruses.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202380064607.3, filed on July 7, 2023, entitled "Oncolytic Vaccine Virus and Recombinant Virus Thereof and Method of Use".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 368,029, filed July 8, 2022, entitled “ONCOLYTIC VACCINIA VIRUSES AND RECOMBINANT VIRUSES AND METHODS OF USE THEREOF,” which is incorporated herein by reference in its entirety for all purposes.

[0004] By referring to the merged sequence list

[0005] This application is submitted together with the accompanying electronic sequence list. The sequence list is provided as a file named 773192000140SeqList.xml, created on July 6, 2023, and is 6,262,578 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0006] This disclosure provides clonal lines of vaccinia virus exhibiting enhanced antitumor properties and / or reduced immunogenicity, as well as recombinant vaccinia viruses derived therefrom. The vaccinia viruses of this disclosure, including recombinant vaccinia viruses, can be used as oncolytic vaccinia virus therapy for the treatment of cancer. This disclosure also provides pharmaceutical compositions, methods, and uses of vaccinia viruses for the treatment of cancer. Background Technology

[0007] Vaccines are oncolytic viruses that accumulate in tumors. In some cases, an oncolytic virus (OV) refers to a virus that replicates selectively or more efficiently in cancer cells than in non-cancer cells. Oncolytic vaccinia viruses include recombinant viruses that are engineered from natural viruses through gene disruption or gene addition to improve their antitumor properties, such as tumor selectivity or preferential replication in tumor cells, host tropism, surface attachment, lysis, and spread. Such recombinant vaccinia viruses include attenuated viruses in which one or more viral genes are modified to result in the loss or reduction of viral gene expression or inactivation of viral proteins. However, the effectiveness of oncolytic viruses is hampered by the strong immune response induced by the virus. Immune factors such as antibodies neutralize the virus by directly binding to it and preventing successful viral infection of cells, or by labeling the virus so that complement or other immune cells can destroy it. Therefore, there is still a need for improved oncolytic vaccinia viruses that have reduced ability to induce antiviral defense while possessing enhanced antitumor activity. Summary of the Invention

[0008] This article provides a recombinant oncolytic vaccinia virus comprising: an inactivating mutation of B2R; a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3); and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines.

[0009] In some embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines comprises a heteronucleotide encoding chemokine ligand 9 (CXCL9) and / or IL-12. In some of any such embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines is a heteronucleotide encoding chemokine ligand 9 (CXCL9). In some of any such embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines is a heteronucleotide encoding IL-12. In some of any embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines is a heteronucleotide encoding both CXCL9 and IL-12. In some embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines is a heteronucleotide encoding both CXCL9 and IL-12.

[0010] In some embodiments: the CXCL9 is human CXCL9. In some embodiments, the CXCL9 comprises the amino acid sequence shown in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 99. In some embodiments, the amino acid sequence of the CXCL9 is shown in SEQ ID NO: 99. In some embodiments, the CXCL9 is mouse CXCL9. In some embodiments, the CXCL9 comprises the amino acid sequence shown in SEQ ID NO: 106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the amino acid sequence of the CXCL9 is shown in SEQ ID NO: 106.

[0011] In some embodiments: the IL-12 is human single-chain IL-12. In some embodiments, the single-chain IL-12 is composed of human IL-12A (p35) and human IL-12B (p40) subunits, optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence shown in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 103. In some embodiments, the amino acid sequence of the single-chain IL-12 is shown in SEQ ID NO: 103. In some embodiments, the IL-12 is mouse single-chain IL-12. In some embodiments, the single-chain IL-12 is composed of mouse IL-12A (p35) and mouse IL-12B (p40) subunits, optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence shown in SEQ ID NO: 102, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 102. In some embodiments, the amino acid sequence of the single-chain IL-12 is shown in SEQ ID NO: 102.

[0012] In some embodiments, at least one heteronucleotide encoding one or more cytokines and / or chemokines comprises a heteronucleotide encoding IL-2. In some embodiments, the IL-2 comprises the amino acid sequence shown in any of SEQ ID NO: 98, 100, 101, 104, and 105, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 98, 100, 101, 104, and 105. In some embodiments, the IL-2 is shown in SEQ ID NO: 105. In some embodiments, the IL-2 is a superfactor of the sequence shown in SEQ ID NO: 105 or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 105.

[0013] In some embodiments, the IL-2 is an IL-2 superfactor. In some embodiments, the IL-2 superfactor is H9, H9T, mDNA11, or mDNA11T. In some embodiments, the H9 IL-2 superfactor comprises the amino acid sequence shown in SEQ ID NO: 100, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100. In some embodiments, the H9T IL-2 superfactor comprises the amino acid sequence shown in SEQ ID NO: 104, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 104. In some embodiments, the mDNA11 IL-2 superfactor comprises the amino acid sequence shown in SEQ ID NO: 101, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 101. In some embodiments, the mDNA11T IL-2 superfactor comprises the amino acid sequence of SEQ ID NO: 98, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 98. In some embodiments, the IL-2 superfactor is mDNA11T, and the mDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 98.

[0014] In some embodiments, the recombinant oncolytic virus further comprises one or more heterologous gene products selected from the group consisting of complement inhibitors, T-cell or NK-cell escapes, immunostimulatory proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0015] In some embodiments, the inactivating mutation of B2R is a deletion of all or part of the B2R locus. In some embodiments, the deletion is sufficient to render the encoded B2R gene product nonfunctional. In some embodiments, the inactivating mutation of B2R is a substitution of one or more amino acids in the encoded gene product. In some embodiments, the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid into the B2R locus, for example, replacing a deletion of all or part of the B2R locus. In some embodiments, the heterologous nucleic acid encodes IRF3 or a cytokine and / or chemokine. In some embodiments, the inactivating mutation of B2R is achieved by inserting a heterologous nucleic acid encoding IRF3 and / or inserting at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines into the B2R locus. In some embodiments, the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12 into the B2R locus.

[0016] In some implementations, a heterologous nucleic acid encoding IRF3 is inserted into the viral genome at the following loci: hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L. In some of any embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is inserted into the HA, J2R, F14.5L, A56R, vaccinia growth factor, A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L locus in the viral genome. In some of any such embodiments, the insertion replaces a deletion of all or part of the corresponding locus.

[0017] In some embodiments, the recombinant oncolytic vaccinia virus genome is derived from a parental vaccinia virus that has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant oncolytic vaccinia virus genome is derived from a parental vaccinia virus that has the nucleic acid genome shown in SEQ ID NO: 1.

[0018] In some of any embodiments, the nucleic acid genome of the parental vaccinia virus is characterized by one or more of the following: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO:57 and containing a polar, uncharged amino acid at position 66, optionally containing a threonine (T) amino acid sequence at position 66; (ii) a variant 038 (K5L) ORF encoding a nucleotide insertion to cause a frameshift mutation, wherein the product of the 038 (K5L) gene is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO:60 and containing a hydrophobic amino acid other than leucine at position 419, optionally containing a phenylalanine (F) amino acid sequence at position 419; (iv) a variant 104 (H4L) ORF encoding a amino acid sequence having at least 95% sequence identity with SEQ ID NO:60 and containing a hydrophobic amino acid other than leucine at position 419; and (iv) a variant 104 (H4L) ORF encoding a amino acid sequence having at least 95% sequence identity with SEQ ID NO:60 and containing a hydrophobic amino acid other than leucine at position 419. (H4L) ORF encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and containing a negatively charged amino acid at position 591, optionally containing an amino acid sequence of aspartic acid (D) at position 591; and (v) variant 182 (A56R) ORF, which contains a two-nucleotide deletion to cause a frameshift mutation, wherein the gene product of the 182 (A56R) ORF is altered.

[0019] In some of any embodiments, the nucleic acid genome of the parent virus is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (viii) the nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (ix) Adenine (A) at position 162715 of SEQ ID NO: 1; (ix) the nucleic acid sequence TACAGACACC at positions 165844 to 185853 of SEQ ID NO: 1; and (x) C at position 187805 of SEQ ID NO: 1.

[0020] In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

[0021] In some of any embodiments: a heterologous nucleic acid encoding IRF3 is inserted into the J2R (thymidine kinase) locus in the viral genome; and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding CXCL9 and IL-12, wherein the heterologous nucleic acid encoding CXCL9 and IL-12 is inserted into the A56R locus in the viral genome.

[0022] In some embodiments, the nucleotide genome of the recombinant oncolytic vaccinia virus comprises the nucleotide sequence of SEQ ID NO: 85, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 85. In some embodiments, the nucleotide genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 85.

[0023] In some of any embodiments, the heterologous nucleic acid encoding IRF3 is inserted into the B2R (viral cGAMP-specific nuclease) locus in the viral genome; and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises heterologous nucleic acids encoding CXCL9 and IL-12, wherein the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus in the viral genome.

[0024] In some embodiments, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding an apoptosis-inducing protein. In some embodiments, the apoptosis-inducing protein is an inducible death effector domain (iDED). In some embodiments, the iDED comprises the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the iDED is shown in SEQ ID NO: 27. In some embodiments, the heterologous nucleic acid encoding the iDED is inserted into or replaces the J2R locus in the viral genome.

[0025] In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises the nucleic acid sequence of SEQ ID NO: 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 86. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 86.

[0026] In some embodiments, the recombinant oncolytic vaccinia virus also includes a heterologous nucleic acid encoding one or more T-cell or NK-cell escape proteins. In some embodiments, the one or more T-cell or NK-cell escape proteins comprise a set of proteins encoded by vaccinia virus ORF 012, 203, and 018 (CPXV012-203-018). In some embodiments, a group of proteins encoded by CPXV012-203-018 comprises: (i) the amino acid sequence shown in SEQ ID NO: 20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; (ii) the amino acid sequence shown in SEQ ID NO: 21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iii) the amino acid sequence shown in SEQ ID NO: 22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. The amino acid sequence shown in 22 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0027] In some embodiments, the recombinant oncolytic vaccinia virus also comprises a heterologous nucleic acid encoding a complement inhibitor. In some embodiments, the complement inhibitor is Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2). In some embodiments, the heterologous nucleic acid encoding CRASP-2 is fused to a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion protein comprises CRASP-2 fused to a viral membrane protein encoded by the viral membrane gene. In some embodiments, the viral membrane protein is F14.5L. In some embodiments, the fusion is performed at the C-terminus of F14.5L.

[0028] In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises the nucleic acid sequence of SEQ ID NO: 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 90. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 90.

[0029] In some of any embodiments, the heterologous nucleic acid encoding IRF3 is inserted into or replaces the B2R (viral cGAMP-specific nuclease) locus in the viral genome; and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines contains a heterologous nucleic acid encoding IL-2, wherein IL-2 is an IL-2 superfactor, which is MDNA11T.

[0030] In some embodiments, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding an immunostimulatory protein and / or a heterologous nucleic acid encoding one or more anti-angiogenic proteins. In some embodiments, the immunostimulatory protein is recombinant LIGHT. In some embodiments, recombinant LIGHT comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, recombinant LIGHT has the sequence shown in SEQ ID NO: 30.

[0031] In some embodiments, one or more anti-angiogenic proteins comprise a VEGF inhibitor, an angiopoietin inhibitor, versikine, or a fusion protein of any two or more of the foregoing. In some embodiments, the one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody. In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 23. In some embodiments, the bispecific anti-VEGF / anti-Ang2 antibody has the sequence shown in SEQ ID NO: 23.

[0032] In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises the nucleic acid sequence of SEQ ID NO: 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 88. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 88.

[0033] In some embodiments, one or more heteronucleotides encoding any of the aforementioned heterogeneous gene products (e.g., IRF3, cytokines, chemokines, or other heterogeneous gene products) are operatively linked to a promoter. In some embodiments, each of the one or more heteronucleotides encoding a heterogeneous gene product is operatively linked to a promoter. In some embodiments, the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, each heteronucleotide encoding a heterogeneous gene product is independently operatively linked to a promoter, optionally wherein each heteronucleotide encoding a heterogeneous gene product is independently operatively linked to a promoter selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter is a poxvirus promoter or a variant or derivative thereof. In some embodiments, the promoter is a vaccinia virus promoter. In some embodiments, the promoter is selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter has the amino acid sequence shown in any one of SEQ ID NO: 29, 53, 55, 68, 69, 70, 71, or 72. In some embodiments, the promoter is a synthetic strong early promoter (SSE). In some embodiments, the promoter comprises the sequence shown in SEQ ID NO: 29. In some embodiments, the promoter is a strong early / late promoter (SEL). In some embodiments, the promoter comprises the sequence shown in SEQ ID NO: 55. In some embodiments, the promoter is mH5. In some embodiments, the mH5 promoter comprises the sequence shown in SEQ ID NO: 53.

[0034] This article also provides a recombinant oncolytic virus comprising: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0035] In some implementations, oncolytic viruses include vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, orf virus, parvovirus, raccoonpox virus, coxsackie virus, reovirus, Newcastle disease virus, Seneca valley virus, Semliki Forest virus, mumps virus, influenza virus, echovirus, and poliovirus (PV).

[0036] In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the nucleotide genome of the recombinant oncolytic vaccinia virus is derived from a parental vaccinia virus that has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide genome of the recombinant oncolytic vaccinia virus is derived from a parental vaccinia virus that has a nucleotide genome having the nucleotide genome shown in SEQ ID NO: 1.

[0037] This article also provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise complement inhibitors, T-cell or NK-cell escapes, immunomodulatory proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0038] This document also provides a recombinant oncolytic virus comprising: a nucleic acid genome modified from a parental vaccinia virus genome having at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1; and a heterologous nucleic acid comprising at least one heterologous nucleic acid encoded by an insertion into the genome, encoding one or more heterologous gene products.

[0039] In some of any embodiments, the nucleic acid genome of the parental vaccinia virus is characterized by one or more of the following: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and containing a polar, uncharged amino acid at position 66, optionally containing a threonine (T) amino acid sequence at position 66; (ii) a variant 038 (K5L) ORF encoding a nucleotide insertion to cause a frameshift mutation, wherein the product of the 038 (K5L) gene is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and containing a hydrophobic amino acid other than leucine at position 419, optionally containing a phenylalanine (F) amino acid sequence at position 419; (iv) a variant 104 (H4L) ORF encoding a amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and containing a hydrophobic amino acid other than leucine at position 419; and (iv) a variant 104 (H4L) ORF encoding a amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and containing a hydrophobic amino acid other than leucine at position 419. (H4L) ORF encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and containing a negatively charged amino acid at position 591, optionally containing an amino acid sequence of aspartic acid (D) at position 591; and (v) variant 182 (A56R) ORF, which contains a two-nucleotide deletion to cause a frameshift mutation, wherein the gene product of the 182 (A56R) ORF is altered.

[0040] In some of any embodiments, the parental vaccinia virus genome is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (viii) the nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (ix) Adenine (A) at position 162715 of SEQ ID NO: 1; (ix) the nucleic acid sequence TACAGACACC at positions 165844 to 185853 of SEQ ID NO: 1; and (x) C at position 187805 of SEQ ID NO: 1.

[0041] In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

[0042] In some of any embodiments, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus is characterized by one or more of the following: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and containing a polar, uncharged amino acid at position 66, optionally containing a threonine (T) amino acid sequence at position 66; (ii) a variant 038 (K5L) ORF containing a nucleotide insertion to cause a frameshift mutation, wherein the product of the 038 (K5L) gene is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and containing a hydrophobic amino acid other than leucine at position 419, optionally containing a phenylalanine (F) amino acid sequence at position 419; (iv) Variant 104 (H4L) ORF, which encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and containing a negatively charged amino acid at position 591, optionally containing an amino acid sequence of aspartic acid (D) at position 591; and (v) Variant 182 (A56R) ORF, which contains a two-nucleotide deletion to cause a frameshift mutation, wherein the gene product of the 182 (A56R) ORF is altered.

[0043] In some embodiments, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (viii) The nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (ix) The nucleic acid sequence TACAGACACC at positions 165844 to 185853 of SEQ ID NO: 1; and (x) C at position 187805 of SEQ ID NO: 1.

[0044] In some of any embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into a non-essential gene or region in the viral genome. In some of any such embodiments, the insertion replaces all or part of the deletion of the gene or region.

[0045] In some of any embodiments, at least one of the heterologous nucleic acids encoding one or more heterologous gene products is inserted into the genome of the virus into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L locus. In some of any embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products from a non-essential gene or region in the viral genome is independently inserted into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L locus in the viral genome. In some of any embodiments, at least one viral gene comprises one or more viral genes selected from hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L, and any combination thereof. In some of any such embodiments, an insertion replaces all or part of the deletion of the corresponding locus.

[0046] In some of any embodiments, at least one viral locus inserted in at least one heterologous nucleic acid is or comprises: (i) B2R; (ii) A35R; (iii) A35R and J2R; (iv) J2R; (v) B2R and J2R; (vi) A35R, B2R and J2R; (vii) B2R, J2R and A56R; or (viii) A35R, B2R, J2R and A56R.

[0047] In some embodiments, one or more inactivating mutations in at least one viral gene are independently achieved by: insertion of at least one heterologous nucleic acid encoding one or more heterologous gene products; deletion of all or part of at least one viral gene; and / or substitution of one or more nucleic acids in at least one viral gene. In some embodiments, the inactivating mutation in one or more of at least one viral gene is achieved by insertion of at least one heterologous nucleic acid encoding one or more heterologous gene products and deletion of all or part of at least one viral gene, wherein the insertion replaces the deletion of all or part of the viral gene.

[0048] In some embodiments, the inactivating mutation is the deletion of all or part of at least one viral gene. In some embodiments, the deletion of at least one viral gene is the deletion of the entire ORF of the viral gene. In some embodiments, the deletion is sufficient to render the encoded viral gene product nonfunctional. In some embodiments, an inactivating mutation of one or more of at least one viral gene is characterized by the insertion of at least one of the heterologous nucleic acids encoding one or more heterologous gene products into a viral locus. In some embodiments, the at least one viral gene comprises B2R. In some embodiments, the at least one viral gene comprises J2R. In some embodiments, the at least one viral gene comprises A35R. In some embodiments, the at least one viral gene comprises A56R. In some embodiments, the at least one viral gene comprises B2R, J2R, and A35R. In some embodiments, the at least one viral gene comprises B2R, J2R, A35R, and A56R. In some embodiments, the at least one viral gene comprises B2R, J2R, and A56R.

[0049] In some of any implementations: at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces F14.5L; and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces A35R; and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces J2R.

[0050] In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more immunomodulatory proteins. In some embodiments, an inactivating mutation in one or more of at least one viral gene is achieved by inserting one or more heteronucleotides each encoding one or more immunomodulatory proteins. In some embodiments, one or more immunomodulatory proteins comprise one or more immunostimulatory proteins. In some embodiments, one or more immunomodulatory proteins comprise one or more cytokines and / or chemokines. In some embodiments, one or more immunomodulatory proteins comprise one or more interferon regulatory factors. In some embodiments, the interferon regulatory factor is IRF3. In some embodiments, one or more interferon regulatory factors are or comprise interferon regulatory factor 3 (IRF3). In some embodiments, one or more immunomodulatory proteins comprise interferon regulatory factor 3 (IRF3) and one or more cytokines and / or chemokines.

[0051] In some embodiments, the one or more immunomodulatory proteins comprise one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments: CXCL9 is human CXCL9. In some embodiments: CXCL9 is human CXCL9 and comprises the amino acid sequence shown in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 99. In some embodiments, CXCL9 is mouse CXCL9. In some embodiments, CXCL9 is mouse CXCL9 and comprises the amino acid sequence shown in SEQ ID NO: 106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 106.

[0052] In some embodiments: IL-12 is human single-chain IL-12. In some embodiments: IL-12 is human single-chain IL-12 and comprises the amino acid sequence shown in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 103. In some embodiments: IL-12 is mouse single-chain IL-12. In some embodiments: IL-12 is mouse single-chain IL-12 and comprises the amino acid sequence shown in SEQ ID NO: 102, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 102.

[0053] In some embodiments, one or more immunomodulatory proteins comprise IRF3. In some embodiments, IRF3 is human IRF3 (hIRF3). In some embodiments, hIRF3 comprises the amino acid sequence shown in SEQ ID NO: 51, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 51. In some embodiments, IRF3 is mouse IRF3 (mIRF3). In some embodiments, mIRF3 comprises the amino acid sequence shown in SEQ ID NO: 52, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 52.

[0054] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 49, 50, 80, 82 and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 49, 50, 80, 82 and 84-93.

[0055] In some embodiments, one or more immunomodulatory proteins comprise IRF3 and one or more immunomodulatory proteins selected from the group consisting of LIGHT, IL-2, IL-12, and CXCL9. In some embodiments, one or more immunomodulatory proteins comprise IL-2. In some embodiments, one or more immunomodulatory proteins comprise IL-12. In some embodiments, one or more immunomodulatory proteins comprise LIGHT. In some embodiments, one or more immunomodulatory proteins comprise CXCL9. In some embodiments, one or more immunomodulatory proteins are or comprise: (i) IRF3; (ii) LIGHT; (iii) IRF3 and LIGHT; (iv) IRF3 and IL-2; (v) IRF3, CXCL9, and IL-12; (vi) IRF3, LIGHT, and IL-2; (vii) IRF3 and CXCL9; or (viii) IRF3, CXCL9, and IL-2.

[0056] In some embodiments, IL-2 is human IL-2. In some embodiments, IL-2 is an IL-2 superfactor. In some embodiments, the IL-2 superfactor is H9, H9T, mDNA11, or mDNA11T. In some embodiments: the H9 IL-2 superfactor comprises the amino acid sequence of SEQ ID NO: 100, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100. In some embodiments, the H9T IL-2 superfactor comprises the amino acid sequence of SEQ ID NO: 104, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 104. In some embodiments, the MDNA11 IL-2 superfactor comprises the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 101. In some embodiments, the MDNA11T IL-2 superfactor comprises the amino acid sequence of SEQ ID NO: 98, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 98. In some embodiments, the IL-2 superfactor is MDNA11 or MDNA11T. In some embodiments, the IL-2 superfactor is MDNA11T, and MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 98.

[0057] In some embodiments, LIGHT is recombinant LIGHT. In some embodiments, recombinant LIGHT is human LIGHT protein or a variant thereof. In some embodiments, recombinant LIGHT comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, recombinant LIGHT is a human LIGHT mutant (hmLIGHT), which is a human LIGHT mutant combining human and mouse LTβR and HVEM. In some embodiments, recombinant LIGHT comprises one or more mutations selected from threonine at position 138, glycine at position 160, glycine at position 221, and lysine at position 222. In some embodiments, recombinant LIGHT comprises the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25. In some of any implementations, the recombinant LIGHT comprises the sequence shown in SEQ ID NO: 25.

[0058] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 11, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 11, 82, 87 and 88.

[0059] In some embodiments, IL-12 is human IL-12. In some embodiments, human IL-12 is human single-chain IL-12 (hscIL-12). In some embodiments, hscIL-12 comprises the amino acid sequence shown in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 103.

[0060] In some embodiments, CXCL9 is human CXCL9. In some embodiments, human CXCL9 comprises the amino acid sequence shown in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 99.

[0061] In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding an apoptosis-inducing protein. In some embodiments, an inactivating mutation in one or more of at least one viral gene is achieved by inserting one or more heteronucleotides each encoding an apoptosis-inducing protein. In some embodiments, the apoptosis-inducing protein comprises a pro-apoptotic molecule fused to an FKBP variant capable of binding a dimerizing chemical inducer (CID). In some embodiments, the FKBP variant is FKBP-F36V. In some embodiments, FKBP-F36V comprises the amino acid sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56.

[0062] In some embodiments, the chemical inducer of dimerization is AP1903 (Rimiducid). In some embodiments, the pro-apoptotic molecule is or comprises Fas, a death effector domain (DED) containing a Fas-associated death domain protein (FADD), or caspase, optionally wherein the caspase is caspase 9. In some embodiments, the apoptosis-inducing protein is an inducible DED (iDED). In some embodiments, the iDED comprises the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 8 or 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 8 or 86.

[0063] In some embodiments, the apoptosis-inducing protein is inducible Fas (iFas). In some embodiments, iFas comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 9, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 9.

[0064] In some embodiments, the apoptosis-inducing protein is inducible caspase 9 (iCas9). In some embodiments, iCas9 comprises the amino acid sequence shown in SEQ ID NO: 26, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 7, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 7.

[0065] In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more T-cell or NK-cell escape proteins. In some embodiments, an inactivating mutation of one or more of at least one viral gene species is achieved by inserting one or more heteronucleotides each encoding one or more T-cell or NK-cell escape proteins.

[0066] In some embodiments, one or more T-cell or NK-cell escape proteins comprise a group of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). In some embodiments, one or more T-cell or NK-cell escape proteins comprise a group of proteins that are or comprise CPXV012, CPXV203, and CPXV018 proteins. In some embodiments, a group of proteins encoded by CPXV012-203-018 comprises: (i) the amino acid sequence shown in SEQ ID NO: 20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; (ii) the amino acid sequence shown in SEQ ID NO: 21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; and (iii) the amino acid sequence shown in SEQ ID NO: 22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. The amino acid sequence shown in SEQ ID NO: 22 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, a set of proteins encoded by CPXV012-203-018 comprises the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 10, 89, and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 10, 89, and 90.

[0067] In some embodiments, at least one heteronucleotide encoding one or more heteronucleotides encoding one or more complement inhibitors comprises one or more heteronucleotides each encoding one or more complement inhibitors. In some embodiments, an inactivating mutation in one or more of at least one viral gene is achieved by inserting one or more heteronucleotides each encoding one or more complement inhibitors.

[0068] In some embodiments, one or more complement inhibitors are Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2) and / or minimizing complement regulatory factor H (miniFH). In some embodiments, one or more complement inhibitors are or comprise CRASP-2. In some embodiments, CRASP-2 comprises the amino acid sequence shown in SEQ ID NO: 18, or has an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, one or more complement inhibitors are or comprise miniFH. In some embodiments, miniFH comprises the amino acid sequence shown in SEQ ID NO: 19, or has an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19.

[0069] In some embodiments, one or more heterologous nucleic acids encoding one or more complement inhibitors are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion protein comprises a complement inhibitor fused to a viral membrane protein encoded by the viral membrane gene. In some embodiments, the viral membrane gene is F14.5L, optionally wherein the fusion is located at the C-terminus of the F14.5L protein. In some embodiments, the fusion protein is incorporated into the outer membrane of an intracellular mature virus (IMV). In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 5, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 5. In some embodiments, the recombinant oncolytic virus genome comprises the nucleic acid sequence shown in SEQ ID NO: 6, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 6. In some embodiments, the recombinant oncolytic virus genome comprises the nucleic acid sequence shown in SEQ ID NO: 89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 89. In some embodiments, the recombinant oncolytic virus genome comprises the nucleic acid sequence shown in SEQ ID NO: 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 90.

[0070] In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more anti-angiogenic proteins. In some embodiments, an inactivating mutation in one or more of at least one viral gene is achieved by inserting one or more heteronucleotides each encoding one or more anti-angiogenic proteins. In some embodiments, the one or more anti-angiogenic proteins are VEGF inhibitors, angiopoietin inhibitors, versikine, or fusion proteins of any two or more of the foregoing. In some embodiments, the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or angiopoietin inhibitors, optionally Ang2 inhibitors. In some embodiments, the one or more anti-angiogenic proteins comprise anti-VEGF antibodies and / or anti-Ang2 antibodies. In some embodiments, the VEGF inhibitor is an anti-VEGF antibody, optionally an anti-VEGF single-chain antibody (scAb). In some embodiments, the angiopoietin inhibitor is an anti-angiopoietin-2 (Ang2) antibody, optionally an anti-Ang2 single-chain antibody (scAb). In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 23. In some embodiments, one or more anti-angiogenic proteins comprise versikine. In some embodiments, versikine comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 24. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 13, 47, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 13, 47, 82, 87 and 88.

[0071] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more therapeutic agents or diagnostic agents. In some embodiments, an inactivating mutation in one or more of at least one viral gene is achieved by inserting one or more heterologous nucleic acids each encoding one or more therapeutic agents or diagnostic agents.

[0072] In some embodiments, one or more therapeutic or diagnostic agents are selected from anticancer agents, antimetastatic agents, antiangiogenic agents, immunomodulatory molecules, antigens, cytostromal degradation genes, genes for tissue regeneration and reprogramming human cells to pluripotency, enzymes that modify substrates to produce detectable products or signals or that can be detected by antibodies, proteins that can bind contrast agents, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging. In some embodiments, one or more therapeutic or diagnostic agents comprise a therapeutic agent selected from hormones, growth factors, cytokines, chemokines, co-stimulatory molecules, ribozymes, transport proteins, single-chain antibodies, antisense RNA, prodrug-converting enzymes, siRNA, microRNA, toxins, antitumor oligopeptides, mitotic inhibitory proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics, angiogenesis inhibitors, tumor inhibitors, cytotoxic proteins, cell suppressor proteins, and tissue factors.

[0073] In some of any embodiments: at least one viral gene is or contains A35R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 3.

[0074] In some of any embodiments: at least one viral gene is or contains A35R and J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 12.

[0075] In some embodiments: at least one viral gene is or contains J2R, and the inactivation mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T-cell or NK-cell escape proteins, optionally wherein the one or more T-cell or NK-cell escape proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and wherein at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids, each encoding one or more complement inhibitors, said heterologous nucleic acids being introduced into the viral membrane gene to produce a fusion gene encoding a fusion protein. In some embodiments, the viral membrane gene is F14.5L. In some embodiments, it is fused at the C-terminus of the F14.5L protein. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 10, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 10.

[0076] In some embodiments: at least one viral gene is or contains J2R. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 4, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 4.

[0077] In some embodiments: at least one viral gene is or comprises J2R and A35R, and the inactivation mutation of A35R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the one or more immunomodulatory proteins are LIGHT. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 11.

[0078] In some embodiments: at least one viral gene is or contains J2R and A35R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some embodiments, the one or more anti-angiogenic proteins contain VEGF inhibitors and / or Ang2 inhibitors. In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 13.

[0079] In some embodiments: at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the one or more immunomodulatory proteins are LIGHT. In some embodiments, the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors. In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 47, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 47.

[0080] In some embodiments: at least one viral gene is or contains J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding an apoptosis-inducing protein. In some embodiments, the apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas), or inducible Cas9 (iCas9). In some embodiments, the nucleotide genome of the recombinant oncolytic virus contains the nucleotide sequence shown in SEQ ID NO: 7, 8, or 9, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, 8, or 9. In some embodiments, the nucleotide genome of the recombinant oncolytic virus contains the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleotide genome of the recombinant oncolytic virus contains the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleotide genome of the recombinant oncolytic virus contains the nucleotide sequence of SEQ ID NO: 9.

[0081] In some embodiments: at least one viral gene is or contains J2R, and the inactivation mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 49, 50, or 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 49, 50, or 93.

[0082] In some embodiments: at least one viral gene is or contains J2R and B2R. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 48, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 48.

[0083] In some of any implementation schemes, at least one viral gene is or contains J2R and B2R.

[0084] In some embodiments: at least one viral gene is or contains J2R and B2R, and the inactivation mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 80, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 80.

[0085] In some embodiments: at least one viral gene is or comprises J2R, B2R, and A35R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. In some embodiments, the one or more anti-angiogenic proteins comprise inhibitors of VEGF and / or Ang2. In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the one or more immunomodulatory proteins are IRF3. In some embodiments, the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, the one or more immunomodulatory proteins comprising LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, one or more immunomodulatory proteins are LIGHT. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 82, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 82.

[0086] In some embodiments: at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IL-2. In some embodiments, IL-2 is an IL-2 superfactor. In some embodiments, the IL-2 superfactor is mDNA11. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 84, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 84.

[0087] In some embodiments: at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, the two or more immunomodulatory proteins comprise IL-12 and CXCL9. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 85.

[0088] In some embodiments: at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9; and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding an apoptosis-inducing protein. In some embodiments, the two or more immunomodulatory proteins comprise IL-12 and CXCL9. In some embodiments, the apoptosis-inducing protein is an inducible DED (iDED). In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 86.

[0089] In some of any embodiments: at least one viral gene is or contains J2R, B2R, A35R, and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins contain inhibitors of VEGF and / or Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 superfactor MDNA11. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 87.

[0090] In some embodiments: at least one viral gene is or comprises J2R, B2R, A35R, and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 superfactor MDNA11T. In some embodiments, MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98. In some implementations, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 88.

[0091] In some of any embodiments: at least one viral gene is or contains J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T-cell or NK-cell escape proteins, optionally wherein one or more T-cell or NK-cell escape proteins comprise a set of vaccinia virus ORFs. Proteins encoded by 012, 203, and 018 (CPXV012-203-018); the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally one or more of which are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally one or more of which are IL-2 superfactors, optionally MDNA11 or MDNA11T; at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors (optionally CRASP-2), said heterologous nucleic acid being introduced into the viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion is performed at the C-terminus of the F14.5L protein. In some implementations, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 89.

[0092] In some of any embodiments: at least one viral gene is or contains J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T-cell or NK-cell escape proteins, optionally wherein one or more T-cell or NK-cell escape proteins comprise a set of proteins derived from vaccinia virus ORFs 012, 203, and 018. The protein encoded by (CPXV012-203-018); the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally two or more of which comprise IL-12 and CXCL9; at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors (optionally CRASP-2), said heterologous nucleic acids being introduced into the viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion is performed at the C-terminus of the F14.5L protein. In some implementations, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 90.

[0093] In some embodiments: at least one viral gene is or contains B2R and J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 91, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 91.

[0094] In some embodiments: at least one viral gene is or comprises B2R, J2R, and A56R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 92, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 92.

[0095] In some embodiments: at least one viral gene is or contains J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence of SEQ ID NO: 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 93.

[0096] In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 85, 86, 88, and 90, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 85. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in SEQ ID NO: 85, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 85.

[0097] In some of any implementations, one or more of the heterologous nucleic acids encoding the heterologous gene product are operatively linked to the promoter.

[0098] In some embodiments, each of one or more heteronucleotides encoding a heterogeneous gene product operably linked to the promoter is selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, each heteronucleotide encoding a heterogeneous gene product is independently operably linked to the promoter, optionally wherein each heteronucleotide encoding a heterogeneous gene product is independently operably linked to a promoter selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter is a poxvirus promoter or a variant or derivative thereof. In some embodiments, the promoter is a vaccinia virus promoter. In some embodiments, the promoter is selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter has the sequence shown in any of SEQ ID NO: 29, 53, 55, 68, 69, 70, 71, or 72. In some embodiments, the promoter is a synthetic strong early promoter (SSE). In some embodiments, the SSE promoter comprises the sequence shown in SEQ ID NO: 29. In some embodiments, the promoter is a strong early / late promoter (SEL). In some embodiments, the SEL promoter comprises the sequence shown in SEQ ID NO: 55. In some embodiments, the promoter is mH5. In some embodiments, the mH5 promoter comprises the sequence shown in SEQ ID NO: 53.

[0099] This document also provides an isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, wherein said nucleic acid genome is characterized by one or more of the following: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and containing a polar uncharged amino acid at position 66, optionally containing a threonine (T) amino acid sequence at position 66; (ii) a variant 038 (K5L) ORF containing a nucleotide insertion to cause a frameshift mutation, wherein the product of said 038 (K5L) gene is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, wherein said ... SEQ ID NO: 60 has at least 95% sequence identity and contains a hydrophobic amino acid other than leucine at position 419, optionally containing phenylalanine (F) at position 419; (iv) variant 104 (H4L) ORF, which encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and containing a negatively charged amino acid at position 591, optionally containing aspartic acid (D) at position 591; and (v) variant 182 (A56R) ORF, which contains a two-nucleotide deletion to cause a frameshift mutation, wherein the gene product of the 182 (A56R) ORF is altered.

[0100] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (i) that variant 017 ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 57. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (ii) that the nucleotide insertion is a guanine (G) inserted after nucleotide 32135 of SEQ ID NO: 1, optionally wherein variant 038 (K5L) ORF is shown in SEQ ID NO: 58. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (ii) that the 038 (K5L) gene product is shown in SEQ ID NO: 59. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 60. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes the amino acid sequence shown in SEQ ID NO: 60. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (iv), and the 104 (H4L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 61. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (iv), wherein variant 104 (H4L) ORF encodes the amino acid sequence shown in SEQ ID NO: 61. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (v), wherein the deletion of two nucleotides is the deletion of two consecutive nucleotides corresponding to the deletion of the nucleotide following nucleotide 165972 of SEQ ID NO: 2, optionally wherein variant 182 (A56R) is shown in SEQ ID NO: 62. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by (v), and the VCV protein is shown in SEQ ID NO: 63.

[0101] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any two of (i)-(v). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any three of (i)-(v). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any four of (i)-(v). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by each of (i)-(v).

[0102] This document also provides an isolated clonal vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, wherein said nucleic acid genome is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (vii) and the nucleic acid sequence CACTTATAT at positions 106870 to 106880 of SEQ ID NO: 1; (viii) The nucleic acid sequence GTTTTCATTA at positions 111267 to 111276 of SEQ ID NO: 1; (ix) The nucleic acid sequence TACAGACACC at positions 165844 to 185853 of SEQ ID NO: 1; and (x) C at position 187805 of SEQ ID NO: 1.

[0103] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any two of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any three of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any four of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any five of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any six of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleotide genome is characterized by any seven of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by any eight items of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by any nine items of (i)-(x). In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is characterized by each item of (i)-(x).

[0104] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 96% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 98% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0105] This article also provides an isolated cloned vaccinia virus (VACV) strain containing a nucleic acid genome that has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0106] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 99.9% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome has at least 99.95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome does not contain the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is not modified to contain a non-viral heteronucleotide with an open reading frame encoding a non-viral heteroprotein. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the nucleic acid genome is as shown in SEQ ID NO: 1.

[0107] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the cloned VCV strain exhibits enhanced production of extracellular enveloped virus (EEV) after cell infection, optionally as determined by a percentage of EEV, wherein the percentage of EEV is determined by the following formula: virus titer in supernatant / (virus titer in supernatant + virus titer in cell lysate) * 100. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, more than 5% of the infectious particles after cell infection are EEVs. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, more than 10% of the infectious particles after cell infection are EEVs. In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, more than 15% of the infectious particles after cell infection are EEVs. In some of any implementations, the recombinant oncolytic virus or cloned VCV strain exhibits enhanced production of extracellular enveloped virus (EEV) after cell infection, as determined by EEV having at least 5%, 10%, or 15% of infectious particles.

[0108] In some embodiments of any recombinant oncolytic virus or any isolated cloned VCV strain, the virus exhibits oncolytic activity to kill tumor cells.

[0109] This article also provides a VACV formulation comprising any isolated clonal VACV strain provided herein.

[0110] This article also provides a VCV formulation containing any recombinant oncolytic vaccinia virus provided herein.

[0111] This article also provides a recombinant oncolytic virus formulation comprising any recombinant oncolytic virus provided herein, wherein at least 70%, 80%, 90%, 95%, or 98% of the virus particles in the formulation have the genomic sequence of a cloned recombinant oncolytic virus.

[0112] In some of any embodiments, the VCV formulation is substantially homogeneous, wherein multiple viral particles in the formulation have genomic sequences of clonal VCV strains.

[0113] In some embodiments, at least 70% of the viral particles in the formulation have the genome sequence of a cloned VCV strain. In some embodiments, at least 80% of the viral particles in the formulation have the genome sequence of a cloned VCV strain. In some embodiments, at least 90% of the viral particles in the formulation have the genome sequence of a cloned VCV strain. In some embodiments, at least 95% of the viral particles in the formulation have the genome sequence of a cloned VCV strain. In some embodiments, at least 98% of the viral particles in the formulation have the genome sequence of a cloned VCV strain.

[0114] This article also provides a pharmaceutical composition comprising any isolated VCV clones provided herein.

[0115] This article also provides a pharmaceutical composition comprising any VACV provided herein.

[0116] This article also provides a pharmaceutical composition comprising any recombinant oncolytic vaccinia virus provided herein.

[0117] This article also provides a recombinant vaccinia virus (VACV) strain comprising the nucleic acid genome of any VACV clone strain provided herein, wherein the nucleic acid genome contains an inactivating mutation in at least one viral gene.

[0118] In some embodiments, the viral gene is selected from hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, the inactivating mutation is the deletion of all or part of at least one viral gene. In some embodiments, the deletion of at least one viral gene is the deletion of the entire ORF of the viral gene. In some of any embodiments, the deletion of at least one viral gene is a deletion of a portion of the ORF of the viral gene, and said deletion is sufficient to render the encoded gene product nonfunctional.

[0119] In some of any embodiments, the at least one viral gene is or contains A35R.

[0120] In some of any embodiments, the nucleic acid genome of the recombinant VCV strain comprises the nucleic acid sequence shown in SEQ ID NO: 3, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 3.

[0121] In some of any implementations, the at least one viral gene is or contains J2R.

[0122] In some of any embodiments, the nucleic acid genome of the recombinant VCV strain comprises the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 4.

[0123] In some of any implementations, the at least one viral gene is or contains B2R.

[0124] In some of any embodiments, the at least one viral gene is or contains A35R and J2R.

[0125] In some of any embodiments, the nucleic acid genome of the recombinant VCV strain comprises the nucleic acid sequence shown in SEQ ID NO: 12, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 12.

[0126] In some of any implementations, the at least one viral gene is or contains B2R and J2R.

[0127] In some of any embodiments, the nucleic acid genome of the recombinant VCV strain comprises the nucleic acid sequence shown in SEQ ID NO: 48, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 48.

[0128] In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 85, 86, 88, and 90, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 85. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in SEQ ID NO: 85, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 85.

[0129] This article also provides a nucleic acid containing the genome of any recombinant oncolytic virus or any isolated VCV clone strain provided herein.

[0130] This article also provides a recombinant oncolytic virus that contains the nucleic acid of any recombinant oncolytic virus provided herein.

[0131] In some embodiments, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus.

[0132] This article also provides a pharmaceutical composition comprising any of the recombinant VCV strains provided herein.

[0133] This article also provides a pharmaceutical composition comprising any of the recombinant oncolytic viruses provided herein, optionally said recombinant oncolytic vaccinia virus.

[0134] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0135] In some embodiments, the pharmaceutical composition is formulated for intravenous, intratumoral, intraperitoneal, or intrapleural administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is lyophilized.

[0136] This article also provides a method for treating a subject with a proliferative disorder, the method comprising administering to the subject any recombinant oncolytic virus provided herein, any isolated oncolytic virus provided herein, or any pharmaceutical composition provided herein.

[0137] In some embodiments, the proliferative disorder is a tumor or metastatic tumor. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is pancreatic cancer, ovarian cancer, lung cancer, colon cancer, prostate cancer, cervical cancer, breast cancer, rectal cancer, kidney (renal) cancer, stomach cancer, esophageal cancer, liver (liver) cancer, endometrial cancer, bladder cancer, brain cancer, head and neck cancer, oral cancer (e.g., oral cavity cancer), cervical cancer, uterine cancer, thyroid cancer, testicular cancer, prostate cancer, skin cancer (e.g., melanoma, such as malignant melanoma), cholangiocarcinoma (cholangiocarcinoma), thymic epithelial carcinoma (e.g., thymoma), leukemia, lymphoma, or multiple myeloma. In some embodiments, the cancer is microsatellite stable (MSS) colorectal cancer.

[0138] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 8.

[0139] In some of any embodiments, the recombinant oncolytic virus or isolated oncolytic virus is in the form of 1x10 5 PFU up to 1x10 14 The dosage of PFU.

[0140] In some embodiments, the method further includes administering a second therapeutic agent to treat the proliferative disorder.

[0141] In some embodiments, the method further includes another treatment selected from surgery, radiotherapy, immunosuppressive therapy, and administration of an anticancer agent. In some embodiments, the other treatment is the administration of an anticancer agent selected from cytokines, chemokines, growth factors, photosensitizers, toxins, anticancer antibiotics, chemotherapeutic compounds, radionuclides, angiogenesis inhibitors, signal transduction modulators, antimetabolites, anticancer vaccines, anticancer oligopeptides, mitotic inhibitory proteins, antimitotic oligopeptides, anticancer antibodies, anticancer antibiotics, immunotherapeutic agents, and any combination thereof.

[0142] In some of any implementations, the recombinant oncolytic virus or isolated oncolytic virus is administered intravenously.

[0143] In some implementations, the method further includes administering AP1903 (Rimiducid) to the subject.

[0144] In some of any implementation schemes, the recombinant oncolytic virus administered to the subject contains a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0145] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 8.

[0146] In some of the implementation schemes, the subjects exhibited severe immunodeficiency and were susceptible to viral infections.

[0147] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93.

[0148] This article also provides a method for inhibiting viral replication, the method comprising contacting cells infected with a recombinant oncolytic virus with AP1903 (Rimiducid), wherein the recombinant oncolytic virus contains a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0149] This article also provides a method for inhibiting viral replication, the method comprising contacting cells with AP1903 (Rimiducid), wherein the cells are infected with any recombinant oncolytic virus provided herein, any isolated oncolytic virus provided herein, or any recombinant oncolytic virus provided herein (e.g., a cloned VCV strain).

[0150] In some embodiments, the contact occurs within the subject. In some embodiments, AP1903 (Rimiducid) has been administered to a subject who has previously been given a recombinant oncolytic virus containing a heterologous nucleic acid encoding an apoptosis-inducing protein. In some embodiments, AP1903 (Rimiducid) has been administered to a subject who has previously been given any recombinant oncolytic virus provided herein or any isolated oncolytic virus provided herein.

[0151] This article also provides a method for inhibiting viral replication in a subject, the method comprising administering AP1903 (Rimiducid) to the subject, wherein the subject has previously been administered a recombinant oncolytic virus containing a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0152] This article also provides a method for inhibiting viral replication in a subject, the method comprising administering AP1903 (Rimiducid) to the subject, wherein the subject has previously been administered any recombinant oncolytic virus provided herein or any isolated oncolytic virus provided herein.

[0153] In some embodiments, the method preferentially inhibits viral replication in non-cancerous cells. In some embodiments, the apoptosis-inducing protein is an inducible death effector domain (iDED). In some embodiments, the iDED comprises the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 27.

[0154] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 8.

[0155] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93. Attached Figure Description

[0156] Figure 1 The percentage of BT-549, A549, LOX-IMVI, HCC-2998, and COLO-205 cells after infection with VIP01-06VACV clone isolates is depicted. For each of VIP01, VIP02, VIP03, VIP04, VIP05, and VIP06, the bars correspond from left to right to BT-549, A549, LOX-IMVI, HCC-2998, and COLO-205 cells.

[0157] Figure 2 The percentage of extracellular enveloped virus (EEV) production of VCP02 and VIP02 is shown in cells infected with 4T1 and B16-F10.

[0158] Figure 3 Tumor volume changes in a post-infected 4T1 mouse breast cancer model were depicted by single intravenous delivery of VCP02 (square), VIP01 (triangular), VIP02 (rhomboid), and a carrier (circular).

[0159] Figures 4A-4B 2D models depicting different cancer cell types infected with VIP02 at MOI = 0.01 (light bars) and MOI = 0.1 (dark bars) were used. Figure 4A ) and 3-D ( Figure 4B Percentage of viable cells in cell culture.

[0160] Figure 5 A series of schematic diagrams depicting the genomic structures of recessive recombinant clones VIR27, VIR37, and VIR46 derived from the parent VIP02 are presented.

[0161] Figure 6 The percentage of host complement inhibition in human and mouse serum after incubation with latent oncolytic virus clones VIR27, VIR37, and VIP02 was depicted.

[0162] Figure 7 Tumor volumes were depicted in a post-infection 4T1 mouse breast cancer model after a single intravenous delivery of VIP02, VIR27, and the vector.

[0163] Figure 8 Tumor volumes in an infected 4T1 mouse model of breast cancer were depicted after a single intravenous delivery of VIR46, VIR52, and the vector.

[0164] Figure 9 A series of schematic diagrams depicting the genomic structures of immune-stimulated oncolytic viruses VIR49 and VIR52 are presented.

[0165] Figure 10 Tumor volumes in a post-infected 4T1 mouse breast cancer model were depicted after a single intravenous delivery of VIR49 and VIR52.

[0166] Figure 11 A series of schematic diagrams depicting the genome structure of the anti-angiogenic oncolytic virus VIR71 are presented.

[0167] Figure 12A and Figure 12B The text describes the delivery of VIR71, VIR52, and a medium via a single intravenous infusion. Figure 12A ) and VIR13, VIR86 and media ( Figure 12B Tumor volume in a 4T1 mouse breast cancer model after infection.

[0168] Figure 13 A series of schematic diagrams depicting the genomic structures of oncolytic viruses VIR40, VIR41, VIR42, and control virus VIR13 that induce apoptosis are presented.

[0169] Figure 14 A series of graphs were drawn up to quantify the viral replication of apoptosis-inducing viral clones VIR13, VIR40, VIR41, and VIR42 in primary healthy HBE, HME, and MME under conditions of MOI of 0.01 and / or 10, in the presence of Rimiducid or DMSO as a control.

[0170] Figures 15A-15G A series of graphs were plotted that quantified the apoptosis-inducing viral clones VIR13, VIR40, VIR41, and VIR42 in BT-549 breast cancer cells initially infected with Rimiducid or DMSO as a control at an MOI of 0.01 and / or 10. Figure 15A Hs578T breast cancer cells ( Figure 15B ), MCF-7 and 4T1 breast cancer cells ( Figure 15C ), A549 and M14 lung cancer and melanoma cells ( Figure 15D HCT-15 MSI colon cancer cells ( Figure 15E HCT-116 MSI colon cancer cells ( Figure 15F ) and KM12 MSI colon cancer cells ( Figure 15G Virus replication in ).

[0171] Figures 16A-16C A series of graphs were plotted that quantified the initial infection of COLO205 cancer cells at MOIs of 0.01 and / or 10, in the presence of Rimiducid or DMSO as a control. Figure 16A HCC-2998 cancer cells ( Figure 16B ) and HT-29 cancer cells ( Figure 16C Virus replication in ).

[0172] Figures 17A-17C A series of graphs were plotted that quantified the apoptosis-inducing viral clones VIR13, VIR40, VIR41, and VIR42 in human primary bronchial / tracheal epithelial cells (HBE) under initial infection with an MOI of 0.01 and / or 0.1, in the presence of Rimiducid or DMSO as a control. Figure 17A Human primary mammary epithelial cells (HME) Figure 17B ), mouse primary mammary epithelial cells (MME) and human primary colonic epithelial cells (HCE), Figure 17C ) cytotoxicity.

[0173] Figures 18A-18K A series of graphs were plotted that quantified the apoptosis-inducing viral clones VIR13, VIR40, VIR41, and VIR42 in BT-549 breast cancer cells initially infected at an MOI of 0.01 and / or 0.1, in the presence of Rimiducid or DMSO as a control. Figure 18A Hs578T breast cancer cells ( Figure 18B ), 4T1 breast cancer cells ( Figure 18C DU-145 prostate cancer cells ( Figure 18D ), PC-3 prostate cancer cells ( Figure 18E ), A549 lung cancer and melanoma cells ( Figure 18F M14 lung cancer and melanoma cells ( Figure 18G COLO 320 DM and HCT-15 MSI colon cancer cells ( Figure 18H HCT-116 and KM12 MSI colon cancer cells ( Figure 18I ), KM12 MSI colon cancer cells ( Figure 18J ) and SW48 MSI colon cancer cells ( Figure 18K ) cytotoxicity.

[0174] Figures 19A-19GA series of graphs were plotted that quantified the apoptosis-inducing viral clones VIR13, VIR40, VIR41, and VIR42 initially infected in COLO205 MSS colon cancer cells at an MOI of 0.01 and / or 0.1, in the presence of Rimiducid or DMSO as a control. Figure 19A HCC-2998 colon cancer cells ( Figure 19B ), HT-29 cells ( Figure 19C ), LS123 cells ( Figure 19D ), LS174T cells ( Figure 19E ), SW620 cells ( Figure 19F ) and WiDR cells ( Figure 19G ) cytotoxicity.

[0175] Figure 20 This study demonstrates complete inhibition of tumor growth in a post-infected SL-4 mouse model of colon adenocarcinoma after a single intravenous injection of VIR13.

[0176] Figures 21A-21F A series of charts depict the effects of applying VIR13, VIR41, or the control ( Figure 21A ); VIR13, VIR86 or control ( Figure 21B ); VIR13, VIR93 or control ( Figure 21C ); VIR13, VIR94 or control ( Figure 21D ); VIR13, VIR96 or control ( Figure 21E Tumor size in mice over time (days after treatment) after treatment. Figure 21F A graph depicting the body weight (g) of mice treated with VIR13, VIR41, VIR86, VIR93, VIR94, VIR96, or the control over time (days after treatment).

[0177] Figures 22A-22H A series of charts were drawn, showing the effects of applying VIR94, VIR100, or the control ( Figure 22A ); VIR94, VIR103 or control ( Figure 22B ); VIR94, VIR105 or control ( Figure 22C ); VIR94, VIR106 or control ( Figure 22D ); VIR94, VIR109 or control ( Figure 22E ); VIR94, VIR113 or control ( Figure 22F ); VIR94, VIR114 or control ( Figure 22G ); VIR94, VIR115 or control ( Figure 22HTumor volume in mice over time (days after treatment) after treatment. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

[0178] Figure 23A A graph depicts tumor size in mice over time (days after treatment) following administration of VIR103, VIR111, or VIR113. Figure 23B A schematic diagram illustrating how mDNA11 and mDNA11T are generated from wild-type human interleukin-2 (wt hIL-2). * = p≤0.05; ** = p≤0.01.

[0179] Figure 24A A graph depicts tumor volume in mice over time (days after treatment) after administration of VIR106 or the control. Figure 24B A graph depicting the body weight (g) of mice over time (days after treatment) following administration of VIR106 or the control is presented. *** = p≤0.001. Figure 24C Images of tumor sites in mice taken on day 8 after administration of VIR106 or the control group are shown, revealing detectable tumors in the control mice and no detectable tumors in the VIR106-treated mice. Figures 24D-24E The figure depicts tumor volume over time (days after treatment) in mice treated with VIR113 or in the control group. Figure 24D ) and body weight (g) Figure 24E (Charts) Figures 24F-24G The figure depicts tumor volume over time (days after treatment) in mice treated with VIR115 or in the control group. Figure 24F ) and body weight (g) Figure 24G (Charts)

[0180] Figures 25A-25F A series of charts were drawn, showing the effects of applying VIR106 or the control ( Figure 25A and Figure 25B ), VIR113 or control ( Figure 25C and Figure 25D VIR115 or control ( Figure 25E and Figure 25F Tumor volume and body weight (g) in mice over time (days after treatment). * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

[0181] Figures 26A-26BA Western blot analysis of proteins was depicted, showing the effects on B16-F10 cells infected with mimics, iVIR13, VIR13, VIR93, VIR94, VIR100, VIR106, VIR113, VIR115, VIR123, or VIR127. Figure 26A ) and Hela S3 cells ( Figure 26B The expression of human phosphorylated IRF3, mouse phosphorylated IRF3, human IRF3, mouse IRF3 and β-actin in ). Detailed Implementation

[0182] This article provides isolated clonal lines that exhibit superior antitumor activity and enhanced evasion of the host immune system compared to other vaccinia viruses. Specifically, the clonal lines provided herein are clonal isolates from parental IHD-J, catalogued at the American Center for Type Culture Collection (ATCC®) with catalog number VR-156™. This article also provides formulations produced by propagation from these isolated clonal lines. This article further provides recombinant vaccinia viruses derived from the isolated clonal lines, which are attenuated by modification to delete or reduce viral gene expression or to inactivate viral proteins. Furthermore, this article provides recombinant viruses further improved to evade host antiviral defenses or to possess further enhanced antitumor activity. For example, such recombinant viruses contain heterologous nucleic acids encoding proteins to evade complement system inhibition, evade natural killer (NK) cell or T cell attack, incorporate immune checkpoint molecules to enhance immunostimulatory activity, or provide anti-angiogenic activity. The recombinant viruses provided herein also include those equipped with viral induction systems to inhibit viral replication as a safety strategy (e.g., by mediating apoptosis in unwanted infected cells, such as healthy cells). Specifically, this document provides a recombinant oncolytic vaccinia virus comprising: an inactivating mutation of B2R; a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3); and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12. This document also specifically provides a recombinant oncolytic virus comprising: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape agents, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof.

[0183] Oncolytic viruses (OVs) are viruses that replicate selectively or more efficiently in cancer cells than in non-cancer cells. In some cases, the ability to selectively infect, replicate, and destroy cancer cells within a cancer cell, and often without harming healthy cells simultaneously, is due to the ability to exploit the biochemical differences between healthy and transformed cells during infection. Cancer cells are characterized by disrupted apoptosis pathways, the acquisition of new abilities to evade the immune system, and the capacity for unlimited proliferation—all characteristics that favor viral replication. Since one of the major challenges in cancer therapy is minimizing toxicity while killing malignant cells, OVs have become an attractive option because they rarely cause off-target toxicity.

[0184] Oncolytic viruses can be divided into three main categories: (1) viruses with a natural tendency to preferentially replicate in cancer cells while being nonpathogenic to humans, such as parvovirus, myxoma virus, Newcastle disease virus, and reovirus; (2) viruses that have been genetically engineered to ensure selective replication in cancer cells, such as adenovirus, HSV, and vesicular stomatitis virus; and (3) viruses that have been attenuated through in vitro propagation to ensure safe use in humans. The last category includes oncolytic viruses derived from vaccinia virus, which are highly favored due to their efficient replication, cell lysis, diffusion, host range, and natural tropism toward tumor tissues (Shen et al. (2004) Mol. Ther., 11:180). For example, vaccinia virus is more efficient than adenovirus vectors in terms of replication and diffusion.

[0185] Vaccinia virus (VV), a typical member of the Orthopoxvirus genus, replicates in the cytoplasm of the host cell. VV is a large, complex enveloped virus with a linear double-stranded DNA genome of approximately 190,000 base pairs, consisting of a single, continuous polynucleotide chain encoding about 250 genes that can potentially express more than 200 proteins. See, for example, McCraith al., (1982) PNAS, 97(9):4879-4884. Typically, the non-segmented, non-infectious genome arrangement makes central genes crucial for viral replication (and therefore conserved), while genes closer to the ends influence more peripheral functions, such as host range and virulence. Vaccinia viruses express differential genes by utilizing groups of open reading frames (ORFs), which are generally non-overlapping. See, for example, Traktman, P., Chapter 27, Poxvirus DNA Replication, pp. 775-798, in DNA Replication in Eukaryotic Cells, Cold Spring Harbor Laboratory Press (1996). The rapid replication capacity of VV leads to efficient lysis of infected cells and spread to other tumor cells during successive rounds of replication, resulting in strong local destruction of the tumor. The VV genome encodes approximately 250 genes and can accept up to 20 kb of external DNA, making it an ideal gene delivery medium. Recombinant VV vectors under development aim to deliver eukaryotic genes, such as tumor-associated antigens, into tumors, thereby promoting the induction of the host immune system to kill cancer cells. However, a limiting factor for using VV as a cancer treatment delivery vector is the strong neutralizing antibody response induced by injecting VV into the bloodstream, which limits the virus's persistence and spread, and hinders the re-administration of the vector. In some cases, neutralizing antibodies recognize and bind to viral glycoproteins with high affinity, preventing the virus from interacting with host cell receptors (leading to viral neutralization).

[0186] Vaccine virus replicates in the cytoplasm of infected cells, where the assembly of progeny viruses begins in specialized regions called viral factories. During replication, three morphologically and antigenically distinct viral forms are produced: intracellular mature viral particles (IMV), intracellular enveloped viral particles (IEV), and extracellular viral particles. A subset of IMV, the first infectious progeny produced, is transported to the trans-Golgi network (TGN), where they are enveloped by two additional membranes to produce IEV. IEV is transported through the cytoplasm to the periphery, where the outermost membrane fuses with the plasma membrane to release a double-membrane form of virus called an EV. EVs that remain on the cell surface are called cell-associated enveloped viral particles (CEV), while those that are no longer attached to the cell surface are called extracellular enveloped viral particles (EEV). IMV is the most abundant infectious form and is thought to be responsible for host-to-host diffusion; CEV is thought to play a role in cell-to-cell diffusion; and EEV is thought to be important for long-range viral dissemination within the host organism. Specifically, EEV is thought to be associated with long-range viral dissemination within the body. See, for example, Blasco et al., (1993) Journal of Virology, 67(6):3319-3325. The outer protein of EEV can induce protective immunity against the virus (Blaso and Moss (1992) J. Virol., 66:4170-4179). However, the amount of EEV produced by vaccinia virus strains varies greatly.

[0187] Attenuated vaccinia virus strains have been developed for therapeutic and diagnostic applications. For example, attenuated viruses include recombinant viruses modified in one or more viral genes, resulting in loss or reduction of viral gene expression, or inactivation of viral proteins. Nevertheless, although vaccinia virus is a well-studied attenuated virus with antitumor properties, many vaccinia virus strains (including recombinant strains) exhibit variability in virulence and safety, making many strains unsuitable for clinical use. Therefore, there is a need for improved vaccinia virus strains with enhanced antitumor properties and low cytotoxicity, as these characteristics are highly desirable for effective oncolytic therapy. The oncolytic viruses and methods described herein meet this need.

[0188] Various approaches have been investigated to improve the antitumor activity of OV, mainly focusing on viral replication and spread, as viral replication is often associated with cancer cell killing efficacy. However, other aspects of viral infection, such as enhancement of the host's antitumor immune response, induction of apoptosis, and control of tumor angiogenesis, are also important aspects of cancer viral therapy (Davola, ME and KL Mossman (2019) Oncoimmunology 8(6): e1581528).

[0189] This article presents an isolated cloned virus derived from a vaccinia virus strain known as IHD-J (ATCC® catalog number: VR-156™). IHD-J is a vaccinia virus strain closely related to the Western Reserve (WR) strain, but which produces 10 to 40 times more EEVs and spreads more efficiently to distant cells (Blasco and Moss, 1992). However, strains exhibiting greater dispersal over longer distances may not demonstrate sufficient antitumor activity for oncolytic virus therapy.

[0190] The implementation scheme presented herein is based on the identification of a specific clonal isolate (named VIP02) from IHD-J, which not only exhibited a high percentage of EEVs but also demonstrated the highest antitumor activity among other clonal isolates from the same lineage. Furthermore, results showed that in a mouse syngeneic tumor model, a single intravenous delivery of the clonal isolate at a low dose significantly inhibited tumor growth, and it also exhibited potent tumor cell killing against a variety of tumor cells in 2-D and 3-D cultures in vitro. This paper also provides vaccinia virus strains possessing the sequence characteristics of the VIP02 clonal isolate.

[0191] The embodiments provided herein also relate to recombinant viruses, wherein heterologous nucleic acids can be introduced into isolated cloned viruses with enhanced antitumor properties to further enhance the antitumor properties of said isolated cloned viruses while minimizing cytotoxicity to healthy cells.

[0192] In some embodiments, the selected clonal lines and their recombinant-derived lines are oncolytic virus candidates for tumor diagnosis and therapy. In some embodiments, isolated clonal lines of vaccinia virus and their recombinant-derived lines are used as therapeutic viruses for the treatment of proliferative disorders, including cancer, hyperplasia, metastasis, and tumors, and for use in other therapeutic and / or diagnostic methods described herein. In some other embodiments, the clonal lines can be used in methods of vaccination. In other embodiments, isolated clonal lines and their recombinant-derived lines are used as parental vaccinia virus to produce recombinant oncolytic viruses.

[0193] All publications cited in this application, including patent documents, scientific articles, and databases, are incorporated herein by reference in their entirety for the same purpose as each individual publication is cited separately. If any definition set forth herein conflicts with or is otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail, and not those incorporated herein by reference.

[0194] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0195] I. Isolated cloned virus strains and their attenuated strains

[0196] This article provides isolated cloned vaccinia virus (VACV) lines from the IHD-J vaccinia virus strain (ATCC® catalog number: VR-156™), or lines exhibiting characteristics of cloned virus lines isolated from it. The parental IHD-J strain is sequence-heterologous. This article reveals that certain vaccinia virus clones with enhanced antitumor properties can be isolated from IHD-J parental vaccinia virus formulations or mixtures.

[0197] In some embodiments, the clonal lines provided herein are present in viral preparations propagated from IHD-J. For example, clonal lines or preparations thereof can be obtained by isolating clonal isolates of IHD-J origin from cell cultures in which parental IHD-J or variants thereof have been propagated. The clonal isolates provided herein were obtained by passage of IHD-J virus in confluent CV-1 cells (from African green monkey kidney fibroblast cultures) grown in 6-well plates and infected with a series of dilutions of vaccinia virus strains.

[0198] In some embodiments, the cloned strain does not contain a nonviral heterologous nucleic acid encoding an open reading frame of a nonviral heterologous protein. In other embodiments, the cloned strain can be used as a parental sequence to generate a recombinant virus, which is modified with a heterologous nucleic acid encoding a nonviral heterologous protein.

[0199] In some embodiments, the IHD-J clone line provided herein is named VIP02 and has the nucleotide sequence shown in SEQ ID NO: 1.

[0200] In some embodiments, this document provides a recombinant oncolytic vaccinia virus comprising: an inactivating mutation of B2R; a heteronucleotide encoding interferon regulatory factor 3 (IRF3); and at least one heteronucleotide encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heteronucleotide encoding one or more cytokines and / or chemokines comprises a heteronucleotide encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0201] In some embodiments, this document also provides a recombinant oncolytic virus comprising: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0202] In some embodiments, the provided vaccinia virus clone has a genome with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.1% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.2% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.3% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.4% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.6% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.7% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.8% sequence identity to SEQ ID NO: 1. In some embodiments, the provided vaccinia virus clone has a genome with at least 99.9% sequence identity to SEQ ID NO: 1.

[0203] In some of these embodiments, the provided vaccinia virus clone does not have a nucleic acid genome containing the amino acid sequence shown in SEQ ID NO:2 (IHD-W1). In some embodiments, the provided clone has a nucleotide sequence having less than 100% sequence identity with SEQ ID NO:2 and having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with SEQ ID NO:2. In some embodiments, the provided clone has a nucleotide sequence differing from SEQ ID NO:2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. The IHD-J cloned viruses described herein comprise viruses that differ in one or more open reading frames (ORFs) compared to the IHD-W1 strain having the nucleotide sequence shown in SEQ ID NO: 2. For example, the IHD-J cloned viruses described herein comprise viruses that differ in one or more ORFs compared to the IHD-W1 strain having the amino acid sequence shown in SEQ ID NO: 2. The IHD-J cloned virus strains described herein may contain nucleotide deletions or mutations in any one or more nucleotides in any ORF compared to SEQ ID NO: 2, or may contain additions or insertions of viral DNA compared to SEQ ID NO: 2.

[0204] In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome that has at least 95% sequence identity with SEQ ID NO: 1 and exhibits the sequence characteristics of SEQ ID NO: 1. For example, as described in Table E1 herein, the exemplary VIP02 clone isolate is characterized by the deletion or mutation of one or more nucleotides compared to SEQ ID NO: 2, including one or more mutations in the ORF of SEQ ID NO: 2. Regarding ORFs, ORFs are numbered sequentially starting from 001. In other embodiments, the vaccinia virus open reading frames may also be named by using a capital letter to represent the HindIII restriction endonuclease fragment, a number to indicate the position within the HindIII fragment, and a letter (L or R) to indicate the direction of transcription, such as K5L. The corresponding protein is represented by a capital letter and a number, such as K5. In some embodiments, the nucleotide changes are in non-ORF regions of the sequence.

[0205] In some embodiments, the provided vaccinia virus clone line comprises or is characterized by a variant 017 open reading frame (ORF) that encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and containing an amino acid other than alanine at position 66. In some embodiments, the amino acid at position 66 is a polar, uncharged amino acid. In some embodiments, the amino acid at position 66 is serine (S), threonine (T), asparagine (N), or glutamine (E). In some embodiments, the amino acid at position 66 is T. In some embodiments, the provided clone line comprises a variant 017ORF having the A66T mutation compared to the 017 ORF shown in SEQ ID NO: 2. In some embodiments, variant 017 ORF encodes an amino acid sequence containing any of the aforementioned amino acid variations at position 66 and having at least 96% sequence identity with SEQ ID NO: 57. In some embodiments, variant 017 ORF encodes an amino acid sequence at position 66 containing any of the aforementioned amino acid variations and having at least 97% sequence identity with SEQ ID NO: 57. In some embodiments, variant 017 ORF encodes an amino acid sequence at position 66 containing any of the aforementioned amino acid variations and having at least 98% sequence identity with SEQ ID NO: 57. In some embodiments, variant 017 ORF encodes an amino acid sequence at position 66 containing any of the aforementioned amino acid variations and having at least 99% sequence identity with SEQ ID NO: 57. In some embodiments, variant 017 ORF has the sequence shown in SEQ ID NO: 57. In some embodiments, such vaccinia virus clones have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity with SEQ ID NO: 1.

[0206] In some embodiments, the provided clonal strain comprises or is characterized by variant 038 (K5L) ORF, which has a nucleotide insertion to cause a frameshift mutation, wherein the 038 (K5L) gene product is altered. In some embodiments, the nucleotide insertion is an insertion of guanine (G) after nucleotide 32135 corresponding to SEQ ID NO: 1. In some embodiments, the full-length sequence of the 038 (K5L) gene product is shown in SEQ ID NO: 59. In some embodiments, variant 038 (K5L) ORF is shown in SEQ ID NO: 58. In some embodiments, such vaccinia virus clonal strains have a nucleic acid genome with at least 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.99% sequence identity with SEQ ID NO: 1. In some embodiments, variant 038 (K5L) ORF is characterized by alterations compared to the nucleic acid shown in SEQ ID NO: 73 or the amino acid sequence shown in SEQ ID NO: 74.

[0207] In some embodiments, the provided clonal line comprises or is characterized by variant 059 (E2L), which encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and containing an amino acid other than leucine at position 419. In some embodiments, the amino acid at position 419 is a hydrophobic amino acid other than leucine. In some embodiments, the amino acid at position 419 is alanine (A), valine (V), isoleucine (I), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some embodiments, the amino acid at position 419 is F. In some embodiments, the provided clonal line comprises variant 059 (E2L) ORF having the L419F mutation compared to 059 (E2L) ORF shown in SEQ ID NO: 2. In some embodiments, variant 059 (E2L) ORF encodes an amino acid sequence containing any of the above-described amino acid variations at position 419 and having at least 96% sequence identity with SEQ ID NO: 60. In some embodiments, variant 059 (E2L) ORF encodes an amino acid sequence at position 419 containing any of the aforementioned amino acid variations and having at least 97% sequence identity with SEQ ID NO: 60. In some embodiments, variant 059 (E2L) ORF encodes an amino acid sequence at position 419 containing any of the aforementioned amino acid variations and having at least 98% sequence identity with SEQ ID NO: 60. In some embodiments, variant 059 (E2L) ORF encodes an amino acid sequence at position 419 containing any of the aforementioned amino acid variations and having at least 99% sequence identity with SEQ ID NO: 60. In some embodiments, variant 059 (E2L) ORF has the sequence shown in SEQ ID NO: 60. In some embodiments, such vaccinia virus clones have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity with SEQ ID NO: 1.

[0208] In some embodiments, the provided clonal strain comprises or is characterized by variant 104 (H4L) ORF, which encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and containing an amino acid other than asparagine (N) at position 591. In some embodiments, the amino acid at position 591 is a negatively charged amino acid. In some embodiments, the amino acid at position 591 is aspartic acid (D) or glutamic acid (E). In some embodiments, the amino acid at position 591 is D. In some embodiments, the provided clonal strain comprises variant 104 (H4L) ORF having the N591D mutation compared to the 104 (H4L) ORF shown in SEQ ID NO: 2. In some embodiments, variant 104 (H4L) ORF encodes an amino acid sequence containing any of the above-described amino acid variations at position 591 and having at least 96% sequence identity with SEQ ID NO: 61. In some embodiments, variant 104 (H4L) ORF encodes an amino acid sequence containing any of the aforementioned amino acid variations at position 591 and having at least 97% sequence identity with SEQ ID NO: 61. In some embodiments, variant 104 (H4L) ORF encodes an amino acid sequence containing any of the aforementioned amino acid variations at position 591 and having at least 98% sequence identity with SEQ ID NO: 61. In some embodiments, variant 104 (H4L) ORF encodes an amino acid sequence containing any of the aforementioned amino acid variations at position 591 and having at least 99% sequence identity with SEQ ID NO: 61. In some embodiments, variant 104 (H4L) ORF has the sequence shown in SEQ ID NO: 61. In some embodiments, such vaccinia virus clones have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity with SEQ ID NO: 1.

[0209] In some embodiments, the provided clonal strain comprises or is characterized by variant 182 (A56R) ORF, which has nucleotide deletions to cause a frameshift mutation, wherein the 182 (A56R) gene product is altered. In some embodiments, the nucleotide deletion is the deletion of two consecutive nucleotides corresponding to the nucleotide following nucleotide 165972 of SEQ ID NO: 2. In some embodiments, the 182 (A56R) gene product is shown in SEQ ID NO: 63. In some embodiments, variant 182 (A56R) ORF is as shown in SEQ ID NO: 62. In some embodiments, such vaccinia virus clonal strains have a nucleic acid genome with at least 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.99% sequence identity with SEQ ID NO: 1. In some embodiments, variant 182 (A56R) ORF is characterized by alterations to the nucleic acid sequence shown in SEQ ID NO: 75 or the amino acid sequence shown in SEQ ID NO: 76.

[0210] In some embodiments, the provided clone line is characterized by a nucleic acid genome comprising at least one of the aforementioned mutations among 017ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clone line is characterized by a nucleic acid genome containing at least two of the aforementioned mutations among 017ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clone line is characterized by a nucleic acid genome containing at least three of the aforementioned mutations among 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided cloned lines are characterized by a nucleic acid genome containing at least four of the aforementioned mutations among 017 ORF, 038(K5L) ORF, 059(E2L) ORF, 104(H4L) ORF, and 182(A56R) ORF. In some embodiments, at least one of the mutations is in 017 ORF. In some embodiments, at least one of the mutations is in 038 (K5L) ORF. In some embodiments, at least one of the mutations is in 059 (E2L) ORF. In some embodiments, at least one of the mutations is in 104 (H4L) ORF. In some embodiments, at least one of the mutations is in 182 (A56R) ORF. In some embodiments, such vaccinia virus cloned lines have a nucleic acid genome with at least 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.99% sequence identity with SEQ ID NO: 1.

[0211] In some embodiments, the provided clone line is characterized by a nucleic acid genome comprising each of the aforementioned mutations among 017ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clone line is characterized by a nucleic acid genome comprising variant 017 ORF encoding the amino acid sequence shown in SEQ ID NO: 57, variant 038 (K5L) ORF encoding the amino acid sequence shown in SEQ ID NO: 59, variant 059 (E2L) ORF encoding the amino acid sequence shown in SEQ ID NO: 60, variant 104 (H4L) ORF encoding the amino acid sequence shown in SEQ ID NO: 61, variant 182 (A56R) ORF encoding the amino acid sequence shown in SEQ ID NO: 62, and variant 182 (A56R) encoding the amino acid sequence shown in SEQ ID NO: 63. In some implementations, such vaccinia virus clones have a nucleic acid genome with at least 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.99% sequence identity with SEQ ID NO: 1.

[0212] In some embodiments, the provided vaccinia virus clone has a nucleic acid genome that has at least 95% sequence identity with SEQ ID NO: 1 and is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT (shown in SEQ ID NO: 77) at positions 106870 to 106880 of SEQ ID NO: 1; (vii) and SEQ ID NO: (viii) The nucleic acid sequence GTTTTCATTA corresponding to positions 111267 to 111276 of SEQ ID NO: 1 (shown in SEQ ID NO: 78); (viii) Adenine (A) corresponding to position 162715 of SEQ ID NO: 1; (ix) The nucleic acid sequence TACAGACACC corresponding to positions 165844 to 185853 of SEQ ID NO: 1 (shown in SEQ ID NO: 79); and (x) C corresponding to position 187805 of SEQ ID NO: 1.

[0213] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any of (i)-(x) above.

[0214] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any two of (i)-(x) above.

[0215] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any three of (i)-(x) above.

[0216] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any four of (i)-(x) above.

[0217] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any five of (i)-(x) above.

[0218] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any six of (i)-(x) above.

[0219] In some embodiments, the vaccinia virus clones provided herein comprise those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any seven of (i)-(x) above.

[0220] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any eight of (i)-(x) above.

[0221] In some embodiments, the vaccinia virus clones provided herein include those vaccinia virus clones having single-point mutations, insertions and / or deletions of nucleotide sequences selected from any nine of (i)-(x) above.

[0222] In some embodiments, the vaccinia virus clones provided herein comprise those vaccinia virus clones having single-point mutations, insertions, and / or deletions of nucleotide sequences selected from each of (i)-(x) above.

[0223] A. Exemplary Features

[0224] In some embodiments, clone lines derived from IHD-J exhibit better antitumor activity and lower pathogenicity / toxicity in in vitro and / or in vivo assays compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains). In some embodiments, clone lines derived from IHD-J exhibit better antitumor properties compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains). In some embodiments, clone lines derived from IHD-J exhibit lower toxicity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains). In some embodiments, clone lines derived from IHD-J exhibit similar antitumor properties and / or similar toxicity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains).

[0225] This document provides IHD-J clonal isolates that exhibit improved properties compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains lacking inserted heterologous DNA). In some embodiments, IHD-J clonal isolates exhibit better antitumor activity and lower toxicity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains lacking inserted heterologous DNA). In some embodiments, IHD-J clonal isolates exhibit improved or better antitumor activity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains lacking inserted heterologous DNA). In some embodiments, IHD-J clonal isolates exhibit lower toxicity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains lacking inserted heterologous DNA). In some embodiments, IHD-J clonal isolates exhibit similar toxicity and / or antitumor activity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains lacking inserted heterologous DNA).

[0226] In some embodiments, clonal isolates exhibiting improved or better antitumor activity compared to a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains) demonstrate, in the determination or method for assessing parameters indicative of antitumor activity, at least 120% to 1000% of the antitumor activity of the reference viral preparation (starting viral preparation or mixture or other reference strains or isolates, including recombinant strains), for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000%, or higher. Antitumor activity can be determined using any in vitro or in vivo test as described herein for parameters indicative of antitumor activity.

[0227] In some implementations, the clonal isolates provided herein exhibit increased production of extracellular enveloped virus (EEV) compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains). Vaccine viruses replicate in cells and produce intracellular viruses (IMV, intracellular mature virus; IEV, intracellular enveloped virus) and extracellular viruses (EEV, extracellular enveloped virus; CEV, cell-associated extracellular virus) (Smith et al. (1998) Adv Exp Med Biol. 440: 395-414). After replication of wild-type vaccinia virus lines, IMV accounts for approximately 99% of viral production. The IMV viral form is relatively stable in the external environment and is primarily responsible for inter-individual diffusion; however, due to low release efficiency within cells and sensitivity to complement and / or antibody neutralization, IMV viruses do not spread effectively within the infected host. In contrast, the EEV form is released into the extracellular environment and typically accounts for only about 1% of viral production (Smith et al. (1998) Adv Exp Med Biol. 440: 395-414). EEVs are responsible for viral spread within the infected host and are relatively easily degraded outside the host. Furthermore, the EEV form has evolved several mechanisms to inhibit its neutralizing effect in the blood. EEVs are relatively resistant to complement due to the incorporation of host cell complement inhibitors into their outer membrane envelope and the secretion of vaccinia virus complement control protein (VCP) into the local extracellular environment (Vanderplasschen et al. (1998) ProcNatl Acad Sci USA. 95(13): 7544-9). Furthermore, EEV exhibits relative resistance to neutralizing antibodies compared to IMV (Smith et al. (1997) Immunol Rev. 159: 137-54; Vanderplasschen et al. (1997) J Gen Virol. 78 (Pt 8): 2041-8). Unlike IMV, which is released only during or after cell death, EEV is released at an earlier time point after infection (e.g., 4–6 hours), thus leading to faster spread of the EEV form (Blasco et al. (1993) J Virol. 67(6):3319-25).

[0228] Because EEVs are relatively resistant to complement action and antibody-mediated neutralization, this viral form exhibits enhanced stability in the bloodstream and maintains its activity for a longer period after intravenous administration when grown in cell types of the same species (Smith et al. (1998) Adv Exp Med Biol. 440: 395-414; Vanderplasschen et al., (1998) Proc Natl Acad Sci US A. (13):7544-9). This is particularly important for repeat administration following elevated neutralizing antibody levels, and cancer therapies often require repeat administration. Therefore, increasing the EEV form of vaccinia virus and other vaccinia viruses can lead to enhanced systemic efficacy.

[0229] In some embodiments, the clonal isolates provided herein exhibit increased production of extracellular enveloped virus (EEV) compared to other clonal isolates derived from IDH-J or Copenhagen strains. In some embodiments, the clonal isolates provided herein exhibit increased production of extracellular enveloped virus (EEV) compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains), such as between 120% and 1000%, for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000%, or higher.

[0230] In some embodiments, after cell infection, greater than or approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20% of the infectious particles are EEVs. In some embodiments, after cell infection, greater than 5% of the infectious particles are EEVs. In some embodiments, after cell infection, greater than 10% of the infectious particles are EEVs. In some embodiments, after cell infection, greater than 15% of the infectious particles are EEVs. In some embodiments, after cell infection, greater than 20% of the infectious particles are EEVs.

[0231] In other embodiments, the clonal isolates provided herein exhibit reduced tumor and / or metastatic growth or increased tumor and / or metastatic shrinkage in in vitro or in vivo assays or models. Tumors can be harvested from subjects, weighed, and compared to the tumor weight harvested from subjects carrying tumors from the initiating viral preparation or mixture, or other reference strains or isolates (including recombinant strains). Tumor weight can also be compared to the tumor weight harvested from subjects treated with controls at the same time post-infection. Weight can be expressed as tumor volume / weight and / or tumor volume / weight ratio (tumor weight of control-treated animals / tumor weight of subjects treated with clonal isolates). It should be understood that, for example, a tumor weight ratio of 1.2 or 5 means, compared to a reference or control, that the virus causes reduced tumor / metastatic weight / growth or increased tumor / metastatic shrinkage, and 120% or 500% antitumor activity.

[0232] In some embodiments, the clonal isolates provided herein exhibit reduced tumor and / or metastatic growth or increased tumor and / or metastatic shrinkage. In some embodiments, the tumor / metastatic volume / weight ratio is greater than 1.0, e.g., greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or higher. In some embodiments, the increased tumor / metastatic shrinkage is at least 120% to 500%, e.g., 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or higher.

[0233] In some embodiments, the clonal isolates provided herein exhibit similar antitumor activity in assays or methods for evaluating indicative toxicity parameters, such as antitumor activity of parental viral preparations, mixtures, or other reference viral strains (including recombinant strains), compared to the starting viral preparation or mixture or other reference strains (including recombinant strains).

[0234] In some embodiments, the clonal isolates provided herein exhibit reduced tumor and / or metastatic volume, size, or weight in in vitro or in vivo assays or models. In some embodiments, the clonal isolates provided herein exhibit reduced tumor and / or metastatic volume, such as 0% to 99% toxicity or tumor and / or metastatic volume, size, or weight, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or lower toxicity or greater tumor and / or metastatic volume, size, or weight.

[0235] Parameters indicating toxicity or viability include, but are not limited to, reduced percentage of cell survival in 2D and 3D cell cultures, decreased subject weight, appearance of fever, rash or other allergic symptoms, fatigue or abdominal pain, tissue distribution of the virus, reduced or decreased subject survival rate, induction of the subject's immune response, amount of tumor antigen released, and reduced rate of acne formation. Toxicity or viability can be determined using any in vitro or in vivo test known to those skilled in the art.

[0236] In some embodiments, the clonal isolates provided herein exhibit lower toxicity compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains), said recombinant strains including, for example, recombinant strains with toxicity of 0% to 99% of starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains), such as less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or lower, including recombinant strains used in the determination or method of parameters indicative of toxicity. In some embodiments, the IHD-J clonal isolates provided herein exhibit toxicity of 0% to 99% compared to other clonal isolates derived from the IHD-J or Copenhagen strains, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or lower. In some embodiments, methods for assessing parameters indicating toxicity include quantifying the percentage of cell survival in cell culture. In some embodiments, methods for assessing parameters indicating toxicity include quantifying the percentage of cell survival in 2D and 3D cell cultures.

[0237] In some embodiments, the clonal isolates provided herein exhibit similar toxicity and / or cytotoxicity compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains), said recombinant strains including recombinant strains with antitumor activity between 70% and 120% of the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains), such as at least or about 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120%, including recombinant strains included in the determination or method for assessing parameters indicative of toxicity, for example.

[0238] In certain embodiments, the clonal isolates provided herein exhibit improved antitumor activity and lower toxicity (i.e., lower virulentness) compared to starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains). For example, the clonal isolates exhibit lower toxicity (i.e., lower virulentness) when administered to subjects at amounts that effectively induce antitumor activity. For the treatment of human subjects or other subjects of similar body size, exemplary therapeutic doses of the clonal isolates range from about or between 1 × 10⁻⁶. 6 Up to 1×10¹ 4 Between pfu, for example, approximately or between 1×10 7 Up to 1×10¹ 0 Between pfu, approximately or between 1×10 9 Up to 1×10¹ 0 Between pfu, for example at least or about 1 × 10 6 1×10 7 1×10 8 1×10 9 2×10 9 3×10 9 4×10 9 Or 5×10 9 PFU. For treatment of mice or other subjects of similar size, exemplary treatment doses of the cloned line range from about or between 1 × 10³ and 1 × 10⁻⁶. 9 Between pfu, for example, approximately or between 1×10 5 Up to 1×10 7 Between pfu, for example, at least or about 1×10³, 1×10 4 1×10 5 1×10 6 2×10 6 3×10 6 4×10 6 Or 5×10 6PFU. These effective doses can be determined empirically by those skilled in the art and depend on a variety of factors, including the subject, the condition or disease being treated, the stage or progression of the disease, the type of cancer, tumor, metastasis, or proliferation, and other factors. Dosing regimens may vary. In some embodiments, the clonal isolates provided herein exhibit 100% survival of the subject throughout the treatment regimen and do not cause weight loss or depletion in the subject during treatment. In one embodiment, the clonal isolates provided herein exhibit increased survival rates when administered to subjects compared to subjects receiving the same or similar therapeutic doses of other clonal isolates. In some embodiments, the clonal isolates provided herein exhibit 100% tumor growth inhibition throughout the treatment regimen.

[0239] The cloned viruses isolated herein can be obtained by plaque isolation of the IHD-J strain, which is propagated through repeated passages in a cell line. In some embodiments, the cloned isolates provided herein can be obtained by passage of the virus in chicken embryo yolk sac cultures, chicken embryo fibroblast (CEF) cells, HeLa S3 cells, confluent CV-1 cells, or BHK-21 cells. In some embodiments, the cloned isolates provided herein can be obtained by passage of the virus in confluent CV-1 (African green monkey kidney fibroblast culture) cells grown in 6-well plates and infected with a series of diluted vaccinia virus strains. The cloned isolates provided herein are sequence-homogeneous. The exemplary cloned viruses provided herein are cloned isolates exhibiting enhanced antitumor properties and reduced virulence.

[0240] III. Attenuated vaccinia virus strains

[0241] This document also provides recombinant vaccinia viruses that exhibit one or more modifications to attenuate viral virulence compared to wild-type or parental strains of the virus (e.g., compared to any isolated cloned virus strains described in Section I of this document). In some embodiments, this document provides a recombinant vaccinia virus that is attenuated (e.g., has reduced virulence) compared to the vaccinia virus strain VIP02. In some embodiments, this document provides a recombinant vaccinia virus that is attenuated (e.g., has reduced virulence) compared to the vaccinia virus strain shown in SEQ ID NO: 1. In some embodiments, the attenuated virus is a virus with low virulence to normal cells, such as low or reduced viral replication, cytolytic activity, or cytotoxicity to normal cells (e.g., non-tumor cells).

[0242] In some embodiments, the attenuated virus is a recombinant oncolytic vaccinia virus comprising: an inactivating mutation of B2R; a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3); and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0243] In some embodiments, the attenuated virus is a recombinant oncolytic virus comprising: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, or apoptosis-inducing proteins, or any combination thereof.

[0244] In some implementations, any vaccinia virus provided herein can be modified to include vaccinia growth factor (VGF) (McCart et al. (2001) Cancer Research 61:8751), thymidine kinase (TK) gene (WO 2005 / 047458), hemagglutinin (HA) gene (WO 2005 / 047458; and Zhang et al. (2007) Cancer Research 67:10038), F3 gene (also known as F14.5L; WO 2005 / 047458; Zhang et al. (2007) Cancer Research 67:10038), ribonucleotide reductase (Gammon et al. (2010) PLoSPathogens 6:e1000984), and serine protease inhibitors (e.g., SPI-1, SPI-2) (Guo et al. (2005) Cancer Research 65:9991; Yang et al.). (2007) Gene Therapy 14:638), ribonucleotide reductase gene F4L or I4L (Child et al. (1990) Virology 174:625; Potts et al. (2017) EMBO Mol. Med. 9:638), B2R (Eaglesham et al. (2019) Nature 566:259-263), B18R (Symons et al. (1995) Cell 81:551; Kirn et al. (2007) PLoS Medicine 4:e353), A48R (Hughes et al. (1991) J. Biol. Chem. 266:20103), B8R (Verardi et al. (2001) J. Virol. 75:11), B15R (Spriggs et al. (1992) Cell 71:145), A41R (Ng et al. (2001) Journal of General Virology 82:2095), A52R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162), F1L (Gerlic et al. (2013) Proc. Natl. Acad. Sci. USA 110:7808), E3L (Chang et al.(1992) Proc. Natl. Acad. Sci. USA89:4825), A44R-A46R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162), K1L (Bravo Cruz et al. (2017) Journal of Virology 91 :e00524), A48R, B18R, C11R and TK (Mejias-Perez et al. (2017) Molecular Therapy: Oncolytics 8:27) and other functional defects can achieve attenuation. In some implementations, it is known that the deletion or disruption of certain non-essential genes, such as J2R (thymidine kinase TK) (Buller et al. 1985), C11R (secreted epidermal growth factor-like) (Buller et al. 1988), A56R (hemagglutinin HA) (Shida et al. 1988), B8R (soluble interferon-γ receptor-like) (Verardi et al. 2001), and F14.5L (WO 2005 / 047458; Zhang et al. (2007) Cancer Research 67:10038), leads to reduced virulence.

[0245] In some embodiments, this document provides a recombinant vaccinia virus strain whose genome contains mutations in any of the genes described above that inactivate the gene, thereby attenuating the virus. In some embodiments, the viral gene is selected from hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, the inactivating mutation is a deletion of all or part of the viral gene. In some embodiments, the inactivating mutation is a deletion of the entire ORF of the viral gene. In some implementations, the inactivating mutation is a deletion of a portion of the ORF of the viral gene, which causes the encoded gene product to lose its function. In some implementations, the deleted ORF portion is a continuous sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides, up to the entire sequence of the viral gene's ORF.

[0246] In some embodiments, a gene region or its encoded gene product may be functionally defective using any of a variety of methods known to those skilled in the art. In some embodiments, the gene region or gene product may be functionally defective due to one or more mutations (e.g., substitution), truncation, or deletion of the gene region. In some embodiments, the gene region or gene product may be functionally defective due to mutations, truncation, or deletion of the promoter region controlling the expression of the gene region. In some embodiments, the gene region or gene product may be functionally defective due to mutations, truncation, or deletion of a polyadenylated sequence, thereby reducing or eliminating the translation of the polypeptide encoded by said gene region.

[0247] In some embodiments, the attenuated recombinant vaccinia virus of this disclosure, which has a defect in a given vaccinia virus gene, exhibits reduced production and / or activity of the gene product (e.g., mRNA gene product; polypeptide gene product). In some embodiments, the amount and / or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the amount and / or activity of the same gene product produced by wild-type vaccinia virus or control vaccinia virus without said gene alteration. For example, in some embodiments, the amount and / or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the amount and / or activity of the same gene product produced by VIP02 or vaccinia virus having the nucleic acid genome shown in SEQ ID NO: 1. In some embodiments, the amount and / or activity of the same gene product produced by the IHD-W1 strain or a vaccinia virus having the nucleic acid genome shown in SEQ ID NO: 2 is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%.

[0248] In some embodiments, the attenuated recombinant vaccinia virus of this disclosure with defects in its viral gene may have deletions in regions comprising designated gene regions or in adjacent gene regions containing the designated gene regions. For example, mutations and / or truncations and / or deletions in promoter regions can lead to reduced transcription of the gene region, resulting in defects. Defecting a gene region by incorporating a transcription termination element reduces or eliminates the translation of the polypeptide encoded by the gene region. Defecting a gene region can also be achieved using gene-editing enzymes or gene-editing complexes to reduce or eliminate transcription of the gene region. Defecting a gene region can also be achieved by utilizing competitive reverse promoter / polymerase occupancy to reduce or eliminate transcription of the gene region. Defecting a gene region can also be achieved by inserting nucleic acids into the gene region, thereby knocking out the gene region. In some cases, heterologous nucleic acids can be inserted into the viral gene, as described in the exemplary recombinant vaccinia virus strains in Section III herein.

[0249] In some embodiments, the OVV provided in this disclosure is thymidine kinase (TK) deficient. In some cases, the OVV of this disclosure contains all or part of the deletion of the vaccinia virus TK coding region, making the replicative recombinant oncolytic vaccinia virus TK deficient. For example, in some cases, the OVV of this disclosure contains a deletion in the J2R gene (i.e., the gene encoding viral thymidine kinase). See, for example, Mejia-Perez et al. (2018) Mol. Ther. Oncolytics8:27. In some cases, the OVV of this disclosure contains an insertion in the J2R region, resulting in reduced vaccinia virus TK expression or activity.

[0250] In some embodiments, any cloned vaccinia virus strain described in Section I herein, such as VIP02 or the vaccinia virus strain shown in SEQ ID NO: 1, may be further modified in its genome to attenuate its virulence. In some embodiments, the vaccinia virus strain is modified in one or more of the TK (J2R), hemagglutinin (HA), A35R, or B2R genes. In some embodiments, the modification renders the gene product encoded by the locus nonfunctional or defective. In some embodiments, all or part of the TK, HA, A35R, or B2R ORF is deleted.

[0251] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as insertion, mutation, or deletion, in the J2R gene encoding thymidine kinase (TK; SEQ ID NO: 66). In some embodiments, the TK locus has been reported to be non-essential for viral replication, and therefore its modification can reduce viral virulence, preventing viral replication in the brain or ovary and preserving the ability to preferentially replicate in tumor tissue (e.g., Buller et al. (1985) Nature, 317:813-815). In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain contains a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the recombinant vaccinia virus is a vaccinia virus named VIR13.

[0252] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as insertion, mutation, or deletion, at the B2R locus encoding the cytoplasmic cGAMP nuclease (poxin) (SEQ ID NO: 54). In some embodiments, the B2R locus has been reported to lead to vaccinia virus attenuation in a skin scratch model (E Eaglesham et al. 2019, Nature 566:259-263). In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as insertion, mutation, or deletion, in the B2R gene, and an inactivating mutation, such as insertion, mutation, or deletion, in the J2R gene. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain contains a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 48. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 48. In some implementations, the recombinant vaccinia virus is a vaccinia virus named VIR94.

[0253] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation at the A35R locus, such as insertion, mutation, or deletion. A35R is a virulence gene that regulates adaptive immune responses; its inactivation (e.g., through deletion) can lead to decreased viral replication capacity and reduced viral virulence (Brennan et al. 2015, J. Virol., 89:9986-9997). In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain contains a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the recombinant vaccinia virus is a vaccinia virus named VIR11. In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation in the A35R gene, such as insertion, mutation, or deletion, and an inactivating mutation in the J2R gene, such as insertion, mutation, or deletion. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 12. In some embodiments, the nucleotide genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 12. In some embodiments, the recombinant vaccinia virus is a vaccinia virus named VIR52.

[0254] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as insertion, mutation, or deletion, at the A56R locus encoding hemagglutinin (HA; SEQ ID NO: 67). In some embodiments, the HA locus has been reported to be non-essential for viral replication, allowing its modification to reduce viral virulence, preventing viral replication in the brain or ovary, and preserving the ability to preferentially replicate in tumor tissue (e.g., Shida et al. (1988) J. Virol., 62:4474-4480).

[0255] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as insertion, mutation, or deletion, in the F14.5L gene (SEQ ID NO: 65). In some embodiments, the attenuated recombinant vaccinia virus provided herein has an insertion, mutation, or deletion of the F3 gene product encoded by the F14.5L gene (SEQ ID NO: 64). In some embodiments, it has been reported that the F14.5L gene (also known as F3) is not essential for viral replication, such that its modification can reduce viral virulence, preventing viral replication in the brain or ovary and preserving the ability to preferentially replicate in tumor tissue (e.g., U.S. Patent Publication No. US2005 / 0031643).

[0256] Multiple methods can be used to assess or determine the attenuation level of a virus. These methods for measuring attenuation levels can be performed in vitro or in vivo and may include assessing changes in any or all of the following viral properties: a) viral mRNA synthesis, b) viral protein expression, c) viral DNA replication, d) viral plaque size, e) viral titer, or f) in vivo toxicity. Methods for assessing viral attenuation levels by in vitro and in vivo methods are known in the art, including, but not limited to, methods such as plaque assays and mouse models of viral pathogenicity. Exemplary methods for studying early, intermediate, and late transcription of vaccinia virus can be found in Broyles et al. Methods Mol. Biol. (2004) 269:135-142 and Wright et al. Methods Mol. Biol. (2004) 269:143-150. Methods for determining viral RNA transcripts and proteins include, but are not limited to, well-known techniques such as RNA hybridization and blotting, and immunohistochemistry.

[0257] IV. Recombinant viral strains with heterologous nucleic acids

[0258] This document provides recombinant viral strains with modified genomic sequences. In some embodiments, this document provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products. The heterologous gene product is not particularly limited, and in some embodiments, it may be a complement inhibitor, a T-cell or NK-cell escape agent, an immunostimulatory protein, an anti-angiogenic protein, an interferon regulator, an apoptosis-inducing protein, or any combination thereof. Therefore, in some embodiments, this document provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are complement inhibitors, T-cell or NK-cell escape agents, immunostimulatory proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0259] This document provides a recombinant oncolytic vaccinia virus comprising: an inactivating mutation of B2R; a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3); and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0260] This document also provides a recombinant oncolytic virus comprising: an inactivating mutation of at least one viral gene; and at least one heteronucleotide encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof. In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more immunomodulatory proteins, such as one or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9; and / or the at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding apoptosis-inducing proteins, such as iDED, iFas, or iCas9; and / or the at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more T cell or NK cell escape proteins, such as a group of vaccinia viruses ORF012, 203, and 018. (CPXV012-203-018) encodes a protein; and / or at least one heteronucleotide encoding one or more heterogeneous gene products comprises one or more heteronucleotides each encoding one or more complement inhibitors, such as CRASP-2 or miniFH; and / or one or more heteronucleotides encoding one or more complement inhibitors are introduced into a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein; and / or the at least one heteronucleotide encoding one or more heterogeneous gene products comprises one or more heteronucleotides each encoding one or more anti-angiogenic proteins, such as VEGF inhibitors, angiopoietin inhibitors, or Versikine; and / or the at least one heteronucleotide encoding one or more heterogeneous gene products comprises one or more heteronucleotides each encoding one or more therapeutic agents or diagnostic agents.

[0261] Inactivation mutations involve altering the expression and / or function of a gene product expressed by an inactivated viral gene through various means, such as gene disruption. Gene disruption can be achieved through, for example, gene deletion, nucleic acid insertion, nucleic acid mutation or substitution, gene knockout, early stop codon, transcription promoter modification, RNAi, or gene editing (such as CRISPR). In some embodiments, inactivation mutations are achieved through gene deletion and / or insertion (also referred to as introduction) of a heterologous nucleic acid encoding one or more gene products. In specific embodiments, inactivation mutations combine gene deletion with heterologous nucleic acid insertion at the locus. For example, in some methods of achieving inactivation mutations (such as through homologous recombination and other methods), the heterologous nucleic acid may be inserted into a deleted gene region. Therefore, it can be understood that in some embodiments, reference to a locus with inserted heterologous nucleic acid refers to a deleted locus of a gene that is inactivated by a complete or partial deletion of the gene. In some embodiments, gene deletion removes the entire sequence of the gene. In other embodiments, gene deletion is a partial deletion, i.e., the removal of a portion of the gene sequence. In one embodiment, the gene deletion is a partial deletion, i.e., removing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the gene sequence. In another embodiment, the gene deletion is a partial deletion, i.e., removing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the protein-coding sequence of the gene. In other embodiments, the gene deletion removes 100% of the gene sequence. In yet another embodiment, the gene deletion removes 100% of the protein-coding sequence of the gene. In one embodiment, the gene deletion removes at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides of the gene sequence. In another embodiment, the gene deletion is a partial deletion, i.e., the removal of at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides from the gene sequence. In one specific embodiment, a partial deletion in a gene results in a partial gene.

[0262] This article also provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise complement inhibitors, T-cell or NK-cell escapes, immunomodulatory proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof.

[0263] This article also provides a recombinant oncolytic virus comprising: a nucleic acid genome having at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, and a heterologous nucleic acid having at least one encoded heterologous gene product inserted into the genome.

[0264] The following subsections describe exemplary heterologous proteins. In addition to recombinant viral strains, any heterologous protein described herein can also be integrated into gene therapy vectors (such as AAV, lentiviruses, and retroviruses) or cell-based therapies (such as T-cell therapy expressing chimeric antigen receptors (CAR-T), natural killer (NK) cells, or tumor-infiltrating lymphocyte (TIL) therapy).

[0265] The viral strains provided herein comprise recombinant viral strains containing at least one heterologous nucleic acid encoding one or more heterologous gene products. In some embodiments, the recombinant viruses include, but are not limited to, vaccinia virus, vesicular stomatitis virus (VSV), Malaba virus (MARAV), measles virus (MV), myxoma virus, sheep pox virus, parvovirus, raccoon pox virus, Coxsackie virus, reovirus, Newcastle disease virus, Seneca Valley virus, Semleeki Forest virus, influenza virus, Echovirus, poliovirus (PV), adenoviruses (such as mammalian adenovirus and avian adenovirus), and herpesviruses (such as herpes simplex virus type 1, herpes simplex virus type 2, herpes simplex virus type 5, and herpes simplex virus type 6). Herpesviruses, Epstein-Barr virus, HHV6-HHV8 and cytomegalovirus), leviviruses (such as light RNA viruses, Enterobacterial bacteriophage MS2, allophytic viruses), poxviruses (such as vertebrate poxviruses, parapoxviruses, fowlpoxviruses, sheep poxviruses, rabbit poxviruses, swine poxviruses, molluscum poxviruses, insect poxviruses), papillomaviruses (such as polyomaviruses and papillomaviruses), paramyxoviruses (such as paramyxoviruses, type 1 parainfluenza virus (such as measles-rubella virus), Rubila virus (such as mumps virus), pneumonia virus (pneumonia) Viruses (human), human respiratory syncytial virus and metapneumovirus (such as avian pneumonia virus and human metapneumovirus)), picornaviruses (such as enteroviruses, rhinoviruses, hepatitis viruses (such as human hepatitis A virus), cardiviruses and oral thrush viruses), reoviruses (such as orthoreoviruses, circoviruses, rotaviruses, cytoplasmic polyhedroviruses, Fijiviruses, plant reoviruses and rice viruses), retroviruses (such as mammalian B retroviruses, mammalian C retroviruses, D retroviruses, BLV-HTLV retroviruses), lentiviruses (such as type 1) Human immunodeficiency virus (HIV) and HIV type 2 (such as HIV gp160), foamy viruses, flaviviruses (such as hepatitis C virus, dengue virus, and West Nile virus), hepatotropic DNA viruses (such as hepatitis B virus), cloacal viruses (such as alpha viruses (such as Sindbis virus) and rubella viruses (such as rubella virus)), rhabdoviruses (such as vesicular viruses, rabies virus, transient fever virus, and cellular rhabdovirus), arenaviruses (such as arenavirus, lymphocytic choriomeningitis virus, ipivirus, and Lassa virus), and coronaviruses (such as coronavirus and circovirus).

[0266] In some implementations, the recombinant virus comprises an oncolytic virus. In some implementations, the recombinant virus is a recombinant oncolytic virus. In some implementations, the recombinant virus (such as a recombinant oncolytic virus) is vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Malaba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, sheep oropharyngeal virus, parvovirus, raccoon pox virus, Coxsackie virus, reovirus, Newcastle disease virus, Seneca Valley virus, Semlikie Forest virus, mumps virus, influenza virus, Echovirus, or poliovirus (PV). In some implementations, the recombinant virus, such as a recombinant oncolytic virus, is a vaccinia virus.

[0267] In some embodiments, the recombinant virus is a non-oncolytic virus. In some embodiments, the recombinant virus is a non-vaccinia virus. In some embodiments, the recombinant virus includes vaccinia virus. In some embodiments, the recombinant virus is derived from the Copenhagen strain.

[0268] In certain embodiments, the recombinant virus is a virus derived from IHD-J. In some embodiments, the recombinant virus is a virus derived from VIP02. In some embodiments, this document provides a recombinant virus (such as a recombinant oncolytic virus) comprising one or more mutations, insertions, deletions, or substitutions (replacements) of nucleic acids, or other modifications to the viral genome sequence. In some embodiments, this document provides a modified VIP02 strain whose genome sequence has been modified compared to the genome sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant virus is derived from a virus having the nucleic acid genome shown in SEQ ID NO: 1, whose genome has been modified by inserting nucleic acids encoding heterologous gene products.

[0269] Methods for generating recombinant viruses using recombinant DNA technology are well known in the art (see, for example, U.S. Patent Nos. 4,769,330; 4,603,112; 4,722,848; 4,215,051; 5,110,587; 5,174,993; 5,922,576; 6,319,703; 5,719,054; 6,429,001; 6,589,531; 6,573,090; 6,800,288; 7,045,313; He et al. (1998) PNAS US A. 95(5): 2509-2514. Racaniello et al., (1981) Science 214:916-919). Methods for generating recombinant vaccinia virus can also be found in the embodiments described herein.

[0270] In some implementations, recombinant viruses have a high capacity to carry insertable foreign genes. For example, the vaccinia virus genome has a high capacity to carry foreign genes, allowing the insertion of foreign DNA fragments up to 25 kb. The genomes of several vaccinia virus strains have been fully sequenced, and many essential and non-essential genes have been identified. Due to the high sequence homology between different strains, genomic information from one vaccinia virus strain can be used to design and generate modified viruses for other strains. Finally, the technology for producing modified vaccinia virus strains through genetic engineering is well-established (Moss, Curr. Opin. Genet. Dev. 3: 86-90 (1993); Broder and Earl, Mol. Biotechnol. 13: 223-245 (1999); Timiryasova et al., Biotechniques 31: 534-540 (2001)).

[0271] The sites for insertion into heterologous nucleic acid molecules are known in the art and have been described for various viral vectors (see, for example, 5,166,057; 5,266,489; 6,338,846; 6,248,320; 6,221,646; 6,841,158; 7,101,685; 7,001,760 and references therein). Heterologous nucleic acid molecules are typically inserted into non-coding regions or coding regions of genes that are not essential for viral replication. For example, in vaccinia virus, the insertion site for a heterologous DNA molecule can be in an intergenic region, a non-coding region, and / or a non-essential gene or gene region, including but not limited to the thymidine kinase (TK) gene, hemagglutinin (HA) gene, F14.5L (see, for example, U.S. Patent Publication No. 2005-0031643), VGF gene (see, for example, U.S. Patent Publication No. 2003-0031681), Hind III F, F13L, or Hind III M (see, for example, U.S. Patent No. 6,548,068); hemorrhagic regions or type A inclusion body regions (ATI) (see, for example, U.S. Patent Nos. 6,265,189 and 6,596,279); A33R, A34R, A36R, or B5R genes (see, for example, Katz et al., (2003) J. Virology 77:12266-12275); SalF7L (see, for example, Moore et al., (1992) EMBO J. 11:1973-1980); N1L (see, for example, Kotwal et al. (1989) Virology 171:579-587); M1 λ (see, for example, Child et al. (1990) Virology. 174:625-629); HR, HindIIII-MK, HindIII-MKF, HindIII-CNM, RR or BamF (see, for example, Lee et al. (1992) J Virol. 66:2617-2630); C21L (see, for example, Isaacset et al. (1992) Proc Natl Acad Sci USA). 89:628-632), host-range region genes K1L and C7L, A35R (see, for example, U.S. Patent Nos. 6,265,189; 7,045,313; U.S. Patent Publication Nos. 2005-0244428; 2006-0159706; Coupar et al. J. Gen. Virol. (2000) 81: 431-439; Smith et al. (1993) Vaccine 11(1): 43-53).If more than one gene expression cassette is inserted, these insertions can occur at the same or different insertion sites. Alternatively, a heterologous nucleic acid molecule can be inserted into the required gene, and the virus can be produced using a cell line used for packaging the virus.

[0272] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces a non-essential gene or region in the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces a hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L locus, or any combination thereof, in the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces the F14.5L locus. The F14.5 locus encodes a viral membrane protein. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces the A35R locus. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces the J2R locus. In some embodiments, "insertion into a locus" means that the locus contains a partial deletion, and the heterologous nucleic acid replaces the deletion. In some embodiments, "insertion into a locus" means that an insertion into a locus is performed, but no part of the endogenous locus is deleted. In some embodiments, "replacement into a locus" means that the entire locus is deleted and replaced by a heterologous nucleic acid.

[0273] Mutations in non-essential vaccinia virus genes can also contribute to increased viral attenuation. Therefore, inserting a heterologous expression cassette into a non-essential gene (such as the TK gene) can attenuate the virus in two ways: through gene mutation and increased transcriptional and / or translational load. For the methods described herein, mutations in non-essential genes are not required; however, one or more non-essential genes can be modified to enhance the attenuation effect of the gene expression cassette. Subsequently, viral attenuation can be reduced (i.e., the virus exhibits increased replication) by removing the expression cassette and replacing it with a non-coding sequence, thereby keeping the gene inactive. Therefore, removing or replacing the gene expression cassette reduces viral transcriptional and / or translational load, resulting in reduced viral attenuation.

[0274] In some embodiments, at least one heteronucleotide encoding one or more heterogeneous gene products is fused to a gene encoding a viral membrane protein in the viral genome. In some embodiments, at least one heteronucleotide encoding one or more heterogeneous gene products is fused to a gene encoding a viral membrane protein to produce a fusion protein. In some embodiments, at least one heteronucleotide encoding one or more heterogeneous gene products is fused to a viral membrane protein to produce a fusion protein. In some embodiments, the gene encoding a viral membrane protein fused to at least one heteronucleotide encoding one or more heterogeneous gene products is F14.5L. In some embodiments, the viral membrane protein is F14.5L. In some embodiments, the viral membrane protein is F14.5L and is fused at the C-terminus of F14.5L. In some embodiments, the fusion protein is incorporated into the outer membrane of an intracellular mature virus (IMV) (e.g., the IMV of vaccinia virus). These fusion proteins containing the viral membrane protein F14.5L are expected to be incorporated into the outer membrane of IMV viral particles, which could give them the ability to resist complement inactivation in the blood.

[0275] Modifications may include mutations, insertions, deletions, or substitutions of nucleic acids, or other modifications to the viral genome sequence. For example, the virus provided herein may be modified to contain one or more heterologous nucleic acid molecules inserted into the viral genome or heterologous nucleic acid molecules substituted into the viral genome. The viral gene may be substituted with a homologous gene or a different gene from another virus. In one embodiment, the modification comprises inserting or substituting one or more nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000, or more nucleotides. In some embodiments, the modification comprises the absence of one or more nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000 or more nucleotides. In some embodiments, the modification comprises the substitution of one or more nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000 or more nucleotides.

[0276] Modification involves inserting and / or substituting (replacing) nucleic acids, or modifying the viral genome sequence with heterologous nucleic acids. Typically, the heterologous gene is a gene encoding a non-viral protein. For example, a heterologous nucleic acid molecule encoding a heterologous gene can be inserted. In some embodiments, the heterologous nucleic acid replaces all or part of the viral gene. In other embodiments, the virus provided herein can be modified by inserting one or more heterologous nucleic acids. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous nucleic acid molecules can be inserted. The heterologous nucleic acid molecule can contain an open reading frame or can be a non-coding sequence. Typically, the inserted heterologous nucleic acid is a continuous nucleotide sequence containing an open reading frame and corresponding to a gene coding region. The inserted or substituted gene can be transcribed and / or translated from the viral genome after infection of host cells (such as tumor cells). As described below, the heterologous nucleic acid can contain regulatory sequences that control gene expression. For example, the heterologous nucleic acid can be operatively linked to a promoter to express an open reading frame. In some embodiments, the promoter has 70%, 80%, 90%, or 100% sequence identity with the sequence shown in SEQ ID NO: 68, 69, 70, 71, or 72.

[0277] Modifications to the viral genome provided herein can result in alterations to viral characteristics or properties. Exemplary modifications include changes to parameters indicative of antitumor activity and / or toxicity. For example, insertions, mutations, or deletions can reduce the pathogenicity of clonal lines, such as reducing the infectivity, toxicity, replication capacity, or accumulation of vaccinia virus in non-tumor organs or tissues. Exemplary nucleic acid insertions, deletions, mutations, and / or substitutions are those that result in better antitumor properties and lower toxicity of vaccinia virus relative to clonal lines and / or starting viral preparations or mixtures or other reference lines or isolates (including recombinant lines) without said modifications. In some embodiments, nucleic acid insertions, deletions, mutations, and / or substitutions are those that result in similar antitumor properties and toxicity of vaccinia virus relative to clonal lines and / or starting viral preparations or mixtures or other reference lines or isolates (including recombinant lines) without said modifications. In some embodiments, modifications to the viral genome reduce virulence relative to clones and / or starting viral formulations or mixtures or other reference strains or isolates (including recombinant strains) that do not contain the modifications. In some embodiments, insertions, mutations, or deletions include, but are not limited to, those that increase viral antitumor activity and reduce viral virulence relative to clones and / or starting viral formulations or mixtures or other reference strains or isolates (including recombinant strains) that do not contain the modifications.

[0278] In some embodiments, insertions, mutations, or deletions include, but are not limited to, those that increase the ability of a clonal virus strain to evade the host's immune system relative to a clonal strain that does not contain the modification and / or a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, insertions, mutations, or deletions include, but are not limited to, alterations that increase the ability of a clonal virus strain to stimulate the host's immune system relative to a clonal strain that does not contain the modification and / or a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, insertions, mutations, or deletions include, but are not limited to, those that increase the host's anti-angiogenic activity relative to a clonal strain that does not contain the modification and / or a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, insertions, mutations, or deletions include, but are not limited to, those that increase the host's apoptotic activity relative to a clonal strain that does not contain the modification and / or a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains).

[0279] In some embodiments, one or more heterologous nucleic acid molecules may encode, for example, an anti-apoptotic gene product or fragment thereof, such as a gene product that modulates the host's apoptotic response; angiogenesis gene product or fragment thereof, such as a gene product that modulates the host's angiogenesis response; or immune system gene product or fragment thereof, such as a gene product that modulates the host's immune response. In some embodiments, the gene product or fragment thereof that modulates the host's immune response may increase the host's immune system's ability to evade complement inhibition, relative to clones and / or initiating viral preparations or mixtures or other reference strains or isolates (including recombinant strains) that do not contain the aforementioned modification. In some embodiments, the gene product that modulates the host's immune response may increase the activity of the host's immune system, relative to clones and / or initiating viral preparations or mixtures or other reference strains or isolates (including recombinant strains) that do not contain the aforementioned modification.

[0280] In some embodiments, the recombinant virus is a vaccinia virus whose genome sequence has been modified compared to the genome sequence shown in SEQ ID NO: 1 or a sequence having at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the recombinant virus is a vaccinia virus whose genome sequence has been modified compared to the genome sequence shown in SEQ ID NO: 1. The large genome size of the vaccinia virus provided herein allows for the insertion of large and / or multiple heterologous DNA nucleotide sequences into the viral genome (Smith and Moss (1983) Gene 25(1):21-28). The virus provided herein can be modified by inserting or substituting one or more nucleotides. In one embodiment, the modification comprises inserting or substituting one or more nucleotides, such as inserting or substituting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000, 4000, 5000 or more nucleotides. In some embodiments, one or more heterologous DNA molecules are inserted into a locus of the viral genome, as described herein. In some embodiments, one or more heterologous DNA molecules are inserted into a non-essential region of the viral genome; for example, the DNA molecule is inserted into a locus that is not essential for viral replication in proliferating cells (such as tumor cells). Exemplary insertion sites are known in the art and are provided herein. In some embodiments, the recombinant vaccinia virus provided herein may contain inactivating mutations in the viral gene, such as any inactivating mutation described herein, such as a complete or partial deletion of the viral gene. In such embodiments, one or more heterologous nucleic acids may be inserted into or replace such loci. In some embodiments, the recombinant virus is a virus modified compared to the genome sequence shown in SEQ ID NO: 1, wherein one or more heterologous nucleic acids are inserted and one or more viral loci are inactivated, such as by gene deletion. The modified recombinant virus may be any virus provided herein having the genome shown in SEQ ID NO: 1, or a genome at least 99% identical to that of SEQ ID NO: 1, or any other virus generated by introducing heterologous DNA as described herein. In some embodiments, the recombinant virus is modified in its genome sequence compared to the genome sequence shown in SEQ ID NO: 1, and its amino acid sequence exhibits at least 85%, 90%, or 95% sequence identity with the sequence shown in SEQ ID NO: 1. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

[0281] In some embodiments, the recombinant virus may be modified to express exogenous or heterologous genes. Exemplary exogenous gene products include proteins involved in apoptosis, angiogenesis, and / or immune system regulation. In some embodiments, the gene product includes proteins that affect the host's apoptosis pathway, such as caspase-9, the death effector domain (DED) of Fas-associated death domain protein (FADD), and Fas. In some embodiments, the gene product includes proteins that affect the host's angiogenesis pathway, such as vascular endothelial growth factor (VEGF) and Versikine (VK). In some embodiments, the gene product includes proteins that affect the host's immune system, such as miniFH complement regulatory factor, CRASP-2 (CRASP-2), vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and the human LIGHT variant (hmLIGHT). The characteristics of such gene products are described herein and elsewhere.

[0282] Specifically, the viruses provided herein can be modified to express genes in vivo and in vitro. In some embodiments, the virus can be modified to express two or more gene products, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products, wherein any combination of two or more gene products can be one or more detectable gene products. In one embodiment, the virus can be modified to express gene products associated with apoptosis. In another example, the virus can be modified to express two or more gene products for generating fusion proteins. In some examples, one or more proteins involved in angiogenesis can be expressed together. When two or more heterologous genes are introduced, these genes can be regulated under the same or different regulatory sequences, and these genes can be inserted into the same or different regions of the viral genome, which can be done through single or multiple gene manipulation steps. In some embodiments, one gene can be under the control of a constitutive promoter, while the second gene can be under the control of an inducible promoter. Methods for inserting two or more genes into a virus are known in the art, and a variety of exogenous genes, regulatory sequences, and / or other nucleic acid sequences can be readily applied to a variety of viruses.

[0283] The viruses described herein can be modified by insertion, deletion, substitution, or mutation as described herein. Standard methods for modifying viruses by inserting, deleting, substituting, and mutating nucleic acids are well known in the art. Such methods include in vitro recombination techniques, synthetic methods, direct cloning, and in vivo recombination methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989), and the embodiments disclosed herein. Techniques for generating recombinant viruses include nucleic acid transfer protocols, various nucleic acid manipulation techniques, nucleic acid amplification protocols, and generally involve the use of standard molecular biology techniques to generate gene cassettes or transfer vectors. See, for example, U.S. Patent Nos. 5,494,807 and 5,185,146, which describe exemplary methods for generating recombinant vaccinia virus and other molecular biology techniques known in the art. Methods for generating recombinant viruses using recombinant DNA technology are well known in the art (see, for example, U.S. Patent Nos. 4,769,330; 4,603,112; 4,722,848; 4,215,051; 5,110,587; 5,174,993; 5,922,576; 6,319,703; 5,719,054; 6,429,001; 6,589,531; 6,573,090; 6,800,288; 7,045,313; He et al. (1998) PNAS 95(5): 2509-2514; Racaniello et al., (1981) Science 214: 916-919; and Hruby et al., (1990) Clin Micro Rev.). Methods for generating recombinant vaccinia virus are well known in the art (see, for example, Hruby et al., (1990) Clin Micro Rev. 3:153-170; U.S. Patent Publication No. 2005-0031643, now U.S. Patent Nos. 7,588,767, 7,588,771, 7,662,398 and 7,045,313).

[0284] In some implementations, homologous recombination can be used to introduce insertions or deletions of nucleic acid molecules into target sequences of interest. Methods using nucleic acid tools (such as vectors, plasmids, promoters, and other regulatory sequences) are well known in the art for a wide variety of viral and cellular organisms. Nucleic acid amplification protocols include, but are not limited to, polymerase chain reaction (PCR), or amplification via viruses or organisms (such as, but not limited to, yeast, bacteria, insect, or mammalian cells). Nucleic acid transfer protocols include electroporation, calcium chloride transformation / transfection, liposome-mediated nucleic acid transfer, or others. A variety of tools for modifying nucleic acids are available from many different sources, including a wide range of commercial sources. For example, point mutations or small insertions or deletions can be introduced into genes of interest using oligonucleotide-mediated site-directed mutagenesis. In another instance, homologous recombination can be used to introduce mutations into nucleic acid sequences or to insert or delete nucleic acid molecules into target sequences of interest. In some instances, positive or negative selection pressure can be used to select for nucleic acid mutations, insertions, or deletions in specific genes. See, for example, *Contemporary Molecular Biology Techniques* (edited., Ausubel et al.). Those skilled in the art, based on their knowledge and design choices, will be able to readily select appropriate tools and methods for gene modification for any specific virus. In some embodiments, plasmids are used for homologous recombination to construct recombinant viruses. In some embodiments, gene splicing is used to connect two fragments to construct plasmids. In some embodiments, the primers used to amplify the two fragments contain 70%, 80%, 90%, or 100% of SEQ ID NO: 14, 15, 16, 17, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 94, 95, 96, or 97.

[0285] Insertion, deletion, substitution, or mutation can specifically target specific sequences in the viral genome. Such sequences in the viral genome include, but are not limited to, intergenetic sequences, regulatory sequences, sequences of unknown function, gene-coding sequences, or non-essential regions of the viral genome. For many viruses, viral genome regions that can be modified are known in the art.

[0286] In some embodiments, the recombinant virus (such as a recombinant oncolytic virus) contains an inactivating mutation in at least one viral gene. The inactivating mutation is not particularly limited, and in some embodiments, it can be any mutation that reduces or eliminates the function of the viral gene's gene product compared to the absence of the inactivating mutation. In some embodiments, the inactivating mutation is a deletion of all or part of at least one viral gene. In some embodiments, the deletion of at least one viral gene refers to the deletion of the entire ORF of the viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of a portion of the ORF of the viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of a portion of the ORF of the viral gene sufficient to render the encoded gene product nonfunctional. In some embodiments, the at least one viral gene is selected from hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, the at least one viral gene comprises two or more viral genes selected from hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, the at least one viral gene is A35R. In some embodiments, the at least one viral gene is J2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene comprises A35R and J2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene comprises B2R and J2R.

[0287] Heterologous nucleic acid molecules are typically inserted into intergenic regions of the viral genome or into loci encoding non-essential viral gene products. Insertion of heterologous nucleic acids at these sites generally does not significantly affect viral infection or replication in target tissues. Examples of insertion sites include, but are not limited to, J2R (thymidine kinase (TK)), A56R (hemagglutinin (HA)), F14.5L, vaccinia growth factor (VGF), A35R, N1L, E2L / E3L, K1L / K2L, superoxide dismutase locus, 7.5K, C7-K1L (host-range gene region), B13R+B14R (hemorrhagic region), A26L (type A inclusion body region (ATI)), or I4L (ribonucleotide reductase large subunit) locus. The viral insertion sites provided herein also include sites corresponding to intragenetic regions described in other vaccinia viruses (such as the Modified Vaccinia Virus Ankara (MVA), an exemplary site shown in U.S. Patent No. 7,550,147) and NYVAC (an exemplary site shown in U.S. Patent No. 5,762,938). In some embodiments, insertion, deletion, substitution, and / or mutation sites include J2R, F14.5L, and / or A35R.

[0288] For example, generating recombinant vaccinia virus expressing a heterologous gene product typically involves using a recombinant plasmid containing a heterologous nucleic acid (optionally operatively linked to a promoter) and flanking the heterologous nucleic acid with vaccinia virus DNA sequences to facilitate homologous recombination and gene insertion into the viral genome. Typically, the viral DNA flanking the heterologous gene is complementary to non-essential segments of the vaccinia virus DNA, allowing the gene to be inserted at a non-essential or any other location. The recombinant plasmid can be grown and purified in *E. coli* and then introduced into suitable host cells, such as, but not limited to, CV-1, BSC-40, BSC-1, and TK-143 cells. The transfected cells are then superinfected with vaccinia virus, which initiates its replication cycle. The heterologous DNA can be incorporated into the vaccinia virus genome via homologous recombination and packaged into infected progeny. The recombinant virus can be identified using methods known in the art, such as detecting the expression of the heterologous gene product, or using positive or negative selection methods (US Patent No. 7,045,313). In some embodiments, recombinant viruses are generated by homologous integration of a plasmid into a region of the viral genome corresponding to the J2R gene. In some embodiments, recombinant viruses are generated by homologous integration of a plasmid into a region of the viral genome corresponding to the A35R gene. In some embodiments, recombinant viruses are generated by homologous integration of a plasmid into a region of the viral genome corresponding to the F14.5L gene. In some embodiments, recombinant viruses are generated by homologous integration of one plasmid into a region of the viral genome corresponding to the J2R gene and another plasmid into a region of the viral genome corresponding to the F14.5L gene. In some embodiments, recombinant viruses are generated by homologous integration of one plasmid into a region of the viral genome corresponding to the J2R gene and another plasmid into a region of the viral genome corresponding to the A35R gene. In some embodiments, recombinant viruses are generated by homologous integration of one plasmid into a region of the viral genome corresponding to the F14.5L gene and another plasmid into a region of the viral genome corresponding to the A35R gene. In some implementations, recombinant viruses are generated by homologously integrating one plasmid into a region of the viral genome corresponding to the J2R gene, and another plasmid into a region of the viral genome corresponding to the A35R gene, and yet another plasmid into a region of the viral genome corresponding to the F14.5L gene.

[0289] In another example, recombinant vaccinia virus expressing a heterologous gene product can be generated by direct cloning (see, for example, U.S. Patent No. 6,265,183 and Scheiflinger et al. (1992) Proc. Natl. Acad. Sci. USA 89: 9977-9981). In this method, the heterologous nucleic acid (optionally operatively linked to a promoter) is flanked by restriction endonuclease cleavage sites for insertion into the unique restriction endonuclease sites of the target virus. Viral DNA is purified using standard techniques and cleaved with sequence-specific restriction endonucleases at unique sites in the viral genome. Any unique site in the viral genome can be used, provided that modification of the site does not interfere with viral replication. Typically, the insertion is at a site located in a non-essential region of the viral genome. For example, exemplary modifications described herein involve inserting a foreign DNA sequence into NotI-digested viral DNA.

[0290] In some instances, heterologous nucleic acids may also contain one or more regulatory sequences to regulate the expression of open reading frames encoding heterologous RNA and / or proteins. Regulatory sequences suitable for functioning, for example, in mammalian host cells are known in the art. Expression may also be influenced by one or more protein or RNA molecules expressed by the virus. Gene regulatory elements (such as promoters and enhancers) possess cell-type-specific activity and can be activated by certain inducible factors (such as hormones, growth factors, cytokines, cell inhibitors, radiation, heat shock) through response elements. Using such regulatory elements as internal promoters to drive gene expression in viral vector constructs allows for controlled and restricted expression of these genes.

[0291] In some embodiments, a heterologous nucleic acid encoding one or more heterologous gene products is operatively linked to a promoter. In some embodiments, one or more heterologous nucleic acids encoding one or more heterologous gene products are operatively linked to a promoter to express heterologous RNA and / or proteins. For example, the heterologous nucleic acid operatively linked to a promoter is also referred to as an expression cassette. Therefore, the virus provided herein can have the ability to express one or more heterologous genes. Gene expression may include expressing a protein encoded by a gene and / or expressing an RNA molecule encoded by a gene. In some embodiments, the virus provided herein can express a foreign gene at a sufficiently high level to allow harvesting the product of the foreign gene from a tumor. The expression of the heterologous gene can be controlled by a constitutive promoter or by an inducible promoter. In other instances, organ- or tissue-specific expression can be controlled by a regulatory sequence. To achieve expression only in a target organ (e.g., a tumor to be treated), the foreign nucleotide sequence can be linked to a tissue-specific promoter and used for gene therapy. Such promoters are well known to those skilled in the art (see, for example, Zimmermann et al., Neuron 12: 11-24 (1994); Vidal et al., EMBO J. 9: 833-840 (1990); Mayford et al., Cell 81: 891-904 (1995); and Pinkert et al., Genes & Dev. 1: 268-76 (1987)).

[0292] Exemplary promoters for expressing heterologous genes are known in the art. Heterologous nucleic acids can be operatively linked to natural promoters or non-viral, naturally occurring heterologous promoters. Any suitable promoter can be used, including synthetic promoters, naturally occurring promoters, and modified promoters. Exemplary promoters include synthetic promoters, including synthetic viral promoters and animal promoters. Natural or heterologous promoters include, but are not limited to, viral promoters, such as vaccinia virus promoters and adenovirus promoters.

[0293] In some embodiments, the promoter is a poxvirus promoter, such as a vaccinia virus promoter. Therefore, in some embodiments, the promoter is a poxvirus promoter or a variant or derivative thereof, such as a vaccinia virus promoter. In some embodiments, the promoter is a vaccinia virus promoter. The vaccinia virus promoter used to express one or more heterologous genes can be a synthetic promoter or a natural promoter, and includes early, intermediate, early / late, and late vaccinia virus promoters. Exemplary vaccinia virus promoters for controlling heterologous gene expression include, but are not limited to, 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, LEO, P7.5k, P11k, PSE, PSEL, PSL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b, or K1 promoters. Therefore, in some implementations, the nucleic acid encoding the heterologous gene product is operatively linked to a promoter selected from the 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, LEO, P7.5k, P11k, PSE, PSEL, PSL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b, and K1 promoters. Other viral promoters include, but are not limited to, adenovirus late promoters, vaccinia ATI promoters, or T7 promoters. Strong late promoters can be used to achieve high-level expression of the heterologous gene. Early and mid-stage promoters can also be used. In one instance, the promoter comprises early and late promoter elements, such as the modified H5 promoter, PmH5, which comprises natural early and late vaccinia promoter regions, a synthetic early / late vaccinia PSEL promoter, and a PSE synthetic early promoter (Hammond et al., Journal of Virological Methods 66:1, 135-138(1997); Stritzker et al., Journal of Virology 88:19, 11556-11567 (2014; Kugler et al., Virol J. 16: 100 (2019)). In some embodiments, the promoter is a synthetic strong early promoter (SSE). In some embodiments, the promoter is a strong early / late promoter (SEL).

[0294] In some embodiments, the promoter is selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter has the amino acid sequence shown in any of SEQ ID NO: 29, 53, 55, 68, 69, 70, 71, or 72. In some embodiments, the promoter has the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the promoter is a synthetic strong early promoter (SSE) and contains the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the promoter has the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the promoter is a strong early / late promoter (SEL) and contains the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the promoter is a poxvirus promoter, and said poxvirus promoter is mH5. In some embodiments, the poxvirus promoter is mH5 and contains the amino acid sequence shown in SEQ ID NO: 53.

[0295] Combinations of different promoters can be used to express different gene products in the same virus or two different viruses. The viruses described in this article may exhibit differences in properties, such as the degree of attenuation, due to the use of stronger and weaker promoters. For example, in vaccinia virus, the early / late and late promoters of synthesis are relatively strong promoters, while the early promoter of synthesis in vaccinia virus is a relatively weak promoter (see, for example, Chakrabarti et al. (1997) BioTechniques23(6) 1094-1097).

[0296] As is known in the art, regulatory sequences can allow constitutive expression of exogenous genes or inducible expression of exogenous genes. Furthermore, regulatory sequences can control the expression level of exogenous genes. In some instances, such as in gene product manufacturing and harvesting, regulatory sequences can lead to constitutive, high-level expression of genes. In some instances, such as in the harvesting of anti-(gene product) antibodies, regulatory sequences can lead to constitutive, low-level expression of genes. In examples of cancer therapy, therapeutic proteins can be controlled by internal or external inducible promoters.

[0297] Therefore, the expression of heterologous genes can be controlled by constitutive or inducible promoters. Inducible promoters can be used to provide tissue-specific expression of heterologous genes, or they can be induced by adding regulatory molecules to provide time-specific induction of the promoter. In some instances, inducible expression can be controlled by cellular or other factors present in tumor cells or virus-infected tumor cells. In other instances, inducible expression can be controlled by an applicable substance, including IPTG, RU486, or other known inducing compounds. Additional regulatory sequences can be used to control the expression of one or more heterologous genes inserted into a virus. Those skilled in the art can use any of a variety of regulatory sequences, depending on known factors and design preferences.

[0298] In some embodiments, one or more heterologous gene products comprise therapeutic agents or diagnostic agents. In some embodiments, one or more heterologous gene products (e.g., therapeutic agents or diagnostic agents) are selected from anticancer agents, antimetastatic agents, antiangiogenic agents, immunomodulatory molecules, antigens, cytokine degradation genes, genes for tissue regeneration and reprogramming human cells to pluripotency, enzymes that modify substrates to produce detectable products or signals or can be detected by antibodies, proteins that can bind contrast agents, genes for optical imaging or detection, genes for positron emission tomography (PET) imaging, and genes for MRI. In some embodiments, one or more heterologous gene products (e.g., therapeutic agents or diagnostic agents) comprise therapeutic agents selected from hormones, growth factors, cytokines, chemokines, co-stimulatory molecules, ribozymes, transport proteins, single-chain antibodies, antisense RNA, prodrug-converting enzymes, siRNA, microRNA, toxins, antitumor oligopeptides, mitotic inhibitor proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics, angiogenesis inhibitors, tumor inhibitors, cytotoxic proteins, cell growth inhibitory proteins, and tissue factors.

[0299] In some of these embodiments, the recombinant virus (such as a recombinant oncolytic virus) comprises a nucleic acid sequence containing at least one heterologous nucleic acid encoding one or more heterologous gene products, such as any heterologous gene product described herein (as in Sections III, A, B, C, and D), comprising, for example, one or more heterologous gene products selected from complement inhibitors, T-cell escape proteins or NK-cell escape proteins, immunostimulatory proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof, and optionally comprising an inactivating mutation of at least one viral gene, such as hemagglutinin (HA) in the viral genome. One or more of the following loci: J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L; optionally, one or more of these viral genes are one or more of B2R, J2R, A35R, and A56R, or any combination thereof.

[0300] In some of these embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in any one of SEQ ID NO: 85, 86, 88, and 90, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 85. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in SEQ ID NO: 85, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 85. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 48, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 48. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 80, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 80. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 82, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 82. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 84, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 84. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 86. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 87.In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 88. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 89. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 90. In some embodiments, the genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 91, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 91. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in SEQ ID NO: 92, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 92. In some embodiments, the nucleotide genome of the recombinant oncolytic virus comprises the nucleotide sequence shown in SEQ ID NO: 93, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 93.

[0301] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escapes, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises A35R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 3, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 3.

[0302] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escapes, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises A35R and J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 12, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 12.

[0303] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of proteins derived from vaccinia viruses ORF012, 203, and 018. The protein encoded by (CPXV012-203-018), wherein at least one heteronucleotide encoding one or more heterogene products comprises one or more heteronucleotides each encoding one or more complement inhibitors, the heteronucleotides being introduced into a viral membrane gene to produce a fusion gene encoding a fusion protein, optionally wherein the viral membrane gene is F14.5L, optionally wherein it is fused at the C-terminus of the F14.5L protein, and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 10, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 10.

[0304] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escapes, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 4.

[0305] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escapes, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 11, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 11.

[0306] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise inhibitors of VEGF and / or Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 13, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 13.

[0307] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is achieved by inserting a heterologous nucleic acid encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises SEQ ID NO: The nucleic acid sequence shown in SEQ ID NO: 47, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 47.

[0308] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding a cell apoptosis-inducing protein, optionally wherein the cell apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas), or inducible Cas9 (iCas9), optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 7, 8, or 9, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 7, 8, or 9.

[0309] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 49, 50, or 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 49, 50, or 93.

[0310] In some of any such embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and B2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 48, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 48.

[0311] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and B2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 80, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 80.

[0312] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A35R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors ... The tube-generating protein is a bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally with IRF3; and the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally with LIGHT; and optionally the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence shown in SEQ ID NO: 82, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 82.

[0313] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IL-2, optionally wherein IL-2 is an IL-2 superfactor, optionally mDNA11; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 84, or the sequence shown in SEQ ID NO: 84. The nucleic acid sequences shown in 84 have at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0314] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 85, or the sequence shown in SEQ ID NO: 85. The nucleic acid sequence shown in 85 has at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0315] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. Optionally, one or more of the immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally including two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally including IL-12 and CXCL9; the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids each encoding an apoptosis-inducing protein, optionally including an inducible DED (iDED); and optionally including the nucleic acid genome of the recombinant oncolytic virus as containing the nucleic acid sequence shown in SEQ ID NO: 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 86.

[0316] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, A35R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors, optionally wherein the one or more anti-angiogenic proteins are Bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IRF3; the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally one or more of which are LIGHT; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IL-2 superfactor MDNA11; and optionally the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence shown in SEQ ID NO:87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:87.

[0317] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, A35R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors, optionally wherein the one or more anti-angiogenic proteins comprise VEGF inhibitors and / or Ang2 inhibitors, and ... The protein is a bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IRF3; the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally one or more of which are LIGHT; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IL-2 superfactor MDNA11T, optionally wherein MDNA11T contains the amino acid sequence shown in SEQ ID NO: 98; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus contains the nucleic acid sequence shown in SEQ ID NO: 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 88.

[0318] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of proteins derived from vaccinia viruses ORF012, 203, and 018. The protein encoded by (CPXV012-203-018); the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of which are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally one or more of which are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally one or more of which are IL-2 superfactors, optionally MDNA11 or MDNA11T; the at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors (optionally CRASP-2), the heterologous nucleic acid being introduced into a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein, optionally fused at the C-terminus of the F14.5L protein; and optionally the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 89, or the sequence shown in SEQ ID NO: 89. The nucleic acid sequences shown in 89 have at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0319] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of proteins derived from vaccinia viruses ORF012, 203, and 018. The protein encoded by (CPXV012-203-018); the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein two or more immunomodulatory proteins comprise IL-12 and CXCL9; the at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors (optionally CRASP-2), the heterologous nucleic acid being introduced into a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein, optionally wherein the fusion is located at the C-terminus of the F14.5L protein; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 90, or the sequence shown in SEQ ID NO: 90. The nucleic acid sequences shown in 90 have at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0320] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises B2R and J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 91, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 91.

[0321] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises B2R, J2R, and A56R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein the one or more heterologous nucleic acids encoding one or more immunomodulatory proteins are... One or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally one or more of the immunomodulatory proteins is IRF3; and the inactivation mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally one or more of the immunomodulatory proteins comprising two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally two or more of the immunomodulatory proteins comprising IL-12 and CXCL9; and optionally the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 92, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 92.

[0322] In some of these embodiments, the recombinant oncolytic virus comprises: an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T-cell or NK-cell escape proteins, anti-angiogenic proteins, interferon regulators, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is via one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 93.

[0323] In some of these embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82 and 84-93; and is characterized by one or more of the following: (i) variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and containing a polar uncharged amino acid at position 66, optionally a threonine (T) amino acid sequence at position 66; (ii) variant 038 (K5L) ORF comprising a nucleotide insertion to cause a frameshift mutation, wherein the 038 (K5L) gene product is altered; (iii) variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57, and containing a polar uncharged amino acid at position 66, optionally a threonine (T) amino acid sequence at position 66; (ii) variant 038 (K5L) ORF comprising a nucleotide insertion to cause a frameshift mutation, wherein the 038 (K5L) gene product is altered; SEQ ID NO: 60 has at least 95% sequence identity and contains a hydrophobic amino acid other than leucine at position 419, optionally the amino acid sequence of phenylalanine (F) at position 419; (iv) variant 104 (H4L) ORF, which encodes an amino acid sequence of at least 95% sequence identity with SEQ ID NO: 61 and contains a negatively charged amino acid at position 591, optionally the amino acid sequence of aspartic acid (D) at position 591; and (v) variant 182 (A56R) ORF, which contains a two-nucleotide deletion to cause a frameshift mutation, wherein the 182 (A56R) ORF gene product is altered.

[0324] In some of these embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93; and is characterized by one or more of the following: (i) guanine (G) at position 7770 of SEQ ID NO: 1; (ii) thymine (T) at position 15261 of SEQ ID NO: 1; (iii) G at position 32136 of SEQ ID NO: 1; (iv) G at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; (vi) guanine (G) at position 49455 of SEQ ID NO: 1; (v) cytosine (C) at position 92969 of SEQ ID NO: 1; and (vi) guanine (G) at position 49455 of SEQ ID NO: 1. The nucleic acid sequence CACTTATAT corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA corresponding to positions 111267 to 111276 of SEQ ID NO: 1; (viii) adenine (A) corresponding to position 162715 of SEQ ID NO: 1; (ix) the nucleic acid sequence TACAGACACC corresponding to positions 165844 to 185853 of SEQ ID NO: 1; and (x) C corresponding to position 187805 of SEQ ID NO: 1.

[0325] A. Latent virus

[0326] In several embodiments, this document provides recombinant viruses comprising heterologous nucleic acids encoding "latent proteins" that can be stably and efficiently expressed in cells infected by a variety of viral types. Such latent proteins can enhance the virus's ability to evade host immune system attacks, such as attacks from evading T cells (e.g., cytotoxic T lymphocytes (CTLs)) or natural killer (NK) cells. In some embodiments, such latent proteins can enhance the recombinant virus's ability to evade host complement cascade / system activation.

[0327] Therefore, in some embodiments, this document provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or contain complement inhibitors or T-cell or NK-cell escapes (sometimes also referred to as recessive proteins).

[0328] In some embodiments, the recombinant virus comprises an oncolytic virus. In some embodiments, the recombinant virus does not include an oncolytic virus. In some embodiments, the recombinant virus includes any virus described herein or incorporated herein by reference. In some embodiments, the recombinant virus includes vaccinia virus. In some embodiments, the recombinant virus comprises a virus derived from VIP02.

[0329] Oncolytic viruses (OVs) can create a favorable microenvironment for the immune system to act against unique cancer cell determinants; however, the antiviral immune response against viral infection is also a key factor in OV-based therapies. In fact, induced antiviral immunity can be detrimental to cancer viral therapies because activating the immune system against the virus itself is expected to limit viral replication and spread, leading to decreased therapeutic efficacy (Lemos de Matos et al., Mol TherMethods Clin Dev. 2020 Jun 12; 17: 349–358). The complement system continuously monitors viruses. Its ability to recognize viruses and virus-infected cells and trigger an immune response leads to viral neutralization and the killing of infected cells. This selective pressure exerted by complement on viruses has prompted viruses to evolve a variety of coping strategies (Agrawal et al., FrontMicrobiol. 2017; 8: 1117).

[0330] In some implementations, the recessive proteins include, but are not limited to, borax complement regulatory acquisition surface protein-2 (CRASP-2), minimized complement regulator H (miniFH), and vaccinia virus ORF012, 203, and 018 (CPXV012-203-018). Further details regarding the recessive proteins and the mechanisms involved in escaped host immune system responses (such as escaped complement or NK or T cell cytotoxicity) can be found in Monrat Chulanetra and Wanpen Chaicumpa, Front. Cell. Infect. Microbiol., 2021, Front. Cell. Infect. Microbiol. 11:702125, which is incorporated herein by reference in its entirety.

[0331] The complement system is an important component of innate immunity, which helps clear pathogens. Therefore, during evolution, pathogens have developed a variety of strategies to avoid being disrupted by complement activation. One such strategy is to acquire proteins that enable pathogens to control the activation steps involved in the host immune response during infection; these are referred to as "latent proteins" (see Kraiczy et al., Infect Immun. 2001 Dec; 69(12): 7800–7809).

[0332] The complement system triggers inflammatory responses, chemotaxis of phagocytes and neutrophils, pathogen neutralization, and subsequent opsonization, as well as the lysis of infected cells, through a complex proteolytic cascade involving more than 30 plasma and cell membrane proteins. Complement activation can be initiated via three independent pathways: (i) the classical pathway, where the first component C1q in the complement cascade binds to the antibody-antigen complex; (ii) the alternative pathway, involving the spontaneous hydrolysis of downstream complement component 3 (C3) convertase and its interaction with the pathogen surface; and (iii) the mannose-binding lectin (MBL) pathway, triggered by the binding of MBL to mannose residues on the pathogen surface. All three pathways converge at the stage where C3 cleaves into the antimicrobial peptide C3a and opsonin C3b, with C3b binding to the pathogen and labeling it for degradation. Because the effector compounds produced in the complement cascade can be delivered to any surface, including the host cell membrane, intact host cells protect themselves by expressing a variety of complement regulatory proteins (Janeway et al., Immunobiology: The Immune System in Health and Disease. 5th edition).

[0333] Inadequate control of the complement system is a potential or aggravating factor in many human diseases. The alternative complement pathway (AP) has the unique characteristic of being continuously and indiscriminately activated, albeit at low levels. In AP, C3b self-proliferates via a positive feedback amplification loop, which requires tight regulation by two key soluble AP regulators, factor H (FH) and their splice product FH-like-1 (FHL-1). The recessive protein miniFH is an engineered version of FH, containing only the N-terminal and C-terminal portions of FH linked by an optimized peptide. Compared to FH, it exhibits approximately 10-fold increased potency in inhibiting complement activation in vitro (FH. Markus J. Harder, *J Immunol. Author manuscript; available in PMC 2017 Jan 15. JImmunol. 2016 Jan 15; 196(2): 866–876. & Christoph Q. Schmidt / J Immunol. Author manuscript; available in PMC 2014 Jun 1. J Immunol. 2013 Jun 1; 190(11): 10.4049 / jimmunol.1203548. Published online 2013 Apr 24. doi: 10.4049 / jimmunol.1203548).

[0334] One microorganism that evolved to evade complement by producing recessive proteins is *Borrelia burgdorferi*, a spirochetal spirochete that causes Lyme disease (LD), the most common tick-borne disease in the Northern Hemisphere. When ticks feed on blood, the spirochetes are exposed to the host's bloodstream, thus facing the first line of defense of the innate immune system, which they must overcome to survive. A key escape mechanism developed by *Borrelia burgdorferi* is the production of complement or CRP-binding proteins, including CRASP, a recessive protein that promotes complement inactivation (see Yi-Pin Lin et al., Front Cell Infect Microbiol. 2020; 10: 1.; US20120142023A1). CRASP-2 (also known as CspZ) binds to FH / FHL-1, thereby conferring serum resistance to gain-functioning Treponema pallidum by inhibiting complement activation on the surface of the spirochetes (Infect Immun. 2001 Dec; 69(12): 7800–7809. Peter Kraiczy. US20200323972A1 Compositions and methods for generating immunity against Treponema pallidum).

[0335] Downregulation of cell surface MHC class I molecule expression is a common immune evasion mechanism among many DNA viruses, including vaccinia virus. CPXV, a member of the genus *Orthopoxvirus*, which includes smallpox virus, camelpox virus, and monkeypox virus, encodes a complex set of immune evasion proteins. CPXV's ability to infect multiple mammalian hosts is likely due to the fact that, among orthopoxviruses, it encodes the most complete set of open reading frames expected to encode immunomodulatory proteins. Its encoded proteins include CPXV012 and CPXV203, which can prevent cytotoxic T cell recognition by interfering with MHC class I molecule-mediated antigen presentation. However, CPXV012 inhibits the transport of antigenic peptides from the cytoplasm to the ER, while CPXV203 blocks the transport of MHC class I molecules to the cell surface (Dina Alzhanova and Klaus Früh* Microbes Infect. 2010 Nov; 12(12-13): 900–909. McCoy et al., Molecular Immunology 55 (2013) 156–158). In addition, the Brighton Red strain produces OMCP (also known as CPXV018), a 171-amino acid protein that is secreted in large quantities from infected cells and can block NKG2D-mediated target cell killing by natural killer cells in vitro (Cell Host & Microbe Volume 6, Issue 5, 19 November 2009, Pages 422-432 / Journal home page for Cell Host & Microbe / Two Mechanistically Distinct Immune Evasion Proteins of Cowpox Virus Combine to Avoid Antiviral CD8 TCells).

[0336] In some embodiments, one or more heterologous gene products comprise a complement inhibitor. In some embodiments, the complement inhibitor is borax complement regulatory acquisition surface protein-2 (CRASP-2) or minimizing complement regulatory factor H (miniFH). In some embodiments, the complement inhibitor is the CRASP-2 gene product (UniProtKB-050665). The CRASP-2 protein can enhance the ability of recombinant viruses to evade host complement. Specifically, in some embodiments, the recombinant virus comprises an expression cassette containing CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia virus F14.5L gene promoter. In some embodiments, the CRASP-2 molecule comprises full-length CRASP-2. In some embodiments, the complement inhibitor is CRASP-2 and has an amino acid sequence having at least 85%, 90%, or 95% sequence identity with the sequence shown in SEQ ID NO: 18. In some embodiments, the complement inhibitor has the sequence shown in SEQ ID NO: 18.

[0337] In some embodiments, the recombinant virus comprises a heterologous nucleic acid encoding a CRASP-2 molecule, which comprises CRASP-2 cDNA fused to the F14.5L locus, wherein CRASP-2 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 18.

[0338] In some embodiments, the recombinant virus comprises an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 18. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 18, but less than 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0339] In some embodiments, the heterologous nucleic acid encoding the CRASP-2 gene product is operatively linked to the F14.5L gene promoter. In some embodiments, the recombinant virus comprising the heterologous nucleic acid encoding the CRASP-2 gene product (e.g., containing the amino acid sequence of SEQ ID NO: 18) is derived from the clone VIP02 strain (containing the nucleic acid sequence of SEQ ID NO: 1) and comprises a nucleotide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with SEQ ID NO: 5 (also known as VIR27). In some embodiments, the heterologous gene product is CRASP-2 and is operatively linked to the F14.5L gene promoter in the viral genome. In some embodiments, the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 5, but less than 100% identical with the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 5. In some embodiments, the heterologous gene product is CRASP-2 and is operatively linked to the F14.5L gene promoter in the viral genome, and the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the heterologous gene product is CRASP-2 and is operatively linked to the F14.5L gene promoter in the viral genome, and the recombinant virus comprises the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the recombinant virus (e.g., a recombinant oncolytic virus) comprises the nucleic acid sequence of SEQ ID NO: 5. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO: 5 is also referred to herein as VIR27.

[0340] In several embodiments, the recombinant virus provided herein exhibits an increased ability to evade the host's immune system. In some embodiments, the recombinant virus provided herein can evade complement inhibition in both in vivo and in vitro systems. In a particular embodiment, when incubated with an effective dose of VIR27 (containing the nucleic acid sequence of SEQ ID NO: 5), VIR27 (containing the nucleic acid sequence of SEQ ID NO: 5) can evade complement inhibition in in vitro complement inhibition systems. Figure 6 In a specific implementation, administration of an effective dose of VIR27 (containing the nucleic acid sequence of SEQ ID NO: 5) to a subject can inhibit tumor, proliferation, or metastatic growth in an in vivo model. Figure 7 ).

[0341] In some embodiments, the complement inhibitor is a miniFH gene product. Specifically, in some embodiments, the recombinant virus comprises an expression cassette containing miniFH cDNA fused to the F14.5L locus under the control of the vaccinia virus F14.5L gene promoter. For more detailed information on miniFH, see Schmidt et al., JImmunol. 2013 Jun 1; 190(11): 10.4049 / jimmunol.1203548., which is incorporated herein by reference in its entirety. In some embodiments, the complement inhibitor is a miniFH gene product comprising an amino acid sequence having at least 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the complement inhibitor is a miniFH gene product comprising the amino acid sequence shown in SEQ ID NO: 19.

[0342] In some embodiments, the recessive protein comprises an FH-based inhibitor, miniFH. In some embodiments, the miniFH gene product can enhance the recombinant virus's ability to evade host complement. In some embodiments, a recombinant virus is provided comprising a polynucleotide encoding a miniFH gene product, comprising miniFH cDNA fused to the F14.5L locus, wherein the miniFH polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the recombinant virus comprises a polypeptide having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 19. For example, in some embodiments, the recombinant virus contains an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 19, but less than 100% identical amino acid sequence with the amino acid sequence of SEQ ID NO: 19.

[0343] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 6. In some embodiments, the heterologous gene product is miniFH and is operatively linked to the F14.5L gene promoter in the viral genome, and the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 6, but less than 100% identical with the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the heterologous gene product is miniFH and is operatively linked to the F14.5L gene promoter in the viral genome, and the recombinant virus comprises the nucleic acid sequence of SEQ ID NO: 6. In some implementations, the recombinant virus (e.g., recombinant oncolytic virus) contains the nucleic acid sequence of SEQ ID NO:6. The recombinant oncolytic virus containing the nucleic acid sequence of SEQ ID NO:6 is also referred to herein as VIR37.

[0344] In some embodiments, the polynucleotide encoding the miniFH molecule is operatively linked to the F14.5L gene promoter. In some embodiments, the recombinant virus comprising the polynucleotide encoding the miniFH molecule (e.g., containing the amino acid sequence of SEQ ID NO: 19) is derived from the clone VIP02 strain (containing the nucleic acid sequence of SEQ ID NO: 1) and comprises a nucleic acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with SEQ ID NO: 6 (also known as VIR37). In some embodiments, the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 6, but less than 100% identical with SEQ ID NO: 6. In some embodiments, the recombinant virus comprises a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 6. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 6.

[0345] In several embodiments, the recombinant virus provided herein exhibits an increased ability to evade host complement. In some embodiments, the recombinant virus provided herein can evade complement inhibition in in vivo and in vitro systems. In a specific embodiment, when an effective dose of VIR37 (containing the nucleic acid sequence of SEQ ID NO: 6) is incubated with human serum and / or BABL / c mouse serum, VIR37 (containing the nucleic acid sequence of SEQ ID NO: 6) can evade complement inhibition in in vitro complement inhibition systems. Figure 6 ).

[0346] In some implementations, one or more heterologous gene products are T-cell escape proteins or NK-cell escape proteins. T-cell escape protein or NK-cell escape protein gene products can enhance the ability of viruses to evade the host's immune system, such as evading attack by evading T cells (e.g., cytotoxic T lymphocytes (CTLs)) or natural killer (NK) cells. Specifically, such T-cell escape protein or NK-cell escape protein gene products can enhance the ability of recombinant viruses to evade the host's complement cascade / system activation.

[0347] In some implementations, T-cell escape proteins or NK-cell escape proteins are a group of proteins encoded by vaccinia virus ORF012, 203, and 018 (CPXV012-203-018). CPXV012-203-018 is a synthetic DNA fragment. In the CPXV012-203-018 synthetic DNA fragment, ORFs 012, 203, and 018 are expressed individually under their respective promoters to encode the CPXV012, CPXV203, and CPXV018 proteins. Vaccinia virus escapes CTLs via CPXV012 and CPXV203. CPXV012 inhibits the transport of antigenic peptides from the cytoplasm to the endoplasmic reticulum (ER), while CPXV203 blocks the transport of MHC class I molecules to the cell surface by utilizing the KDEL receptor cycling pathway. CPXV018 encodes a soluble NKG2D ligand called orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity.

[0348] In several embodiments, recombinant viruses expressing the CRASP-2 gene product (UniProtKB-050665) and vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) have been generated herein. Specifically, in some embodiments, the recombinant virus (e.g., recombinant oncolytic virus) comprises an expression cassette containing CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia virus F14.5L gene promoter, and a series of polynucleotide sequences comprising open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) with their respective promoters inserted into the J2R locus.

[0349] In some embodiments, the recessive protein comprises borax complement regulatory acquisition surface protein-2 (CRASP-2). In some embodiments, expression of the CRASP-2 protein can enhance the ability of the recombinant virus to evade host complement. In some embodiments, a recombinant virus is provided comprising a polynucleotide encoding a CRASP-2 molecule, said polynucleotide comprising CRASP-2 cDNA fused to the F14.5L locus, wherein the CRASP-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:18. In some embodiments, the recessive protein comprises vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) having respective promoters. In some embodiments, expression of vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) can enhance the ability of the recombinant virus to evade host T cells and NK cells. In some implementations, expression of recessive proteins, including CRASP-2 and vaccinia virus open reading frames (ORFs) 012, 203, and 018, can enhance the ability of recombinant viruses to escape host complement, as well as T and NK cells.

[0350] In some embodiments, the T-cell escape protein or NK-cell escape protein is a group of proteins encoded by vaccinia virus ORF012, 203, and 018 (CPXV012-203-018) comprising the amino acid sequences shown in SEQ ID NO: 20, 21, and 22, or amino acid sequences having at least 70%, 80%, 85%, 90%, or 95% sequence identity with the amino acid sequences of SEQ ID NO: 20, 21, and 22. In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV012 having an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 20. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 20, but less than 100% identical with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV203 having an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 21. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 21, but less than 100% identical with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the recombinant virus comprises a polypeptide sequence encoding CPXV018, said polypeptide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 22.For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 22, but less than 100% identical amino acid sequence. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO: 22.

[0351] In some embodiments, the T-cell escape protein or NK-cell escape protein is a group of proteins encoded by vaccinia virus ORF012, 203, and 018 (CPXV012-203-018), and this group of proteins encoded by CPXV012-203-018 comprises amino acid sequences exhibiting at least 85%, 90%, or 95% sequence identity with the sequence shown in SEQ ID NO: 20 (CPXV012), amino acid sequences exhibiting at least 85%, 90%, or 95% sequence identity with the sequence shown in SEQ ID NO: 21 (CPXV0203), and amino acid sequences exhibiting at least 85%, 90%, or 95% sequence identity with the sequence shown in SEQ ID NO: 22 (CPXV018). In some embodiments, the group of proteins encoded by CPXV012-203-018 comprises the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0352] In some embodiments, the recombinant virus comprises a polypeptide encoding CRASP-2 having an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with SEQ ID NO: 18. For example, in some embodiments, the recombinant virus comprises a nucleic acid sequence encoding a polypeptide having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 18, but less than 100% identical with the amino acid sequence of SEQ ID NO: 18. In some embodiments, the recombinant virus comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the CRASP-2 molecule is operatively linked to the F14.5L gene promoter. In some implementations, nucleotide sequences encoding CPXV012, CPXV203, and CPXV018 are inserted into the J2R genome region.

[0353] In some embodiments, the recombinant virus comprising nucleic acid sequences encoding CRASP-2 (e.g., SEQ ID NO: 18), CPXV012 (e.g., SEQ ID NO: 20), CPXV203 (e.g., SEQ ID NO: 21), and CPXV018 (e.g., SEQ ID NO: 22) is derived from the VIR27 strain (containing the nucleic acid sequence of SEQ ID NO: 5) and comprises a nucleotide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with SEQ ID NO: 10 (also known as VIR46). For example, in some embodiments, the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 10, but less than 100% identical with the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the recombinant virus comprises the nucleic acid sequence of SEQ ID NO: 10. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO: 10 is also referred to herein as VIR46.

[0354] In several embodiments, the recombinant virus provided herein exhibits an increased ability to evade host complement. In some embodiments, the recombinant virus provided herein can evade complement inhibition in in vivo and in vitro systems. In specific embodiments, administration of an effective dose of VIR46 (containing the nucleic acid sequence of SEQ ID NO: 10) to a subject can inhibit tumor, hyperplastic, or metastatic growth in an in vivo model. Figure 8 ).

[0355] B. Immunomodulatory viruses

[0356] In several embodiments, this document provides recombinant viruses comprising heterologous nucleic acids encoding immunomodulatory proteins that can be stably and efficiently expressed in cells infected with various types of viruses. In some embodiments, the immunomodulatory proteins include cytokines, chemokines, immune receptors, antigens of immune receptors, proteins in immune cell activation pathways, intracellular signaling proteins that stimulate immune cell activation or cytokine secretion, and antigens. In some embodiments, the immunomodulatory protein comprises one or more cytokines and / or chemokines. In some embodiments, the one or more cytokines and / or chemokines comprise one or more of chemokine ligand 9 (CXCL9), IL-2, and IL-12. In some embodiments, the immunomodulatory protein is a member of the tumor necrosis factor superfamily 14 (LIGHT). In some embodiments, the immunomodulatory protein is an interferon regulator that activates the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. In some embodiments, the immunomodulatory protein is interleukin-12 (IL-12). In some embodiments, the immunomodulatory protein is chemokine ligand 9 (CXCL9). In some embodiments, the immunomodulatory protein is IL-2 or IL-2 superfactor. In some embodiments, the immunomodulatory protein is IL-2 superfactor. In some embodiments, the immunomodulatory protein is mDNA11. In some embodiments, mDNA11 has been mutated to enhance the antitumor efficacy of the recombinant virus, and the immunomodulatory protein is mDNA11T. In some embodiments, the recombinant virus comprises a heterologous nucleic acid encoding one or more of the following immunomodulatory proteins: LIGHT, IRF3, IL-12, CXCL9, mDNA11, mDNA11T, and other immunomodulatory proteins. In some embodiments, one or more immunomodulatory proteins are immunostimulatory proteins, such as LIGHT.

[0357] In some embodiments, at least one heteronucleotide encoding one or more heterologous gene products comprises one or more heteronucleotides each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are or comprise one or more cytokines and / or chemokines. In some embodiments, the one or more immunomodulatory proteins are or comprise one or more interferon regulators, such as IRF3. In some embodiments, the one or more immunomodulatory proteins comprise one or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, any of which will be discussed in detail below.

[0358] 1. Tumor necrosis factor superfamily member 14 (LIGHT)

[0359] In some embodiments, at least one heteronucleotide encoding one or more heterogeneous gene products comprises one or more heteronucleotides each encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins comprise LIGHT.

[0360] LIGHT has been studied in preclinical research for over a decade and has proven to be a promising approach for treating a variety of cancers. LIGHT has been successfully used to clear established solid tumors and treat metastatic events. When expressed in tumors, LIGHT molecules cause significant changes in the tumor microenvironment, primarily driven by angiogenesis and the formation of tertiary lymphoid structures (TLS). The formation of these lymphoid structures can be induced by activation of the tumor necrosis factor superfamily receptor lymphotoxin β receptor (LTBR / TNFRSF3), a TNF superfamily receptor that can be activated by LIGHT (see Schrama et al., Immunity 14:111-121 (2001); Tang et al., Cell. Mol. Immunol. 14:809-18 (2017)). The presence of TLS in the tumor microenvironment is often associated with immune infiltration and better prognosis, suggesting that TLS is involved in antitumor immune responses (see Dieu-Nosjean et al., J. Clin. Oncol. 26:4410-17 (2008) and Weinstein and Storkus, Adv. Cancer Res. 128:197-233 (2015)). For example, a homotrimeric single-chain LIGHT variant with improved stability and cross-reactivity in humans and mice, called 3×hmLIGHT, fused with an antibody targeting EGFR-specific tumors, induced antitumor immunity in mouse and human tumor models by increasing lymphocyte infiltration. Therefore, activation of LTBR has the potential to promote the formation of TLS in the tumor microenvironment, induce antitumor immune responses, and improve current cancer immunotherapies. More detailed information on the antitumor properties of LIGHT can be found in U.S. Publication No. 2021 / 0188990, which is incorporated herein by reference in its entirety.

[0361] In some embodiments, this document provides a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are or comprise an immunostimulatory protein.

[0362] In some embodiments, the recombinant virus comprises at least one heterologous nucleic acid encoding an immunostimulatory protein that can be stably and efficiently expressed in cells infected with various types of viruses. Such immunostimulatory proteins can enhance the antitumor properties of the virus. Specifically, such immunostimulatory proteins can enhance the ability of the recombinant virus to induce tumor lymphocyte infiltration in the host immune system. In some embodiments, the recombinant virus comprises an oncolytic virus. In some embodiments, the recombinant virus does not include an oncolytic virus. In some embodiments, the recombinant virus comprises any virus described herein or incorporated herein by reference. In some embodiments, the virus comprises vaccinia virus. In some embodiments, the virus comprises a recombinant virus derived from the VIP02 virus strain.

[0363] In some embodiments, the recombinant virus comprises at least one heterologous nucleic acid encoding an immunostimulatory protein that can be stably and efficiently expressed in various types of virus-infected cells and enhance the antitumor properties of the virus. Therefore, in some embodiments, the immunostimulatory protein increases or enhances the antitumor properties of the virus. In some embodiments, the immunomodulatory protein includes, but is not limited to, tumor necrosis factor superfamily member 14 (LIGHT), such as hmLIGHT. In some embodiments, the immunomodulatory protein includes, but is not limited to, any immunomodulatory protein incorporated herein by reference.

[0364] In some embodiments, the recombinant virus expresses the hmLIGHT gene product (NP_001363816 XP_016882906). Specifically, in some embodiments, the resulting recombinant virus comprises an expression cassette containing a full-length hmLIGHT cDNA controlled by the vaccinia virus PSE gene promoter and inserted into the A35R genomic region. In some embodiments, the immunomodulatory protein includes hmLIGHT. In some embodiments, the hmLIGHT protein can enhance the antitumor properties of the recombinant virus.

[0365] In some embodiments, the immunostimulatory protein is LIGHT. In some embodiments, the immunostimulatory protein is recombinant LIGHT. In some embodiments, the recombinant LIGHT is a human protein or a variant thereof.

[0366] In some embodiments, the recombinant LIGHT comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 30. In some embodiments, the recombinant LIGHT comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 30.

[0367] In some embodiments, the recombinant LIGHT is hmLIGHT, which is a human LIGHT variant that combines human and mouse LTβR and HVEM. In some embodiments, the recombinant LIGHT contains one or more mutations selected from threonine at position 138, glycine at position 160, glycine at position 221, and lysine at position 222.

[0368] In some embodiments, the recombinant virus comprises a nucleic acid sequence encoding hmLIGHT, wherein hmLIGHT comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25. In some embodiments, the recombinant LIGHT comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the recombinant LIGHT comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 25. In some embodiments, the recombinant LIGHT comprises the sequence shown in SEQ ID NO: 25.

[0369] In some embodiments, the recombinant virus comprises a polypeptide having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with the amino acid sequence of SEQ ID NO: 25.

[0370] In some embodiments, the polynucleotide encoding the hmLIGHT molecule is operatively linked to the PSE gene promoter. In some embodiments, the recombinant virus containing the polynucleotide encoding the hmLIGHT gene product (e.g., containing the amino acid sequence of SEQ ID NO: 25) is derived from the VIR13 strain (containing the nucleic acid sequence of SEQ ID NO: 4) and contains a nucleotide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity with SEQ ID NO: 11 (also known as VIR49). For example, in some embodiments, the recombinant virus contains a nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the nucleic acid sequence of SEQ ID NO: 11, but less than 100% identical to SEQ ID NO: 11. In some implementations, the recombinant virus comprises the nucleic acid sequence of SEQ ID NO: 11. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO: 11 is also referred to herein as VIR49.

[0371] In some embodiments, the recombinant viruses provided herein, such as VIR49 (containing the nucleic acid sequence of SEQ ID N...

Claims

1. A recombinant vaccinia virus comprising a heterologous nucleic acid encoding an inducible apoptosis protein, wherein the apoptosis protein is a death effector domain (DED) containing a Fas-associated death domain, Fas, or caspase.

2. The recombinant vaccinia virus according to claim 1, wherein the apoptosis protein is DED.

3. The recombinant vaccinia virus according to claim 1, wherein the apoptosis protein is caspase, and the caspase is caspase 9 (Cas9).

4. The recombinant vaccinia virus according to claim 1, wherein the apoptosis protein is Fas.

5. The recombinant vaccinia virus according to claim 1, wherein the heterologous nucleic acid encoding the induced apoptosis protein is inserted into a locus in the genome of the vaccinia virus consisting of the group consisting of: hemagglutinin (HA), J2R, F14.5L, A56R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L.

6. The recombinant vaccinia virus according to claim 1, wherein the heterologous nucleic acid encoding the induced apoptosis protein is inserted into the B2R, J2R, A35R, A56R or F14.5L locus in the genome of the vaccinia virus.

7. The recombinant vaccinia virus of claim 1, wherein the heterologous nucleic acid encoding the induced apoptosis protein is inserted into the J2R locus in the genome of the vaccinia virus.

8. The recombinant vaccinia virus of claim 1, wherein the inducible apoptosis protein comprises an apoptosis protein fused with an FKBP variant capable of binding a dimerizing chemical inducer (CID).

9. The recombinant vaccinia virus of claim 8, wherein the FKBP variant is an FKBP containing the mutant F36V.

10. The recombinant vaccinia virus of claim 9, wherein the FKBP variant comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 56.

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