RECOMBINANT VACCINIA VIRUS

1 . A replication-competent recombinant oncolytic vaccinia virus (RVV) which comprises modifications in the viral genome relative to the corresponding wild-type virus, wherein the modifications comprises, in the viral genome: (1) an inserted nucleotide sequence encoding an immunostimulatory cytokine polypeptide; and (2) an inserted nucleotide sequence encoding a heterologous thymidine kinase (TK) polypeptide.

2 . The RVV of claim 1 , wherein the RVV comprises a further modification that renders the vaccinia TK deficient.

3 . The RVV of claim 2 , wherein the further modification results in lack of J2R expression and/or function.

4 . The RVV of claim 2 , wherein the RVV is a Copenhagen strain vaccinia virus.

5 . The RVV of claim 2 , wherein the RVV is a WR strain vaccinia virus.

6 . The RW of any one of claims 1-5 , wherein the immunostimulatory cytokine polypeptide is an interleukin-2 (IL-2) polypeptide.

7 . The RVV of any one of claims 1-6 , wherein the immunostimulatory cytokine polypeptide is a wild-type IL-2 polypeptide.

8 . The RVV of any one of claims 1-6 , wherein the immunostimulatory cytokine polypeptide is a variant IL-2 (IL-2v) polypeptide having reduced undesirable biological activity as compared to the corresponding wild-type IL-2 polypeptide.

9 . The RVV of any one of claims 1-8 , wherein the heterologous TK polypeptide is an HSV-TK polypeptide.

10 . The RW of any one of claims 1-9 , wherein the RW comprises an A34R gene comprising a K151E substitution.

11 . The RVV of any one of claims 8-10 , wherein the IL-2v polypeptide comprises substitutions of one or more of F42, Y45, and L72, based on the amino acid numbering of the IL-2 amino acid sequence of SEQ ID NO: 1.

12 . The RVV of any one of claims 8-11 , wherein the IL-2v polypeptide comprises substitution an F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42D, F42R, or F42K substitution, based on the amino acid numbering of the IL-2 amino acid sequence of SEQ ID NO: 1.

13 . The RVV of any one of claims 8-12 , wherein the IL-2v polypeptide comprises a Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K substitution, based on the amino acid numbering of the IL-2 amino acid sequence of SEQ ID NO: 1.

14 . The RVV of any one of claims 8-13 , wherein the IL-2v polypeptide comprises a L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72R, or L72K substitution, based on the amino acid numbering of the IL-2 amino acid sequence of SEQ ID NO: 1.

15 . The RVV of any one of claims 8-14 , wherein the IL-2v polypeptide comprises a F42A, Y45A, and L72G substitutions, based on the amino acid numbering of the IL-2 amino acid sequence of SEQ ID NO: 1.

16 . The RW of any one of claims 8-14 , wherein the IL-2v polypeptide-encoding nucleotide sequence is operably linked to a regulatable promoter.

17 . The RVV of claim 16 , wherein the regulatable promoter is regulated by tetracycline or a tetracycline analog or derivative.

18 . The RVV of any one of claims 1-17 , wherein heterologous TK polypeptide is capable of catalyzing phosphorylation of deoxyguanosine.

19 . The RVV of any one of claims 1-18 , wherein the heterologous TK polypeptide is a variant herpes simplex virus (HSV) TK polypeptide.

20 . The RVV of claim 19 , wherein the variant HSV TK polypeptide comprises an amino acid sequence having at least 80% amino acid sequence identity to a wild-type HSV TK, and comprises a substitution of one or more of L159, I160, F161, A168, and L169, based on the amino acid numbering of wild-type HSV TK amino acid sequence of SEQ ID NO: 25.

21 . The RVV of claim 20 , wherein the variant HSV TK polypeptide comprises an A168H substitution.

22 . The RVV of claim 20 , wherein the variant HSV TK polypeptide comprises an L159I substitution, an I160L substitution, an F161A substitution, an A168Y substitution, and an L169F substitution.

23 . The RVV of claim 20 , wherein the variant HSV TK polypeptide comprises an L159I substitution, an I160F substitution, an F161L substitution, an A168F substitution, and an L169M substitution.

24 . The RW of claim 20 , wherein the variant HSV TK polypeptide comprises the amino acid sequence of SEQ ID NO: 26, 27, or 28.

25 . A composition comprising:

a) the RW of any one of claims 1 24 ; and

b) a pharmaceutically acceptable carrier.

26 . A method of inducing oncolysis in an individual having a tumor, the method comprising administering to the individual an effective amount of the RVV of any one of claims 1-24 , or the composition of claim 25 .

27 . The method of claim 26 , wherein the tumor is a brain cancer tumor, a head and neck cancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lung cancer tumor, a thymic cancer tumor, a stomach cancer tumor, a colon cancer tumor, a liver cancer tumor, an ovarian cancer tumor, a uterine cancer tumor, a bladder cancer tumor, a testicular cancer tumor, a rectal cancer tumor, a breast cancer tumor, or a pancreatic cancer tumor.

28 . The method of claim 26 , wherein the tumor is a colorectal adenocarcinoma, a non-small cell lung carcinoma, or a triple-negative breast cancer.

29 . The method of any one of claims 26-28 , wherein the tumor is recurrent.

30 . The method of any one of claims 26-28 , wherein the tumor is a primary tumor.

31 . The method of any one of claims 26-28 , wherein the tumor is metastatic.

32 . The method of any one of claims 26-31 , further comprising administering to the individual a second cancer therapy.

33 . The method of claim 32 , wherein the second cancer therapy is selected from chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, antivascular therapy, cryotherapy, toxin therapy, oncolytic virus therapy, a cell therapy, and surgery.

34 . The method of claim 32 , wherein the second cancer therapy comprises an anti-PD1 antibody or an anti-PD-L1 antibody.

35 . The method of any one of claims 26-34 , wherein the individual is immunocompromised.

36 . The method of any one of claims 26-35 , wherein said administering of the RVV or the composition is intratumoral.

37 . The method of any one of claims 26-35 , wherein said administering of the RVV or the composition is peritumoral.

38 . The method of any one of claims 26-35 , wherein said administering of the RVV or the composition is intravenous.

39 . The method of any one of claims 26-35 , wherein said administering of the RVV or the composition is intra-arterial, intrabladder, or intrathecal.

40 . The method of any one of claims 26-39 , further comprising administering to the individual ganciclovir in an amount that is effective to reduce an adverse side effect of the vaccinia virus.

41 . A replication-competent, recombinant oncolytic vaccinia virus (RVV), comprising, in its genome: (1) a nucleotide sequence encoding a variant interleukin-2 (IL-2v) polypeptide comprising SEQ ID NO: 9; (2) a nucleotide sequence encoding a heterologous TK polypeptide comprising SEQ ID NO:28; and (3) a K151E substitution in the A34R gene, wherein the RVV is a Copenhagen strain vaccinia virus and is vaccinia thymidine kinase deficient.

42 . The RVV of claim 41 , wherein the IL-2v polypeptide further comprises a signal peptide.

43 . The RVV of claim 42 , wherein the signal peptide comprises SEQ ID NO:22.

44 . The RVV of any one of claims 8-24 , wherein the nucleotide sequence encoding the variant IL-2v polypeptide comprises SEQ ID NO: 10.

45 . The RVV of any one of claims 8-24 , wherein the nucleotide sequence encoding the variant IL-2v polypeptide comprises SEQ ID NO: 12.

46 . A composition, comprising: (i) the RVV of any one of claims 41 to 45 and (ii) a pharmaceutically acceptable carrier.