AU2024336449A1 - Recombinant vaccinia virus and methods of use thereof - Google Patents

Abstract

The present disclosure provides a replication-competent, recombinant oncolytic vaccinia virus; and compositions comprising the replication-competent, recombinant oncolytic vaccinia virus. The present disclosure also provides use of the vaccinia virus or composition for inducing oncolysis in an individual having a tumor.

Description

RECOMBINANT VACCINIA VIRUS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/580,606, filed September 5, 2023 and U.S. Provisional Patent Application Serial No.

63/651,594, filed May 24, 2024, the entire contents of each of which are incorporated herein by reference.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable XML file, entitled “019912-5001 WO Sequence Listing” created on August 29, 2024, with a file size of about 66,000 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Oncolytic viruses (OVs) are viruses that selectively replicate in cancer cells. Live replicating OVs have been tested in clinical trials in a variety of human cancers. OVs can induce anti-tumor immune responses, as well as direct lysis of tumor cells. Common OVs include attenuated strains of Herpes Simplex Virus (HSV), Adenovirus (Ad), Measles Virus (MV), Coxsackie virus (CV), Vesicular Stomatitis Virus (VSV), and Vaccinia Virus (VV).

[0004] Vaccinia virus replicates in the cytoplasm of a host cell. The large vaccinia virus genome codes for various enzymes and proteins used for viral DNA replication. During replication, vaccinia produces several infectious forms which differ in their outer membranes: the intracellular mature virion (IMV), the intracellular' enveloped virion (IEV), the cell- associated enveloped virion (CEV) and the extracellular enveloped virion (EEV). IMV is the most abundant infectious form and is thought to be responsible for spread between hosts; the CEV is believed to play a role in cell-to-cell spread; and the EEV is thought to be important for long range dissemination within the host organism. SUMMARY OF THE INVENTION

[0005] Disclosed are nucleic acids that include a recombinant vaccinia virus genome comprising one or more nucleotide sequences encoding heterologous polypeptides(s). Also disclosed are, replication-competent recombinant oncolytic viruses comprising such nucleic acids. In certain aspects, the replication-competent recombinant oncolytic virus is an oncolytic vaccinia virus comprising, in its genome, nucleotide sequence(s) encoding one or more of the following polypeptides: CD80 or a fusion protein comprising a CD80 polypeptide, IL-2 or a variant thereof, IL-12, IL-21 and IL-7. In a preferred embodiment, the replication-competent recombinant oncolytic vaccinia virus comprises, in its genome, nucleotide sequence(s) encoding soluble CD80 or a fusion protein comprising a soluble CD80 polypeptide, IL- 12, IL-21 and IL-7, wherein each nucleotide sequence is operably linked to an expression control sequence. In another preferred embodiment, the replication-competent oncolytic vaccina virus comprises, in its genome, nucleotide sequence(s) encoding a soluble CD80 polypeptide or a fusion protein comprising a soluble CD80 polypeptide, and nucleotide sequence encoding IL-2 or a variant thereof. In related embodiments, the replication-competent recombinant oncolytic vaccinia virus further comprises, in its genome, a nucleotide sequence encoding a heterologous thymidine kinase (TK) gene, preferably a herpes simplex virus (HSV) TK gene. In some aspects, the expression control sequence comprises a vaccinia virus promoter, a synthetic promoter, a promoter that directs transcription during at least the early phase of infection, and/or a promoter that directs transcription during at least the late phase of infection. In certain aspects, the replication-competent recombinant oncolytic vaccinia virus comprises an inactive M2L gene and comprises an inactive J2R (thymidine kinase) gene. In preferred embodiments, the replication- competent recombinant oncolytic vaccinia virus comprises one or more modifications that increase virus spreading, such as those described in U.S. Patent Application Publication No. 2023/0002740, the entire contents of which are incorporated herein by reference. In particularly preferred embodiments, the replication-competent recombinant oncolytic virus comprises a KI 5 IE amino acid substitution in the A34R gene in order increase virus spreading. Also disclosed are pharmaceutical compositions comprising a replication-competent recombinant oncolytic virus as described herein and the use of such pharmaceutical compositions for the treatment of cancer, as a monotherapy or as a component of a combination therapy with a cancer therapeutic. DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1A-C provides schematic representation of full genomes for (A) OVV-014, OVV-004, OVV-015, OVV-016, OVV-005, OVV-006 and OVV-013, (B) OVV-008, OVV-009, OVV-010 and OVV-011 and (C) OVV-007 and OVV-003. Abbreviations: pSEL synthetic early late promoter, pF17 = promoter from the F17R gene, pA14 = promoter from the A14R gene, hCD80-Fc = human secreted CD80 molecule fused with an immunoglobulin Fc fragment, hCD80-FL = human foil length CD80 molecule (not secreted), mIL-12 ^:= mouse fused interleukin-12 A and interleukin- 12B, mIL-21 ^:=: mouse interleukin-21, mil, -7 = mouse interleukin-7. * = mutation encoding substitution of lysine to glutamate at position 151 of A34 protein.

[0007] FIG. 2A-D. Provides data of transgene expression analysis following infection of cells with recombinant oncolytic vaccinia viruses. Total cell lysate of the infected cells harvested after 18 hours was analyzed by western blotting as described in materials and methods. (A) human secreted CD80 protein fused with an immunoglobulin Fc fragment (expressed by OVV-004 and OVV-016 minus and plus M2L viral gene respectively); and non-secreted human CD80 full length (expressed by OVV-015 with M2L viral gene deletion) detection analysis following infection of cells with recombinant oncolytic vaccinia viruses. (B) mouse fused interleukin- 12A and interleukin- 12B detection analysis following infection of cell with recombinant oncolytic vaccinia vims OVV-013 (mIL-12 expression driven by synthetic early late promoter), OVV-005 (mIL-12 expression driven by Fl 7 promoter), OVV-006 (mIL-12 expression driven by Al 4 promoter), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by F17 promoter), OVV-011 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by A14 promoter) and P70 a recombinant IL- 12 protein as a control. (C) mouse interleukin-7 analysis following infection of cells with recombinant oncolytic vaccinia virus OVV-007 (mIL-7), OV V- 008 (hCD80-Fc, mIL-21 and mIL-7), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mil .-12 expression driven by F17 promoter) and OVV-011 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by Al 4 promoter). (D) mouse interleukin-21 analysis following infection of cells with recombinant oncolytic vaccinia virus OVV-003 (mIL-21), OVV-008 (hCD80-Fc, mlL- 21 and mIL-7), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by F17 promoter) and OVV-Ol 1 (hCD80-Fc, mIL-21 , mIL-7 and mIL-12 expression driven by A14 promoter).

[0008] FIG. 3A-D. Provides data of the concentration of the secreted transgenes expressed follo wing infection of producer cells with recombinant oncolytic vaccinia virus. Supernatants of the infected cells collected after 24 hours were analyzed by ELISA using two different dilutions as mentioned in materials and methods. (A) detection of CD80 concentrations in the supernatant of cells infected with recombinant oncolytic vaccinia virus OVV-004 (hCD80-Fc with M2L viral gene deletion), OVV-Ol 6 (hCD80-Fc containing M2L viral gene), OVV-008 (hCD80-Fc, mlL- 21 and mIL-7), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by Fl 7 promoter) and OVV-Ol 1 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by A14 promoter). (B) detection of IL- 12 concentration in the supernatant of cells infected with recombinant oncolytic vaccinia virus OVV-013 (mIL-12 expression driven by synthetic early late promoter), OVV-005 (mIL-12 expression driven by F17 promoter), OVV-006 (mIL-12 expression driven by Al 4 promoter), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by Fl 7 promoter), OVV-Ol 1 (hCD80-Fc, mIL-21, niIL-7 and mIL-12 expression driven by A14 promoter) and OVV-007 (mIL-7) as a negative control. (C) detection of IL-7 concentration in the supernatant of cells infected with recombinant oncolytic vaccinia virus OVV-007 (mIL-7), OVV-008 (hCD80-Fc, mIL-21 and mIL-7), OVV-010 (hCD80-Fc, mIL-21 , mIL-7 and mIL-12 expression driven by F17 promoter), OVV-Ol 1 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by A14 promoter) and OVV-003 (mIL-21) as a negative control. (D) detection of IL-21 concentration in the supernatant of cells infected with recombinant oncolytic vaccinia virus OVV-003 (mIL-21), OVV-008 (hCD80-Fc, mIL-21 and mIL-7), OW-OIO (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by Fl 7 promoter), OVV-Ol 1 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by A14 promoter) and OVV-007 (mIL-7) as a negative control. The concentration for all the transgenes is in ng/mL.

[0009] FIG. 4A-E. Provides data on the effect of secreted hCD80-Fc (plus and minus M2L gene), non-secreted hCD80-FL and mIL-12 (driven by different viral promoters) expression on vaccinia virus replication in representative human cancer cell lines. Cancer cell lines (A) A549 (lung adenocarcinoma), (B) HT-29 (colorectal adenocarcinoma), (C) Colo 741 (colorectal adenocarcinoma), (D) HCT-116 (colorectal carcinoma), (E) U-2 OS (osteosarcoma), were infected with recombinant vaccinia viruses; backbone not expressing any transgenes (OVV-014) as a control for the experiment, an irrelevant vaccinia virus expressing GM-CSF and LacZ similar to JX-594 (OVV-009) as a comparator, OVV-004 (secreted hCD80-Fc with M2L viral gene deletion), OVV-015 (non-secreted CD80-FL with M2L viral gene deletion), OVV-016 (secreted hCD80-Fc containing M2L viral gene), OVV-005 (mIL-12 expression driven by F17 promoter), OVV-006 (mIL-12 expression driven by A14 promoter) and OVV-013 (mIL-12 expression driven by synthetic early late promoter); at an MOI of 1 in triplicate. The infected cells were harvested 24 hours post-infection. The viral titer of each sample was determined by viral plaque assay and represented by plaque-forming units (PFU) produced per cells. Error bars indicate SD^^S). Asterisks indicate statistical significance against the control not expressing any transgenes (OW-014) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001) one-way Anova test followed by Tukey’s multiple comparisons test.

[0010] FIG. 5. Provides data on the effect of tran sgene expression on recombinant vaccinia virus potency to kill HCT-116 colorectal cancer cell line, as assessed by an in vitro cytotoxicity assay. HCT-116 cells were infected in quadruplicate with recombinant vaccinia virus OVV-014 (control not expressing any transgenes), OVV-004 (secreted hCD80-Fc with M2L viral gene deletion), OVV-005 (mIL-12 expression driven by F17 promoter), and OVV-006 (mIL-12 expression driven by A14 promoter) with a range of different MOIs. The infected cells were washed after 1 hour of absorption and incubated for 72 hours. Cytotoxicity was determined by counting cells alive with the colorimetric CCK-8 assay. Error bars indicate SD (n^:=4). Graphs adjusted to fit using a 4-parameter logistic regression with GraphPad Prism 10.

[0011] FIG. 6. Provides data of the functional acti vity of secreted IL-12 expressed follo wing infection of cells with recombinant oncolytic vaccinia virus. Supernatants of the infected cells with OVV-013 (mIL-12 expression driven by synthetic early late promoter), OVV-005 (mIL-12 expression driven by F17 promoter), OVV-006 (mIL-12 expression driven by A14 promoter), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by F17 promoter), and OVV-011 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression driven by Al 4 promoter), were collected after 24 hours and serial dilutions of the supernatants were measured as described in the materials and methods. The EC50 (ng/inL) of functional IL-12 w'as calculated and plotted for the infection with each recombinant oncolytic vaccinia virus.

[0012] FIG. 7A-H. Provides results of assessment of virotherapy-induced tumor growth inhibition, toxicity and survival on C57BL/6J female mice implanted SC with MC38 tumor cells.

(A) Tumor growth trajectories are shown for average mice in groups (n=10) treated intravenously with vehicle only or WR vaccinia virus containing the A34R KI 5 IE substitution not armed with M2L and J2R viral gene deletions (OW-014) as a control, OVV-004 expressing human secreted CD80 protein fused with an immunoglobulin Fc fragment (hCDSO-Fc) wdth M2L viral gene deletion, OVV-015 expressing non-secreted human CD80 full length (11CD80- FL ) with M2L viral gene deletion and OVV-016 expressing hCD80-FC with M2L viral gene intact. Average tumor volumes (mm³) ±95% confidence intervals for each treatment group are shown through day 28 post-tumor implant, which was the last tumor measurement time point when all animals in each group were still ali ve. Asterisk indicate statistical significance against the vehicle (*p<0.05); two-way Anova test followed by Tukey’s multiple comparison test. (B) Percentage of average of body weight change of the same mice groups listed in section A. Average percent body weight change ±95% confidence intervals per group are shown through day 25 post-tumor implant as there were no difference after that day in the study. (C-G) Tumor growth trajectories are shown for individual mice in groups treated as described in section A. The dashed vertical line on each graph represents the time point when animals received intravenous injections of vehicle and viruses. Individual tumor volumes (mm⁵) for each treatment group are shown through day 42 post-tumor implant, which was the last tumor measurement time point before the study was terminated. (H) Survival data of the same mice groups listed in section A. Mice were designated daily as deceased upon reaching tumor burden > 2000 mm³. The point of intersection between each group’s curve and the horizontal dashed line indicates the median (50% survival threshold for group).

[0013] FIG. 8A-H. Provides results of assessment of virotherapy-induced tumor growth inhibition, toxicity and survival on C57BL/6J female mice implanted SC wdth MC38 tumor cells. (A) Tumor growth trajectories are shown for average mice in groups (n 10) treated intravenously with vehicle only or WR vaccinia virus containing the A34R KI 5 IE substitution not armed with M2L and J2R viral gene deletions (OVV-014) as a control, a comparator vaccinia virus expressing GM-CSF and LacZ similar to JX-594 (OVV-009), a recombinant oncolytic vaccinia virus armed with hCD80-Fc, mIL-21, mIL-7 and mIL-12 driven by A14 promoter (OVV-Ol 1), a recombinant oncolytic vaccinia virus armed with hCD80-Fc, mIL-21, mIL-7 and mIL-12 driven by Fl 7 promoter (OVV-OIO) and a recombinant oncolytic vaccinia virus armed with hCD80-Fc, mIL-21 and mIL-7 (OVV-008). Average tumor volumes (mm³) ±95% confidence intervals for each treatment group are shown through day 28 post-tumor implant, which was the last tumor measurement time point when all animals in each group were still alive. Asterisks indicate statistical significance against the vehicle group (*p<0.05); two-way Anova test followed by Tukey’s multiple comparison test. (B) Percentage of average of body weight change of the same mice groups listed in section A. Average percent body weight change ±95% confidence intervals per group are shown through day 25 post-tumor implant as there were no difference after that day in the study. (C-H) Tumor growth trajectories are shown for individual mice in groups treated as described in section A. The dashed vertical line on each graph represents the time point when animals received intravenous injections of vehicle and viruses. Individual tumor volumes (mm³) for each treatment group are shown through day 42 post-tumor implant, which was the last tumor measurement time point before the study was terminated. (I) Survival data of the same mice groups listed in section A. Mice were designated daily as deceased upon reaching tumor burden > 2000 mm³. The point of intersection between each group’s curve and the horizontal dashed line indicates the median (50% survival threshold for group).

[0014] FIG. 9 provides schematic representation of full genomes for OVV-029, OVV-030, OVV-031, OVV-032, and OVV-033, engineered in Copenhagen vaccinia virus strain. Abbreviations: pSEL = synthetic early late promoter, pF17 = promoter from the F17R gene, hTL- 2gv -= human interleukin glycovariant, hCD80-Fc = human secreted CD80 molecule fused with an immunoglobulin Fc fragment, Luc-2A-GFP = Luciferase, self-cleaving 2A. peptide and green fluorescent protein reporter, HSV-TK.007 = herpes simplex virus thymidine kinase gene with mutation encoding alanine to histidine substitution at position 168, hIL-12 = human fused interleukin-12A and interleukin-12B, hIL-21 =^: human interleukin IL-21, and * = mutation encoding substitution of lysine to glutamic acid at position 151 in A34 protein. [0015] FIG. 10A-C. Provides data on the effect of hIL-12, secreted hCD80-Fc, hIL-21, hlL- 2g v and HSV-TK.007 transgenes combination expression on vaccinia virus replication in representative human cancer cell lines. Cancer cell lines (Fig. 10A) HT-29 (colorectal adenocarcinoma), (Fig. 10B) A549 (lung adenocarcinoma), and (Fig. 10C) Colo 741 (colorectal adenocarcinoma), were infected with recombinant vaccinia viruses: backbone with J2R and M2L genes are deleted, substitutions of KI 5 IE of A34 protein and armed with Luciferase-2A-GFP reporter (OVV-029) as a control for the experiment, OVV-030 (expressing a combination of secreted hCD80-Fc and hIL-12), OVV-031 (expressing a combination of secreted hCD80-Fc, hIL-12 and HSV-TK.007), OW-032 (expressing a combination of secreted hCD80-Fc, hIL-21 and ML- 12) and OVV-033 (expressing a combination of hIL-2gv and HSV-TK.007): at a MOI of 1 in triplicate. The infected cells were harvested 48 hours post-infection. The viral titer of each sample was determined by viral plaque assay and represented by plaque-forming units (PFU) produced per cells (PFU/cell). Error bars indicate SD (n^:=3). Asterisks indicate statistical significance against the control not expressing any therapeutic transgenes (OVV-029) (*p<0.05; **p<0.01) one-way Anovatest followed by Tukey’s multiple comparisons test.

[0016] FIG. 11A-C. Provides data on the effect of transgene expression on recombinant vaccinia virus potency to kill (Fig. 11 A) HCT 116 (colorectal carcinoma cells), (Fig. 1 IB) Colo 741 (colorectal adenocarcinoma) and (Fig. 11 C) A549 (lung adenocarcinoma cells), as assessed by an in vitro cytotoxicity assay. Cells were infected in quadruplicate with recombinant vaccinia virus backbone with J2R and M2L genes are deleted, substitutions of KI 5 IE of A34 protein and armed with Luciferase-2A-GFP reporter (OVV-029) as a control for the experiment, OV V-030 (expressing a combination of secreted hCD80-Fc and ML- 12), OVV-031 (expressing a combination of secreted hCD80-Fc, hIL-12 and HSV-TK.007), OVV-032 (expressing a combination of secreted hCD80-Fc, hIL-21 and hIL-12) and OVV-033 (expressing a combination of hIL-2gv and HSV-TK.007); with a range of different MOIs. The infected cells were washed after 1 hour of absorption and incubated for 72 hours with HCT 116 cells and 96 hours with Colo 741 and A549 cells. Cytotoxicity was determined by counting cells alive with the colorimetric CCK-8 assay. Error bars indicate SD (n=4). Graphs adjusted to fit using a 4- parameter logistic regression with GraphPad Prism 10. [0017] FIG. 12. Provides schematic representation of full genomes for OVV-034, OVV-035, OVV-036, OVV-037, OVV-038, OVV-039 and OV V-040 engineered in Western reserve vaccinia virus strain. Abbreviations: pSEL = synthetic early iate promoter, pF 17 ^:= promoter from the F17R gene, mIL-2v = mouse interleukin variant, hCD80-Fc = human secreted CD80 molecule fused with an immunoglobulin Fc fragment, HSV-TK.007 = herpes simplex virus thymidine kinase gene with mutation encoding alanine to histidine substitution at position 168, * mutation encoding substitution of lysine to glutamic acid at position 151 in A34 protein, # = mutation encoding substitution of methionine to arginine at position 63 in A33 protein, ^{A ::::} mutation encoding substitution of methionine to threonine at position 66 in A34 protein, & = mutation encoding substitutions of asparagine to threonine at position 241, glutamic acid to valine at position 243, valine to serine at position 247, glycine to arginine at position 250 and alanine to phenylalanine at position276 in B5 protein.

[0018] FIG. 13A-C. Provides data of transgene expression analysis following infection of cells with recombinant oncolytic vaccinia viruses. Total cell lysate of the infected cells harvested after 22 hours was analyzed by western bloting as described in materials and methods. (Fig.

13A) mouse IL-2v protein (expressed by OVV-036 to OVV-040); (Fig. 13B) human secreted CD80 protein fused with an immunoglobulin Fc fragment (expressed by OVV-035, OVV-037, OVV-039 and OVV-040); and (Fig. 13C) mouse interleukin-21 analysis following infection of cells with recombinant oncolytic vaccinia virus OVV-038.

[0019] FIG. 14A-C. Provides data on the effect of IL-2v, secreted hCD80-Fc, and mIL-21 , singles and in combinations expression on vaccinia virus replication in representative mouse and human cancer cell lines. Cancer cell lines (Fig. 14A) MC38 (murine colorectal carcinoma), (Fig. 14B) A549 (lung adenocarcinoma), (Fig. 14C) HT-29 (colorectal adenocarcinoma), were infected with recombinant vaccinia viruses; backbone with J2R and M2L genes are deleted, substitutions of K151E of A34 protein, expression of HSV-TK.007 (OVV-034) as a control for the experiment, OVV-035 (expressing secreted hCD80-Fc), OVV-036 (expressing mIL-2v), OVV-037 (expressing a combination of secreted hCD80-Fc and mIL-2v) and OVV-038

(expressing a combination of mIL-21 and mIL-2v); at an MOI of 1 in triplicate. The infected cells were harvested 48 hours post-infection. The viral titer of each sample was determined by viral plaque assay and represented by plaque-forming units (PFU) produced per cells. Error bars indicate SD (n=3). Asterisks indicate statistical significance against the control not expressing any therapeutic transgenes (OVV-034) (*p<0.05; **p<0.01) one-way Anovatest followed by Tukey’s multiple comparisons test.

[0020] FIG. 15A-B. Provides data on the effect of transgene expression on recombinant vaccinia virus potency to kill (Figure 15 A) MC38 (murine colorectal carcinoma cells) and (Figure 15 B) A549 (lung adenocarcinoma cells), as assessed by an in vitro cytotoxicity assay. Cells were infected in quadruplicate with recombinant vaccinia virus OVV-034 (control backbone not expressing therapeutic transgenes), OVV-035 (expressing secreted hCD80-Fc), OVV-036 (expressing mIL-2v), OVV-037 (expressing a combination of secreted bCD80-Fc and ml'L-2v), and OVV-038 (expressing a combination of mIL-21 and mIL-2v) with a range of different MOIs. The infected cells were washed after 1 hour of absorption and incubated for 72 hours with MC38 cells and 96 hours with A549 cells. Cytotoxicity was determined by counting ceils alive with the colorimetric CCK-8 assay. Error bars indicate SD (n^::::4). Graphs adjusted to fit using a 4-parameter logistic regression with GraphPad Prism 10.

[0021] FIG. 16A-L. Provides results of assessment of virotherapy-induced tumor growth inhibition, toxicity and survival on C57BL/6J female mice implanted subcutaneous (SC) with MC38 tumor cells. (Fig. 16A) Tumor growth trajectories are shown for average mice in groups (n=l 0) treated intravenously with vehicle only or WR vaccinia virus containing the A34 KI 51 E substitution, with M2L and J2R viral gene deletions and expression of HSV-TK.007 transgene (OVV-034) as a control, a recombinant oncolytic vaccinia virus armed with hCD80-Fc (OVV- 035), a recombinant oncolytic vaccinia virus armed with mIL-2v (OW-036), a recombinant oncolytic vaccinia virus armed with a combination of hCD80-Fc and m!L-2v (OW-037), a recornbinant oncolytic vaccinia virus armed with a combination of IL-21 and mIL-2v (OVV- 038) a recombinant oncolytic vaccinia virus armed with a combination of hCD80-Fc and mlL- 2v, containing the A33 M66R, A34 M66T substitutions (OVV-039) and a recombinant oncolytic vaccinia virus armed with a combination of hCD80-Fc and mIL-2v, containing the B5 N241T, E243V, V247S, G250R and A276F substitutions (OW-040). Average tumor volumes (mm³) ± 95% confidence intervals for each treatment group are shown through day 56 post-tumor implant, which was the last tumor measurement time point before the study was terminated.

Asterisks indicate statistical significance against the vehicle group and control group (OVV-034) (****p<0.0001); two-way Anova. test followed by Tukey’s multiple comparison test. (Figs. 16B- I) Tumor growth trajectories are shown for individual mice in groups treated as described in section A. The dashed horizontal line on each graph represents a tumor size of 1500 mnr. Individual tumor volumes (mm³) for each treatment group are shown through day 56 post-tumor implant, which was the last tumor measurement time point before the study was terminated. (Fig. 16J) Percentage of average of body weight change of the same mice groups listed in section A. Average percent body weight change ± 95% confidence intervals per group are shown through day 56 post-tumor implant. (Fig. 16K) Survival data of the same mice groups listed in section A. Mice were designated daily as deceased upon reaching tumor burden > 2000 mm³. The point of intersection between each group’s curve and the horizontal dashed line indicates the median (50% survival threshold for group). (Fig. 16L) Provides data of the concentration of the hCD80 transgene expressed in the serum collected from the animals of the indicated groups after 3 days of virus treatment and quantified by ELISA. The concentration is the average of hCD80 in ng/mL ± SD. Asterisk indicate statistical significance against the group expressing hCD80 as a single (OVV-035) (**p<0.01, ****p<0.0001); one-way Anova test followed by Sidak’s multiple comparison test.

[0(122] FIG. 17. Provides data on virus spreading of the indicated virus containing A33/A34 or B5 substitutions. Representative images of comets formed following infection of BSC-40 cells demonstrate that vaccinia viruses with a combination of substitutions in A33 M63R/A34 M66T and the A34 K151E (OVV-039) as well as the vaccinia virus with a combination substitution in B5 N241T, E243V, V247S, G250R, A276F and the A34 K151E (OVV-040), results in longer comet tails and more satellite smaller plaques than OVV-037 containing only A34 KI 51 substitutions. Fewer comets are observed in the infection with the comparator vaccinia virus expressing GM-CSF and LacZ similar to JX-594 and lacking A34 KI 5 IE substitution (OVV- 009). [0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Definitions

[0025] The term "isolated" designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated."

[0026] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then that a single vector can contain just a single coding region, or comprise two or more coding regions.

[0027] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5^! non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory' regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. [0028] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.

[0029] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[00301 "Transcriptional control sequences” refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissuespecific promoters and enhancers as well as lymphokine-inducible promoters (e.g,, promoters inducible by interferons or interleukins),

[0031] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence), and "self-cleaving” 2A peptides ” of about 20 amino acids that produce equimolar levels of multiple genes from the same mRNA and may be used in place of IRES elements in multicistronic vectors, non-limiting examples include T2A, P2A, E2A and F2A peptides sequences.

^0032] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0033] A "vector" refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion, of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0034] Vectors can be engineered to encode selectable markers or reporters that pro vide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: HSV-TK, luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0035] “Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissuespecific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

£0036] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular doublestranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0037] A polynucleotide or polypeptide has a certain percent ’’sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. .Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USzA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needlernan and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0038] Oncolytic Viruses

[0039] Any oncolytic virus may be modified to contain heterologous nucleic acid encoding CD80 (e.g., soluble CD80 such as CD80-Fc), IL-2 or a variant thereof, IL-12 (comprising both subunits or single chain such as IL-12p70 or IL-12p40), IL-21, IL-7 or any combination or subcombination thereof.

[0040] In some embodiments, the virus comprises nucleotide sequences encoding human CD80 (preferably human CD80-Fc) and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B16R gene

[0041] In some embodiments, the virus comprises nucleotide sequences encoding human CD80 (preferably human CD80~Fc) and human IL-12 and optionally an HSV-TK and comprises non-functional M2L and J2R genes. [0042] In some embodiments, the virus comprises nucleotide sequences encoding human CD80 (preferably human CD80-Fc) and human IL-2 (preferably a human IL -2 variant) and optionally an HSV-TK and comprises non- functional M2L and J2R genes and optionally comprises a non-functional B16R gene

[0943] In some embodiments, the virus comprises nucleotide sequences encoding human CD80 (preferably human CD80-Fc), human IL- 12 and human IL-21 and optionally an HSV-TK and comprises non-functional M2L and J2R genes

[0044] In some embodiments, the virus comprises nucleotide sequences encoding human IL-2 (preferably a human IL-2 variant) and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B16R gene.

[0045] In some embodiments, the virus comprises nucleotide sequences encoding human IL- 12 and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B 16R gene.

[0046] In some embodiments, the virus comprises nucleotide sequences encoding human IL-2 (preferably a human IL-2 variant) and human IL-21 and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B16R gene.

[0047] In some embodiments, the virus comprises nucleotide sequences encoding human IL-7 and optionally an HSV -TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B16R gene.

[0048] In some embodiments, the virus comprises nucleotide sequences encoding human CD80 (preferably human CD80-Fc), human IL-21 and human IL-7 and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a non-functional B 18R gene.

[0049] In some embodiments, the virus comprises nucleotide sequences encoding human

CD80 (preferably human CD80-Fc), human IL- 12, human IL-21 and human IL-7 and optionally an HSV-TK and comprises non-functional M2L and J2R genes and optionally comprises a nonfunctional B18R gene and/or a non-functional B16R gene.

[0050] In some embodiments, the oncolytic virus is a native or genetically modified vesicular stomatitis vims (VSV), Maraba virus, a herpesviridae virus such as HS V-1 or HSV-2, a Cytomegalovirus (CMV), a Measles virus, a reovirus (e.g. reolysin), Newcastle disease virus (NDV), seneca valley virus, poliovirus or parvovirus.

[0051] In preferred embodiments, the oncolytic virus is a Poxviridae virus and is most preferably a vaccinia virus. Vaccinia virus is a large and complex enveloped virus having a linear double-stranded DNA genome of about 190K bp and encoding approximately 250 genes.

[0052] In some aspects, the oncolytic vaccinia virus is a genetically modified replication- competent oncolytic virus derived fromWestem Reserve strain (ATCC VR-119; GenBank accession number NC_006998), Wyeth strain (ATCC VR-325), Lister strain, Copenhagen strain (accession number M35027), modified vaccinia Ankara (MV A) strain, Tian Tan strain, CL strain (ATCC VR-117), IHD-J strain or Lederle-Chorioallantoic (ATCC VR-325) strain or any other known vaccinia strain; most of these strains are available from the American Type Culture Collection (Manassas, Va.). In particularly preferred embodiments, oncolytic vaccinia virus is derived from Copenhagen strain.

[0053] The oncolytic vaccinia virus may be engineered to lack one or more functional genes, e.g., in order to increase the cancer selectivity of the virus. In one preferred aspect, the oncolytic vaccinia virus may be engineered to lack thymidine kinase (TK) activity. A TK-deficient vaccinia virus requires thymidine triphosphate for DNA synthesis, which leads to preferential replication in dividing cells (particularly cancer cells). In another aspect, the oncolytic vaccinia virus may be engineered to lack one or both copies of vaccinia virus growth factor (VGF). This secreted protein is produced early in the infection process, acting as a mitogen to prime surrounding cells for infection. In some aspects, the oncolytic vaccinia virus is engineered to lack both VFG and TK activity. Additional genes that can be modified include: E3L, C2L, C1L, NIL, N2L, MIL, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B5R, B14R, B8R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G,

1 o B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In some preferred embodiments, the oncolytic vaccinia virus lacks J2R and M2L activity⁷ (e.g., by foil or partial deletion or insertion). In other embodiments, a replication-competent recombinant oncolytic vaccinia virus as herein described comprises a modification of A45R, B13R and A53R and optionally further comprises a modification of one or more of NIL, A44L-A46R and A49R. Such modification encompasses complete or partial deletion, insertion of heterologous nucleic acid, substitution of a portion of the gene with heterologous nucleic acid and the like.

[0(154] In some preferred embodiments, the oncolytic vaccinia virus is engineered to lack a functional M2L gene. In related aspects, the oncolytic vaccinia virus is engineered to lack a functional M2L gene and to lack a function thymidine kinase gene.

[0055] In some aspects, the oncolytic vaccinia virus comprises a mutation in the M2L and/or J2R genes that results in a negative M2L phenotype and/or a negative thymidine kinase phenotype. In some aspects the negative M2L and/or negative thymidine kinase phenotypes result from an M2L and/or J2R gene containing a deletion of nucleic acid sequence (e.g., a full or partial deletion of the M2L and/or J2R genes). In other aspects, the negative M2L and/or negative thymidine kinase phenotypes result from an insertion of nucleic acid sequence in the M2L and/or J2R genes. In other aspects, the negative M2L and/or negative thymidine kinase phenotypes result from a substitution of nucleic acid sequence in the M2L and/or J2R genes.

[0056] In other preferred embodiments, the oncolytic vaccinia virus comprises a mutation in the A45R, B13R. A53R and/or B18R genes that results in a negative A45R, B13R, zk53R and/or B 18R phenotype. In some aspects the negative A45R, B 13R, A53R and/or B 18R phenotypes result from an A45R, B13R, A53R and/or Bl 8R gene containing a deletion of nucleic acid sequence (e.g., a full or partial deletion of the A45R, B13R, A53R and/or Bl 8R genes). In other aspects, the negative A45R, B13R, A53R and/or Bl 8R phenotypes result from an insertion of nucleic acid sequence in the A45R, B13R, A53R and/or B18R genes. In other aspects, the negative A45R, BI3R, A53R and/or B18R phenotypes result from a substitution of nucleic acid sequence in the A45R, B13R, A53R and/or B18R genes. [0057] In particularly preferred embodiments, the oncolytic vaccinia virus is a TK-deficient and M2L-deficient Western Reserve or Copenhagen strain vaccinia virus. In related embodiments, the oncolytic vaccinia virus is further deficient, in one or more of, and preferably all of, the A45R, B13R, A5R3 and Bl SR genes.

[0058] Vaccinia virus may be propagated using the methods described by Earl and Moss (Ausubel et al. (1994) Current Protocols in Molecular Biology, pages 16.15.1 to 16.18.10) or the methods described in WIPO Publication No. W02013/022764, both of which are incorporated herein by reference.

[0059] In some embodiments, the vaccinia virus thymidine kinase gene in any of the recombinant oncolytic vaccinia viruses herein described is replaced with a herpes simplex virus thymidine kinase (HSV-tk) gene, allowing for viral replication control and li ve imagining via an anti-viral agent such as a 2 ’-deoxy guanosine analog (e.g., ganciclovir). In some aspects, the HSV-tk gene encodes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to or 100% identical to SEQ ID NO:1 :

MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRl’EQKMPTLLRVYIDGPH GMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDA AVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHP1AHLLCYPAAR YLMGSMTPQAVLAFVALIPP'FLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIR RVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRA PELLAPNGDLYNATAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMIQTHV TTPGSIPTICDLARTFAREMGEAN (SEQ ID NO:1)

[0060] In related aspects, the HSV-tk gene comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to or 100% identical to SEQ ID NO:2:

ATGGCGTCTTATCCGGGACATCAACATGCGTCTGCTTTTGATCAAGCGGCGAGATCT

AGAGGACACTCCAATAGAAGAACAGCGCTAAGACCGAGAAGACAGCAAGAAGCGA

CAGAAGTTAGACCCGAACAAAAGATGCCGACCTTGCTAAGAGTCTACATCGATGGA CCACACGGAATGGGAAAGACAACAACAACCCAACTACTAGTCGCGCTAGGATCCAG AGATGATATCGTATATGTACCCGAGCCGATGACCTATTGGAGAGTATTGGGAGCGTC TGAGACAATCGCGAACATCTACACAACACAGCACAGACTAGATCAGGGAGAAATCT CTGCGGGAGATGCTGCTGTAGTAATGACATCTGCGCAAATCACAATGGGAATGCCG TACGCTGTAACCGATGCTGTACTAGCTCCACATATTGGTGGAGAAGCGGGATCTTCT CATGCTCCACCACCAGCTCTAACCCTAATCTTTGATAGACATCCGATCGCGCACCTA CTATGTTATCCAGCTGCGAGATACCTAATGGGATCTATGACACCACAAGCGGTACTA GCGTl’CGTAGCTCTAATTCCACCAACACTACCGGGAACGAACATAGTTTTGGGAGCG CTACCAGAGGATAGACACATTGATAGACTAGCGAAGAGACAAAGACCGGGAGAAA GACTAGATTTGGCTATGCTAGCGGCGATCAGAAGAGTGTACGGACTACTAGCGAAC ACCGTCAGATATC J ACAAGG rGGTGGAlCTTGGAGAGAGGATTGGGGACAATTATC TGGAACAGCAGTACCACCACAAGGTGCTGAACCACAATCTAATGCTGGACCAAGAC CGCATATCGGAGATACCCTATTCACACTATTCAGAGCGCCAGAATTGCTAGCTCCAA ACGGAGACTTGTACAACGTATTCGCTTGGGCGCTAGATGTCCTAGCCAAAAGACTAA GACCCATGCACGTGTTCATCCTAGACTACGATCAATCTCCAGCGGGATGTAGAGATG CGCTACTACAAC IAACCTCCGGAATGATCCAGACGCACGTAACAACACCAGGATCT ATCCCGACCATTTGTGATCTAGCGAGAACCTTCGCTAGAGAGATGGGAGAAGCTAA T (SEQ ID NO:2)

[0061] A replication-competent, recombinant oncolytic vaccinia virus of the present disclosure preferably comprises a modification that enhances virus spreading, e.g., a mutation in the A34R, A33R and/or B5R genes. In particularly preferred embodiments, the replication- competent, recombinant oncolytic vaccinia virus of the present disclosure comprises an A34R gene comprising a KI 5 IE substitution (i.e., comprising a modification that provides for a K151E substitution in the encoded polypeptide). See, e.g., Blasco et al. (1993) J. Virol. 67(6):3319- 3325; and Thirunavukarasu et al. (2013) Mol. Ther. 21 :1024. The A34R gene encodes vaccinia virus gp22-24.

((M)€s2| In some embodiments, the vims comprises an A33 gene comprising an M63R substitution. In some embodiments, the A33 gene encodes the following amino acid sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto and comprising an M63R substitution:

MMTPENDEEQTSVFSAWYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLN QCRSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYQL FSDAKANCTAESSTLPNKSDVLITWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRK YFCVKTMN (SEQ ID N0:3)

[0063] In related aspects, the A33 gene encodes an A33 protein comprising an M63R substitution and comprises the following nucleotide sequence or a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto:

ATGATGACACCAGAAAACGACGAAGAGCAGACATCTGTGTrCTC-CGCTACTGTTTAC GGAGACAAAATTCAAGGAAAGAATAAACGCAAACGCGTGATTGGTCTATGTATTAG AATATCTATGGTTATTTCACTACTATCTATGATTACCATGTCCGCGTTTCTCATAGTG CGCCTAAATCAATGCAGATCTGCTAACGAGGCTGCTATTACTGACGCCGCTGTTGCC

GTTGCTGCTGCATCATCTACTCATAGAAAGGTTGCGTCTAGCACTACACAATATGAT CACAAAGAAAGCTGTAATGGTTTATATTACCAGGGTTCTTGTTATATATTACATTCA GACTACCAGTTATTCTCGGATGCTAAAGCAAATTGCACTGCGGAATCATCAACACTA CCCAATAAATCCGATGTCTTGATTACCTGGCTCATTGATTATGTTGAGGATACATGG

GGATCTGATGGTAATCCAATTACAAAAACTACATCCGATTATCAAGATTCTGATGTA TCACAAGAAGTTAGAAAGTATTTTTGTGTTAAAACAATGAAC (SEQ ID NO:4)

[0064] In other aspects, the virus comprises an A34 gene comprising a K151E and an M66T substitution. In some embodiments, the A34 gene encodes the following amino acid sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto and comprising KI 5 IE and M66T substitutions:

MKSLNRQTVSRFKKLSVPAAIMMILSTIISGIGTFLHYKEELMPSACANGWIQYDKHCYL DTNIKTSTDNAVYQCRKLRARLPRPDTRHLRVLFSIFYKDYWSLKKTNDKWLDINND roiDISKLTNFKQLNSTTDAEACYIYKSGKLVETVCKSTQSVLCVKKFYK (SEQ ID NO:5) [0065] In related aspects, the A34 gene encodes an A34 protein comprising KI 51E and M66T substitutions and comprises the following nucleotide sequence or a nucleotide sequence at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

ATGAAATCGCTTAATAGACAAACTGTAAGTAGGTTTAAGAAGTTGTCGGTGCCGGCC GCTATAATGATGATACTCTCAACCATrATTAGTGGGATAGGAACATrTCTGCATTAC AAAGAAGAACTGATGCCTAGTGCTTGCGCCAATGGATGGATACAATACGATAAACA TTGTTATTTAGATACTAACATTAAAACATCTACAGATAATGCGGTTTATCAGTGTCGT AAATTACGAGCCAGATTGCCTAGACCGGATACTAGACATCTGAGAGTATTGTTTAGT ATTTTTTATAAAGATTATTGGGTAAGTTTAAAAAAGACCAATGATAAATGGTTAGAT ATTAATAATGATAAAGATATAGATAlTAGTAAATTAACAAATTTrAAACAACTAAAC AGTACGACGGATGCTGAAGCGTGTTATATATACAAGTCTGGAAAACTGGTTGAAAC AGTATGTAAAAGTACTCAATCTGTACTATGTGTTAAAAAATTCTACAAG (SEQ ID N0:6)

[0066] In some embodiments, the virus comprises a B5R gene comprising N241T E243V V247S G250R and A276F amino acid substitutions. In some embodiments, the B5R gene encodes the following amino acid sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto and comprising N241T E243V V247S G250R and A276F amino acid substitutions:

MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDQGYHSSDPNA VCETDKWTKYENPCKKMCTVSDYISELYNKPLYEWSTMTLSCNGETKYFRCEEKNGNT SWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGASYISCTANSW NVIPSCQQKCDMPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPVLPICV RTTEVFDPSDDRPDDETDLSKLSKDVVQYI2QE1ESLEFTYHIIIVALTIMGVIFL1SVIVLVC SCDKNNDQYKFHKLLP (SEQ ID NO:7)

|0067! In related aspects, the B5R gene encodes a B5R protein comprising N241T E243V V247S G250R and A276F amino acid substitutions and comprises the following nucleotide sequence or a nucleotide sequence at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto: ATGAAAACGATTTCCGTTGTTACGTTGTTATGCGTACTACCTGCTGTTGTTTATTCAA CATGTACTGTACCCACTATGAATAACGCTAAATTAACGTCTACCGAAACATCGTTTA ATGATAAACAGAAAGTTACGTTTACATGTGATCAGGGATATCATTC'FTCGGATCCAA ATGCTGTCTGCGAAACAGATAAATGGAAATACGAAAATCCATGCAAAAAAATGTGC ACAGTTTCTGATTACATCTCTGAATTATATAATAAACCGCTATACGAAGTGAATTCC ACCATGACACTAAGTTGCAACGGCGAAACAAAATA riTTCGTI GCGAAGAAAAAAA TGGAAATACTTCTTGGAATGATACTGTTACGTGTCCTAATGCGGAATGTCAACCTCT TCAATTAGAACACGGATCGTGTCAAC-CAGTTAAAGAAAAATACTCATTTGGGGAAT ATATGACTATCAACTGTGATGTTGGATATGAGGTTATTGGTGCTTCGTACATAAGTT GTACAGCTAATTCTTGGAATGTTATTCCATCATGTCAACAAAAATGTGATATGCCGT CTCTATCTAATGGATTAATTTCCGGATCTACATTTTCTATCGGTGGCGTIATACATCT TAGTTGTAAAAGTGGTTTTACACTAACGGGGTCTCCATCATCCACATGTATCGACGG TAAATGGAATCCCGTACTCCCAATATGTGTACGAACTACCGAAGTGTTCGATCCGTC

TGATGATAGACCGGATGACGAAACCGACCTATCCAAGCTATCTAAGGATGTCGTCC AGTACGAGCAAGAGATCGAGTCTCTAGAATTTACTTATCATATAATCATAGTGGCGT TAACAATTATGGGCGTCATATTTTTAATCTCCGTTATAGTATfAGTnGTTCCTGTGA CAAAAATAATGACCAATATAAGTTCCATAAATTGCTACCG (SEQ ID N0:8)

[0968] In some embodiments, the recombinant oncolytic vaccinia virus of the present disclosure comprises one or more modifications that enhance virus spreading selected from an amino acid substitution at position M63 of the A33R gene, an amino acid substitution at positions M66 and KI 51 of the A34R gene, and an amino acid substitution at position(s) N241, E243, V247, G250 and/or A276 of the B5R gene.

[0069] In some preferred embodiments, the recombinant oncolytic vaccinia virus of the present disclosure comprises an M63R substitution in the A33R gene, M66 and KI 5 IE substitutions in the A34R gene, and N241T, E243V, V247S, G250R and A276F substitutions in the B5R gene.

[0070] Heterologous sequence (e.g., encoding one or more of CD80 (e.g., soluble CD80 such as CD80-Fc), IL -2 or a variant thereof, IL-12 (comprising both subunits or single chain such as IL-12p70 or IL-12p40), IL-21 and IL-7, as herein described) can be placed under the control of a vaccinia virus promoter and integrated into the genome of the vaccinia virus. Alternatively, expression of the heterologous sequence can be achieved by transfecting a shutle vector or plasmid such as those found in Table 1 of Current Techniques in Molecular Biology, (Ed. Ausubel, et al.) Unit 16.17.4 (1998) containing the vaccinia promoter-controlled sequence into a cell that has been infected with vaccinia virus and introducing the heterologous sequence by homologous recombination. Strong late vaccinia virus promoters may be utilized when high levels of expression are desired. In preferred embodiments, the heterologous sequence is under the control of a vaccinia virus promoter containing early and late promoter elements. Suitable early late promoters include, without limitation, a promoter of vaccinia virus gene coding for 7.5K polypeptide. Suitable late promoters include, without limitation, a promoter of vaccinia virus gene coding for 1 IK or 2SK polypeptide. In some embodiments, the heterologous sequence is inserted into a TK, M2L, and/or Bl 8R gene to inactivate the TK, M2L, and/or B18R sequence.

[0071] CD80

[0072] CD80 is a costimulatory molecule that binds CD28 on T cells in trans, leading to increased T cell activation as a classic “signal 2”, T cell proliferation, and cytokine production. The present inventors created a secreted version of the costimulatory molecule by removing the transmembrane domain, allowing the factor to have immunoenhancing effects beyond the tumor (i.e.. in the tumor draining lymph nodes). Furthermore, an immunoglobulin Fc fragment was fused onto the molecule to enhance CD80 pharmacokinetic properties and stability. Since vaccinia virus naturally expresses an inhibitor of CD80 encoded by the M2L gene (Kleinpeter et al., 2019), that non-essential gene was deleted from the viral backbone. The present inventors have demonstrated that expressing secreted, stabilized CD80 in the vicinity' of tumor lysis results in enhanced immune response to released tumor antigens, and that by deleting the viral M2L gene, the antitumor activity of CD80-FC expressed from oncolytic vaccinia virus is enhanced.

[0073] Thus, in some aspects, a replication-competent recombinant oncolytic vaccinia virus is provided comprising, in its genome, nucleotide sequence encoding a CD80 (B7-1) polypeptide. In preferred aspects, the nucleotide sequence encoding a CD80 polypeptide encodes a soluble human CD80 polypeptide, which may be fused to Fc IgGl. In some aspects, the encoded CD80 polypeptide has the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MGHTRROGTSPSKCPYLNFFOLLVLAGLSHFCSGVIKVTKEVKEVATLSCGHNVSVEEL AQTRIYWQKEKKMVLTMMSGaWTO

LKYEKDAFRREFn..AEViXSVKADFPdlPSlSDFEIFrSNIRRIICSTSGGFFEPHLSWLE^GEE LNAINTrVSQDPET^ELYAVSSKLDFNMCTHFISFMCLIKYGI-ILRVN0TFN:V/N''r'rKOEHFP DNGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEA^TTCW^rVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTVRVVSVLTVIuHQDWLNGKEYKCKV SNKAJ__JPAPIEKTISKAKGQPREPQVYTL.PPSRDELTKNQVSLTCLVKGFYPSI)1AVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO:9)

[0074] SEQ ID NO:9 is the amino acid sequence of soluble human CD80 fused to an immunoglobulin Fc fragment (hCD80-Fc). The underlined portion of SEQ ID NO:9 corresponds to soluble CD80 and the remaining amino acids correspond to the Fc fragment and the bolded portion of SEQ ID NO:9 corresponds to the Fc fragment (they are connected by a GG linker). In some embodiments, the encoded CD80 polypeptide has a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the underlined portion of SEQ ID NO:9.

[0075] The amino acid sequence of full length human CD80 is provided below:

MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEEL AQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVV LKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGE ELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHF PDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:10)

[0076] In some aspects, a replication-competent recombinant oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding a soluble human CD80 (e.g., fused to an immunoglobulin Fc fragment as in SEQ ID NO:9, said nucleotide sequence operably linked to an expression control sequence. In some preferred embodiments, the expression, control sequence comprises a pSEL promoter. In some aspects, the replication-competent recombinant oncolytic vaccinia virus lacks functional M2L and J2R genes and comprises a K151E substitution in the A34 gene. In related aspects, the nucleotide sequence encoding soluble human CD80 is inserted into the M2L or J2R gene.

[0077] IL-12

[0078] The IL- 12 gene encodes the IL- 12 protein. IL- 12 is a heterodimer of a 35~kD subunit (p35) and a 40-kD subunit (p40) linked through a disulfide linkage to make fully functional IL- 12p70. The IL, -12 gene encodes both the p35 and p40 subunits. The encoded IL-12 protein induces IFNg and TNFa production from T cells and significantly alters the TME by promoting Thl polarization, (Lasek et al., 2014, Vignaly & Kuchroo, 2012) cytotoxic T lymphocyte (CTL) activation, NK activation, and killing of tumor. Studies expressing IL- 12 from a vector or recombinant protein demonstrated a reduction in tumor-growth rates in preclinical and clinical trials (Berraondo et al, 2018, Colombo & Trinchieri, 2002, Noguchi et al., 1996). Nonetheless, the potential lethal liver toxicities associated with systemic administration and the inability to control systemic dissemination even with local administration have limited the application of those studies. Different strategies have been developed to attenuate 11,-12. toxicity like the removal of N-terminal signal peptide to prevent IL-12 secretion (Wang et al., 2017), anchoring IL-12 to the membrane of the tumor (Ge et al., 2022) or conditional expression using a synthetic small molecule ligand (Barret et al., 2000). However, the time and level of expression within an Oncolytic virus (OV) has not been evaluated. The present inventors have discovered that by modulating the time and level of expression of IL-12 using specific VV late promoters like Al 4 and Fl 7, the toxicity of IL- 12 can be dampened, while maintaining the therapeutic effect of IL- 12.

[0079] Thus, in other aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding an IL- 12 polypeptide. In some aspects, the encoded IL- 12 polypeptide has the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto: MCPQKLTISWFAIVLLVSPLMAMWELEKDVYWEVDWTPDAPGETVNLTCDTPEEDDI

TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK

NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV

TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKN

LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV

EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVI

PVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDI-IEDI’rRDQTSTLKTCLPLE

LHKNESCLATRETSSTFRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNH NHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRW TINRVMGYLSSA (SEQ ID NO:11)

[0080] SEQ ID NO:11 corresponds to the amino acid sequence of murine IL-12. In some preferred aspects, the encoded IL- 12 polypeptide has the following sequence of human IL- 12 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDIL KDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLS AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWS1THSYFSLTFCVQVQGKSKREKKDRVFTDKTS ATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPG MFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKN ESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQ

IFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKL.CILLHAFRIRAVTIDRVM SYLNAS (SEQ ID NO:12)

[0081] SEQ ID NO: 12 is the amino acid sequence of human IL-12. Mouse IL-12 of SEQ ID NO:11 acts on both mouse and human cells. Human IL-12 of SEQ ID NO:12 acts only on human cells. [0082] In other aspects, the nucleotide sequence encoding IL-12 encodes IL-12p70, a single chain fusion protein comprising the two subunits of IL- 12 (p40 and p35) optionally including linker amino acids between subunits. In other aspects, the nucleotide sequence encoding IL- 12 encodes IL-12p40, the p40 subunit of IL- 12.

]0083] In some aspects, a replication-competent recombinant oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human or mouse IL-12, said nucleotide sequence operably linked to an expression control sequence. In some preferred embodiments, the expression control sequence comprises an SEL, Fl 7 or Al 4 promoter. In some aspects, the replication-competent recombinant oncolytic vaccinia virus lacks a functional J2R gene and comprises a K151E substitution in the A34 gene. In related aspects, the nucleotide sequence encoding human or mouse IL-12 is inserted into the B15R-B17L intergenic region in a Western Reserve strain vaccinia virus or is inserted into the BRI 6 gene region in a Copenhagen strain vaccinia virus, in which case the remaining portion of the partially deleted BRI 6 gene may be deleted.

[0084] IL-21

[0085] IL -21 is a cytokine produced by follicular helper T (Tfh) cells, T helper 17 (Thl7), and natural killer T (NKT) cells. IL-21 signals via heterodimerization with the IL-21 receptor (IL-21R) (Ozaki et al., 2000, Parrish-Novak et al., 2000) and the common cytokine receptor g- chain. It has a role in T and NK/NKT cell expansion, activation and memory . It also blunts Treg expansion by suppressing Foxp3 expression and favors the enrichment of antigen- stimulated CD8+ T cells (Li Y, Yee C, 2008). Systemic deli very of IL-21 alone fails to reach the tumor microenvironment (TME) in sufficient concentrations to activate T cells due to its short half-life and peripheral consumption (Xue et a., 2021). Previous reports have proven that expressing IL- 21 from VV has an anti-tumor effect in preclinical models. The present inventors have found that arming oncolytic vaccinia virus with IL-21 in combination with additional immune cytokines may result in a strong response on Tfh that may promote tertiary' lymphoid structures (TLS) within the tumor microenvironment (TME). [0086] 'Thus, in other aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding an IL-21 polypeptide. In some aspects, the encoded IL -21 polypeptide has the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVK GHCEHAAFACFQKAKLKJ’SNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLS (SEQ ID NO: 13)

[0087] SEQ ID NO: 13 corresponds to the amino acid sequence of murine IL-21. In some preferred aspects, the encoded IL-21 polypeptide has the following sequence of human IL-21 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MRSSPGNMERLVICLMV1FLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEF LPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKI^KRKPPSTNAGRRQKHRL TCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 14)

[0088] In some aspects, a replication-competent recombinant oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human or mouse IL-21, said nucleotide sequence operably linked to an expression control sequence. In some preferred embodiments, the expression control sequence comprises an F 17 promoter. In some aspects, the replication- competent recombinant oncolytic vaccinia virus lacks functional J2R, M2L, B18R genes and comprises a KI 5 IE substitution in the A34 gene and optionally lacks functional A45R, Bl 3R, and A53R genes. In related aspects, the nucleotide sequence encoding human or mouse IL-21 is inserted into the B 18R gene.

[0089] IL-7

[0090] IL-7 promotes T-cell expansion, homeostasis/stemness, antigen recognition and reduces exhaustion. IL-7 is an essential cytokine for both survival and homeostatic proliferation of naive and memory T-cells (Surh and Sprent, 2008). [0091] Thus, in other aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding an IL-7 polypeptide. In some aspects, the encoded IL-7 polypeptide has the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MFHVSFRYIFGIPPLILVLLPVTSSECHIKDKEGKAYESVLMISIDELDKMTGTDSNCPNN EPNFFRKHVCDDTKEAAFLNRAARKLKQFLKMNISEEFNVHLLTVSQGTQTLVNCTSKE EKNVKEiQKKNDACFLKRl^LREIKTCWNKILKGSI (SEQ ID NO: 15)

[0092] SEQ ID NO: 15 corresponds to the amino acid sequence of murine IL-7. In some preferred aspects, the encoded IL-7 polypeptide has the following sequence of human IL-7 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLN NEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQ VKGRKPAALGEAQPTKSLEENK.SLKEQKKLNDLCFLKRLLQEIKTCWKILMGTKEH (SEQ ID NO: 16)

[0093] In some aspects, a replication-competent recombinant oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human or mouse IL-7, said nucleotide sequence operably linked to an expression control sequence. In some preferred embodiments, the expression control sequence comprises an SEL promoter. In some aspects, the replication- competent recombinant oncolytic vaccinia virus lacks functional J2R, M2L and B18R genes and comprises a KI 5 IE substitution in the A34 gene. In related aspects, the nucleotide sequence encoding human or mouse IL-7 is inserted into the B18R gene.

[0094] Codon-Optimized Nucleotide Sequences

[0095] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding soluble human CD80 fused to an immunoglobulin Fc fragment (hCD80-Fc), wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 17 or is identical to SEQ ID NO: 17: ATGGGCCACACAAGAAGGCAGGGCACAAGCCCTAGCAAGTGCCCCTACCTGAACTT CrTCCAGCTGCTGGTGCTGGCCGGCCTGAGCCACTITTGTlCTGGCGrGATCCACGTG ACCAAAGAAGTGAAAGAGGTCGCCACACTGAGCTGCGGCCACAATGTGTCCGTGGA AGAACTGGCCCAGACCAGAATCTACTGGCAGAAAGAAAAGAAAATGGTGCTGACCA TGATGAGCGGCGACATGAACATCTGGCCCGAGTACAAGAACCGGACCATCTTCGAC ATCACCAACAACCTGAGCATCGTGATCCTGGCTCTGAGGCCTTCTGACGAGGGCACC TATGAGTGCGTGGTGCTGAAGTACGAGAAGGACGCCTTCAAGCGGGAACACCTGGC CGAAGTGACACTGAGCGTGAAGGCCGACTTTCCCACACCTAGCATCAGCGACTTCG AGATCCCCACCAGCAACATCCGGCGGATCATCTGTTCTACCAGCGGCGGCTTTCCTG AGCCTCACCTGAGCTGGCTTGAGAACGGCGAGGAACTGAACGCCATCAACACCACC GTGTCTCAGGACCCCGAGACAGAGCTGTATGCCGTGTCCAGCAAGCTGGACTTCAA CATGACCACCAACCACAGCTTCATGTGCCTGATTAAGTACGGCCACCTGAGAGTGA ACCAGACCTTCAACTGGAATACCACCAAGCAAGAGCACTTCCCCGACAACGGCGGA CCCAAGAGCTGCGATAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTC GGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGC AGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGT GAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTA GAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCAC CAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCC TGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGG

TTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCT GCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCC AGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTC TTCCTGTACAGCAAACTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTT

CAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGA GCCTGTCTCCTGGCAAA (SEQ ID NO: 17)

^0096] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding full length human CD80, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 18 or is identical to SEQ ID NO:18: ATGGGCCACACAAGAAGGCAGGGCACAAGCCCTAGCAAGTGCCCCTACC1 GAAC IT

CTTCCAGCTGCTGGTGCTGGCCGGCCTGAGCCACTTTTGTTCTGGCGTGATCCACGTG

ACCAAAGAAGTGAAAGAGGTCGCCACACTGAGCTGCGGCCACAATGTGTCCGTGGA

AGAACTGGCCCAGACCAGAATCTACTGGCAGAAAGAAAAGAAAATGGTGCTGACCA

TGATGAGCGGCGACATGAACATCTGGCCCGAGTACAAGAACCGGACCATCTTCGAC

ATCACCAACAACCTGAGCATCGTGATCCTGGCTCTGAGGCCTTCTGACGAGGGCACC

TATGAGTGCGTGGTGCTGAAGTACGAGAAGGACGCCTTCAAGCGGGAACACCTGGC

CGAAGTGACACTGAGCGTGAAGGCCGACTTTCCCACACCTAGCATCAGCGACTTCG

AGATCCCCACCAGCAACATCCGGCGGATCATCTGTTCTACCAGCGGCGGCTTTCCTG

AGCCTCACCTGAGCTGGCTTGAGAACGGCGAGGAACTGAACGCCATCAACACCACC

GTGTCTCAGGACCCCGAGACAGAGCTGTATGCCGTGTCCAGCAAGC FGGACTTCAA

CATGACCACCAACCACAGCTTCATGTGCCTGATTAAGTACGGCCACCTGAGAGTGA

ACCAGACCTTCAACTGGAATACCACCAAGCAAGAGCACTTCCCCGACAACCTGCTTC

CTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGA

CCTACTGCTTCGCCCCTAGATGCAGAGAGCGGCGGAGAAACGAACGGCTGAGAAGA

GAA'FCTGFGCGGCCCGTT (SEQ ID NO: 18)

[0097] In some aspects, the oncolytic vaccinia vims comprises, in its genome, nucleotide sequence encoding murine IL- 12, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 19 or is identical to SEQ ID NO: 19

ATGTGCCCTCAGAAGCTGACCATCAGTTGGTTCGCCATCGTGCTGCTGGTGTCCCCA

CTGATGGCTATGTGGGAACTCGAGAAGGACGTGTACGTGGTGGAAGTGGACTGGAC

CCCTGATGCTCCTGGCGAGACAGTGAACCTGACCTGCGACACACCTGAAGAGGACG

ACATCACCTGGACCAGCGATCAGAGACACGGCGTGATCGGCTCTGGCAAGACCCTG

ACAATTACCGTGAAAGAGTTCCTGGACGCCGGCCAGTACACCTGTCACAAAGGCGG

AGAGACACTGAGCCACTCTCATCTGCTGCTGCACAAGAAAGAGAACGGCA TCTGGT

CCACCGAGATCCTGAAGAACTTCAAGAACAAGACCTTCCTGAAGTGCGAGGCCCCT

AACTACAGCGGCAGATTCACCTGTAGCTGGCTGGTGCAGAGAAACATGGACCTGAA

GTTCAACATOAAGTCCTCCAGCAGCAGCCCCGACAGCAGAGCTGTGACA I'GTGGCA TGGCTAGCCTGAGCGCCGAGAAAGTGACACTGGACCAGAGAGACTACGAGAAGTAC AGCGTGTCCTGCCAAGAGGACGTGACCTGTCCTACCGCCGAGGAAACACTGCCTAT CGAGCTGGCCCTGGAAGCCAGACAGCAGAACAAATACGAGAACTACTCTACCAGCT TCTTCATCCGGGACATCATCAAGCCCGATCCTCCAAAGAACCTGCAGATGAAGCCTC TGAAGAACAGCCAGGTCGAGGTGTCCTGGGAGTACCC TGACAGCTGGTCTACCCCT

CACAGCTACTTCAGCCTGAAATTCTTCGTGCGGATCCAGCGCAAGAAAGAAAAGAT GAAGGAAACCGAGGAAGGCTGCAACCAGAAAGGCGCTTTCCTGGTGGAAAAGACC AGCACCGAGGTGCAGTGCAAAGGCGGCAATGTCTGTGTGCAGGCCCAGGACCGGTA CTACAACAGCAGCTGTAGCAAGTGGGCCTGCGTGCCATGCAGAGTCAGATCTGGTG

GCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGATCTAGAGTGATTCCTGTG TCTGGCCCTGCCAGATGCCTGAGCCAGTCTAGAAACCTGCTGAAAACCACCGACGA CATGGTCAAGACCGCCAGAGAGAAGCTGAAGCACTACTCCTGCACAGCCGAGGACA TCGACCACGAGGATATCACCAGGGACCAGACAAGCACCCTGAAAACCTGCCTGCCT CTGGAACTGCATAAGAACGAGAGCTGGCTGGCCACCAGAGAAACCAGCTCTACCAC

AAGAGGCAGCTGTCTGCCTCCTCAGAAAACCAGCCTGATGATGACCCTGTGCCTGG GCAGCATCTACGAGGATCTGAAGATGTACCAGACCGAGTTCCAGGCCATCAACGCC GCTCTGCAGAACCACAACCACCAGCAGATCATCCTGGACAAGGGCATGCTGGTGGC TATCGACGAGCTGATGCAGAGCCTGAACCATAACGGCGAGACACTGCGGCAGAAGC

CTCCAGTTGGAGAGGCCGATCCTTACAGAGTGAAGATGAAGCTGTGCATCCTGCTGC ACG CCTTC AGC ACC AGAGTGGTC ACCzATC AAC AGAGTG ATGGGCT AC CTG AGO AGC GCC (SEQ ID NO: 19)

^0098] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human IL-12, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:20 or is identical to SEQ ID NO:20:

ATGTGTCACCAGCAGCTGGTCATCAGCTGGTTCAGCCTGGTGTTCCTGGCCTCTCCTC TGGTGGCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTGGAACTGGACTGGTAT CCCGATGCTCCTGGCGAGATGGTGGTGCTGACCTGCGATACCCCTGAAGAGGACGG CATCACCTGGACACTGGATCAGTCTAGCGAGGTGCTCGGCAGCGGCAAGACCCTGA CCATCCAAGTGAAAGAGTTTGGCGACGCCGGCCAGTACACCTGTCACAAAGGCGGA GAAGTGCTGAGCCACAGCCTGCTGCTGCTCCACAAGAAAGAGGATGGCATTTGGAG CACCGACATCCTGAAGGACCAGAAAGAGCCCAAGAACAAGACCTTCCTGAGATGCG AGGCCAAGAACTACAGCGGCCGGTTCACATGTTGGTGGCTGACCACCATCAGCACC GACCTGACCTTCAGCGTGAAGTCCAGCAGAGGCAGCAGTGATCCTCAGGGCGTTAC ATGTGGCGCCGCTACACTGTCTGCCGAAAGAGTGCGGGGCGACAACAAAGAATACG

AGTACAGCGTGGAATGCCAAGAGGACAGCGCCTGTCCAGCCGCCGAAGAGTCTCTG CCTATCGAAGTGATGGTGGACGCCGTGCACAAGCTGAAGTACGAGAACTACACCTC CAGCTTTTTCATCCGGGACATCATCAAGCCCGATCCTCCAAAGAACCTGCAGCTGAA GCCTCTGAAGAACAGCAGACAGGTGGAAGTGTCCTGGGAGTACCCCGACACCTGGT

CTACACCCCACAGCTACTTCAGCCTGACCTTTTGCGTGCAAGTGCAGGGCAAGTCCA AGCGCGAGAAAAAGGACCGGGTGTTCACCGACAAGACCAGCGCCACCGTGATCTGC AGAAAGAACGCCAGCATCAGCGTCAGAGCCCAGGACCGGTACTACAGCAGCTCTTG GAGCGAATGGGCCAGCGTGCCATGTTCTGGTGGCGGAGGATCTGGCGGAGGTGGAA GCGGCGGAGGCGGATCTAGAAATCTGCCTGTGGCCACTCCTGATCCTGGCATGTTCC CTTGTCTGCACCACAGCCAGAACCTGCTGAGAGCCGTGTCCAACATGCTGCAGAAG GCCAGACAGACCCTGGAATTCTACCCCTGCACCAGCGAGGAAATCGACCACGAGGA

CATCACCAAGGATAAGACCAGCACCGTGGAAGCCTGCCTGCCTCTGGAACTGACCA AGAACGAGAGCTGCCTGAACAGCCGGGAAACCAGCTTCATCACCAACGGCTCTTGC CTGGCCAGCAGAAAGACCTCCTTCATGATGGCCCTGTGCCTGAGCAGCATCTACGAG GACCTGAAGATG1 ACCAGGTGGAATTCAAGACCATGAACGCCAAGCTGCTGATGGA

CCCCAAGCGGCAGATCTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGAT GCAGGCCCTGAACTTCAACAGCGAGACAGTGCCCCAGAAGTCTAGCCTGGAAGAAC CCGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCA GAGCCGTGACCATCGACAGAGTGATGAGCTACCTGAACGCCTCCTAATGA (SEQ ID

NO:20)

[0099] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding murine IL-21 , wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:'21 or is identical to SEQ ID NO:21: ATGGAAAGAACCCTCGTGTGCCTGGTGGTCATCTTCCTGGGAACAGTGGCCCACAAG TCTAGCCCTCAGGGACCTGACAGACTGCTGATCAGACTGAGACACCTGATCGACATC GTGGAACAGCTGAAGATCTACGAGAACXJATCTGGACCCCGAGCTGCTGAGCGCTCC TCAGGATGTGAAGGGACACTGTGAACACGCCGCCTTCGCCTGTTTCCAGAAGGCCA AGCTGAAGCCTAGCAACCCCGGCAACAACAAGACCTTCATCATCGACCTGGTGGCC CAGCTGAGAAGAAGGCTGCCTGCTAGAAGAGGCGGCAAGAAGCAGAAACATATCG CCAAGTGTCCCAGCTGCGACAGCTACGAGAAGAGAACCCCTAAAGAGTTCCTGGAA AGGCTGAAGTGGCTGCTGCAGAAGATGATCCATCAGCACCTGAGC (SEQ ID N0:21)

[00100] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human IL-21, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:22 or is identical to SEQ ID NO:22:

ATGAGAAGCAGCCCCGGCAACATGGAACGGATCGTGATCTGCCTGATGGTCATCTTC CTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCG GATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGG TGCCTGAGTTCCTGCCTGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCT

TCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGG AT'CATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGG AAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGC CTCCAAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAG

CACCTGAGCAGCAGAACCCACGGCTCTGAGGATTCTTAATGA (SEQ ID NO:22)

[00101] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding murine IL-7, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:23 or is identical to SEQ ID NO:23:

ATGTTCCACGTGTCCTTCCGGTACATCTTCGGCATCCCTCCACTGATCCTGGTGCTGC

TGCCTGTGACAAGCAGCGAGTGCCACATCAAGGACAAAGAAGGCAAGGCCTACGA

GAGCGTGCTGATGATCTCCATCGACGAGCTGGACAAGATGACCGGCACCGACAGCA ACTGCCCCAACAACGAGCCCAACTTCTTCAGAAAGCACGTGTGCGACGATACCAAA

GAGGCCGCCTTCCTGAACAGAGCCGCCAGAAAGCTGAAGCAGTTCCTGAAGATGAA

CATCAGCGAAGAGTTCAACGTGCACCTCCTGACCGTGTCTCAGGGCACACAGACCCT

GGTCAACTGCACCAGCAAAGAGGAAAAGAACGTCAAAGAGCAGAAGAAGAACGAC

GCCTGCTTCCTGAAGCGGCTGCTGAGAGAGATCAAGACCTGCTGGAACAAGATCCT

GAAGGGCAGCATC (SEQ ID NO:23)

[00102] In some aspects, the oncolytic vaccinia virus comprises, in its genome, nucleotide sequence encoding human IL-7, wherein said nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:24 or is identical to SEQ ID NO:24:

ATGTTCCACGTGTCCTTCCGGTACATCTTCGGCCTGCCTCCACTGATCCTGGTGCTGC

TGCC1 G TGGCCAGCAGCGAC1 G TG ATATCGAGGGCAAAGACGGCAAGCAGTACGAG

AGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAAATCGGCAG

CAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAA

AGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGC TGCGGCAGTTCCTGAAGATGA

ACAGCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCTGAGGGCACCACCATCC

TGCTGAATTGCACCGGCCAAGTGAAGGGCAGAAAGCCTGCTGCTCTGGGAGAAGCC

CAGCC I ACCAAGAGCC1 GGAAGAGAACAAGT CCCTGAAAGAGCAGAAGAAGCTGA ACGACCTCTGCTTCCTGAAGCGGCTGCTGCAAGAGATCAAGACCTGCTGGAACAAG ATCCTGATGGGCACCAAAGAGCACTAATGA (SEQ ID NO:24)

[00103] In some embodiments, an isolated nucleic acid is provided comprising a nucleotide sequence selected from any one of SEQ ID NOs:17 to 24 or a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99% identical to any one of SEQ ID Nos: 17 to 24, optionally linked to a promoter. In related embodiments, a vector comprising such a nucleic acid is provided. In other related embodiments, a pharmaceutical composition comprising such a nucleic acid or vector is provided. [00105] In some aspects, the nucleic acid sequence encoding CD80, IL-12, IL-21 and/or IL -7 is operably linked to a natural, synthetic, or modified vaccinia virus early, intermediate, late (e.g. Pl 1, 1 IL) or early/late promoter (e.g. p7.5, F7L or H5R). In preferred embodiments, vaccinia virus promoter is a natural or modified vaccinia virus early/late promoter (e.g. P7.5 or mH5) or is a synthetic vaccinia virus early/late promoter (e.g. pSEL). In a preferred embodiment, the nucleic acid sequence encoding SI domain and/or M protein and/or N protein, and/or immunogenic fragment thereof and/or fusion protein thereof is inserted within the vaccinia virus thymidine kinase gene and is operably linked to a synthetic early/late promoter (pSEL) as described in Hammond et ai., J. Virol. Methods, 66(1):135-138 (1997).

[00106] In some aspects, the nucleic acid sequence encoding CD80, IL- 12, IL-21 and/or IL-7 is operably linked to a synthetic early-late promoter (pSEL) and comprises the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical thereto:

TATAAAAATTGAAATTTTATTTTTTTTTTITGGAATATAAATA (SEQ ID NO:25)

[00107] In some aspects, the nucleic acid sequence encoding CD80, IL- 12, IL-21 and/or IL-7 is operably linked to a (late) Fl 7 promoter (promoter from the Fl 7R gene) comprising the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical thereto:

ATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATA (SEQ ID NO:26)

[00108] In some aspects, the nucleic acid sequence encoding CD80, IL- 12, IL-21 and/or IL-7 is operably linked to a (late) Al 4 promoter comprising the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical thereto:

GTTTATATTCCACTTTGTTCATTCGGCGATTTAAAATTTTTATTAGTTAAATA (SEQ ID NO:27) [00109] In some aspects, the nucleic acid sequence encoding CD80, IL-12, IL-21 and/or IL-7 is operably linked to an A45 promoter comprising the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical thereto:

TTGCTGCCACTCATAATATCAGACTACTTATTCTATTTTACTAAATA (SEQ ID NO:28)

[00110] Other suitable promoters for driving expression of CD80, IL-12, IL -21 and/or IL-7 in oncolytic vaccinia virus is provided below

[00111] In some preferred embodiments, a replication-competent recombinant oncolytic vaccinia virus is provided having a genome corresponding to any one of OVV-014, OW-004, OVV-015, OVV-016, OVV-005, OVV-006, OVV-013, OVV-008, OVV-009, OVV-010, OVV- 011, OVV-007 and OVV-003 as illustrated at Figure I.

[ 00112] In one embodiment, a replication-competent recombinant oncolytic vaccinia virus is provided lacking functional M2L and J2R genes, comprising a K151E amino acid substitution in the A34 gene and comprising, in its genome, nucleotide sequence encoding soluble human CD80 operably linked to a promoter, preferably an SEL promoter, preferably wherein said nucleotide sequence is inserted into the M2L gene. [00113] In another embodiment, a replication-competent recombinant oncolytic vaccinia virus is provided lacking a functional J2R gene, comprising a KI 5 IE amino acid substitution in the A34 gene and comprising, in its genome, nucleotide sequence encoding soluble mouse or human IL- 12 operably linked to a promoter, preferably an SEL, Fl 7 or Al 4 promoter, preferably wherein said nucleotide sequence is inserted into the B15R-B17L intergenic region in the case of a Western Reserve strain and is inserted into the B16 gene in the case of a Copenhagen strain, in which case the remaining portion of the partially deleted B16 gene may be deleted.

[00114] In another embodiment, a replication-competent recombinant oncolytic vaccinia virus is provided lacking a functional J2R and B18R genes, comprising a KI 5 IE amino acid substitution in the A34 gene and comprising, in its genome, nucleotide sequence encoding soluble mouse or human IL-7 operably linked to a promoter, preferably an SEL promoter, preferably wherein said nucleotide sequence is inserted into the B18R gene.

[00115] In another embodiment, a replication-competent recombinant oncolytic vaccinia virus is provided lacking a functional J2R gene, comprising a K151E amino acid substitution in the A34 gene and comprising, in its genome, nucleotide sequence encoding soluble mouse or human IL-21 operably linked to a promoter, preferably an Fl 7 promoter, preferably wherein said nucleotide sequence is inserted into the J2R gene.

[00116] In another embodiment, a. replication-competent recombinant oncolytic vaccinia vims is provided lacking functional M2L, J2R and B18R genes, comprising a KI 51 E amino acid substitution in the A34 gene and comprising, in its genome, (i) nucleotide sequence encoding soluble human CD80 operably linked to promoter, preferably an SEL promoter (ii) nucleotide sequence encoding mouse or human IL-21 operably linked to a promoter, preferably an Fl 7 promoter and (iii) mouse or human IL -7 operably linked to a promoter, preferably an SEL promoter, preferably wherein the nucleotide sequence of (i) is inserted into the M2L gene, the nucleotide sequence of (ii) is inserted into the J2R gene and the nucleotide sequence of (iii) is inserted into the B18R gene.

[00117] In yet another embodiment, a replication-competent recombinant oncolytic vaccinia virus is provided lacking functional M2L, J2R and B18R genes, comprising a K1.51E amino acid substitution in the A34 gene and comprising, in its genome, (i) nucleotide sequence encoding soluble human CD80 operably linked to promoter, preferably an SEL promoter (ii) nucleotide sequence encoding mouse or human IL-21 operably linked to a promoter, preferably an F 17 promoter and (iii) mouse or human IL-7 operably linked to a promoter, preferably an SEL promoter and (iv) mouse or human IL-12 operably linked to a promoter, preferably an Al 4 or F17 promoter, preferably wherein the nucleotide sequence of (i) is inserted into the M2L gene, the nucleotide sequence of (ii) is inserted into the J2R gene, the nucleotide sequence of (iii) is inserted into the B18R gene and the nucleotide sequence of (iv) is inserted into the intergenic B15R-B17L region of Western Reserve strain or is inserted into the Bl 6 gene of Copenhagen strain.

[00118] Additional Therapeutic Genes

[00119] The oncolytic virus, in addition to expressing one or more of, and preferably all of, CD80, IL- 12, IL-21 and IL-7, may further express one or more additional genes.

[00120] In some aspects, the oncolytic virus further expresses a cytokine. Representative examples of cytokines that may be co-expressed with CD80, IL- 12, IL-21 and IL-7, include GM- CSF, IL-2, IL-4, IL-5, IL-15, type I IFN (a and p), type II IFN (IFN-y), and TNF-a.

[00121] In some preferred embodiments, the vaccinia virus comprises, in its genome, (i) a nucleotide sequence encoding a soluble human CD80 polypeptide (ii) a nucleotide sequence encoding IL-2, or a variant thereof, (iii) non- functional M2L and J2R genes and (iv) a K I 51 E substitution in the A34R gene. In some aspects, the IL-2 variant is IL-2v as described in U.S. Patent Application Publication No. 2020/0222520, the entire contents of which are incorporated herein by reference. In one embodiment, the IL-2 variant is a murine IL-2 variant and comprises the amino acid sequence of SEQ ID NO:36 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:36:

MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQEL LSRMENYRNLKLPRMLTAKFALPKQATELKDLQCLEDELGPLRHVLDGTQSKSFQLED AENFISNIRVTWKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ (SEQ ID NO:36)

[00122] In a related aspect, the murine IL -2 variant is encoded by the following nucleotide sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

ATGTACAGCATGCAGCTGGCCTCTTGCGTGACCCTGACACTGGTGCTGCTGGTCAAC AGCGCCCCTACCAGCAGCTCTACAAGCAGCAGCACAGCCGAGGCTCAGCAGCAACA ACAACAGCAACAGCAACAACAGCAGCATCTGGAACAGCTGCTGAIGGACCTGCAAG AACTGCTGAGCAGGATGGAAAACTACCGGAACCTGAAGCTGCCCAGAATGCTGACC

GCCAAGTTCGCCCTGCCTAAGCAGGCTACAGAGCTGAAGGATCTGCAGTGCCTGGA AGATGAGCTGGGCCCTCTGAGACACGTGCTGGATGGCACACAGAGCAAGAGCTTCC AGCTCGAGGACGCCGAGAACTTCATCAGCAACATCAGAGTGACCGTGGTCAAGCTG AAGGGCAGCGACAACACCTTCGAGTGCCAGTTCGACGACGAGAGCGCTACCGTGGT GGACTTCCTGAGAAGATGGATCGCCTrCTGCCAGAGCATCATCAGCACAAGCCCTCA G (SEQ ID NO:37)

[00123] In a preferred embodiment, the IL-2 variant is a human IL-2 variant (hIL-2v) and comprises SEQ ID NO:38 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:38 (signal sequence is underlined; differences from wild type human IL-2 are in bold - F42A, Y45A, L72G):

MYRMOt^GlAESM^VWACTSSSTKKTOLOLEHLLLDLQMILNGINNYKNPKLTRM

LTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISN1NVIVLELKGS

ETTFMCEYADETATIVEFLNRWITFCQSnSTLT (SEQ ID NO:38)

[00124] In a related aspect, the human IL -2 variant is encoded by one of the following nucleotide sequences or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto: ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGTCACAAAT AGCGCCCCTACCAGCAGCAGCACCAAGAAAACACAGCTGCAACTGGAACACCTCCT GCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGA CCCGGATGCTGACCGCCAAGTTTGCCATGCCTAAGAAGGCCACCGAGCTGAAGCAC

CTCCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACGGCGCCCA

GAGCAAGAACTTCCATCTGCGGCCCAGAGATCTGATCAGCAACATCAACGTGATCG TGCTGGAACTGAAAGGCAGCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACA GCTACCATCGTGGAATTTCTGAACCGGTGGATCACCTrCTGCCAGAGCATCATCAGC ACCCTGACC (SEQ ID NO:39)

[00125] ATGTACAGAATGCAGCTACTATCCTGTATCGCGCTATCCTTGGCGCTAGTA ACAAATTCTGCGCCGACATCCTCGTCCACAAAGAAAACACAACTACAGCTAGAGCA CCTACTACTAGACCTACAGATGATCCTAAACGGAATCAACAACTACAAGAACCCGA AGCTAACCAGAATGCTAACCGCGAAATTTGCCATGCCGAAAAAGGCGACAGAGCTA

AAGCACTTGCAGTGCCTAGAAGAAGAGTTGAAGCCGCTAGAAGAGGTCTTGAACGG TGCCCAATCTAAGAACTTCCACCTAAGACCGAGAGACTTGATCTCCAACATCAACGT CATCGTCTTGGAGCTAAAGGGATCCGAAACCACCTTCATGTGTGAATACGCTGATGA GACAGCGACCATCGTCGAGTTTCTAAACAGATGGATCACCTTCTGCCAGTCCATCAT

CTCTACACTAACA (SEQ ID NO:40)

[00126] SEQ ID NOs:39 and 40 are codon optimized nucleotide sequences encoding an IL~2 variant of SEQ ID NO:38. SEQ ID NO:39 is codon optimized for mammalian expression. SEQ ID NO:40 is codon optimized for vaccinia virus expression.

[00127] In another preferred embodiment, the IL~2 variant is a human IL-2 variant (hIL-2gv) and comprises SEQ ID NO:41 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:41 (signal sequence is underlined; differences from wild type human IL-2 are indicated in bold - R38N, L40T, K43N and Y45T):

MYRMOLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLOMILNG1NNYKNPKLTNM

TTFNFTMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE

TTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:41) [00128] In a related aspect, the human IL-2 variant is encoded by the following nucleotide sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto:

ATGTATCGTATGCAGCTGCTGAGCTGCATCGC3TTATCTTTAGCTTTAGTGACCAACA GCGCCCCTACCAGCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGC TGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTA ATATGACCACCn CAACT1 CACTAI GCCCAAGAAGGCCACCGAGCTGAAGCACCTCC AGTGTTTAGAGGAGGAGCTGAAGCCTTTAGAGGAGGTGCTGAATTTAGCCCAGAGC AAGAATTTCCAT1TAAGGCCTCGTGATTTAATCAGCAACATCAACGTGATCGTGCTG GAGCTGAAAGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCAC CATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGAGCATCATCAGCACTTTA ACC (SEQ ID NO:42)

[00129] In some embodiments, the oncolytic virus also expresses a tumor antigen selected from BAGE, GAGE-1, GAGE-2, CEA, AIM2, CDK4, BMI1, COX-2, MUM-1, MUC-1, TRP-1 TRP-2, GP100, EGFRvIIL EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2 , SOX2, human papillomavijrus-E6, human papillomavirus-E7, ART1, ART4, SART1, SART2, SART3, B-cyclin, p-catenin, Glil. Cav-i, cathepsin B, CD74, E-cadherin, EphA2/Eck, Fra-1/Fosl 1, Ganglioside/GD2, GnT-V, pi ,6-N, Her2/neu, Ki67, Ku70/80, IL-13Ra2, MAGE-1, MAGE-3, NY-ESO-1, MART-1, PROXI, PSCA, SOXIO, SOX11, Survivin, caspase-8, UP AR, CA-125, PSA, pl85HER2, CDS, IL-2R, Fap-a, tenascin, melanoma-associated antigen p97, and WT-1.

[00130] In some preferred embodiments, the oncolytic virus (e.g,, vaccinia virus) comprises a nucleotide sequence encoding an immune checkpoint, inhibitor and/or is co-administered to a subject with cancer with an immune check point inhibitor.

[00131] Immune checkpoint proteins interact with specific ligands which send a signal into T cells that inhibits T cell function. Cancer cells exploit this by driving high level expression of checkpoint proteins on their surface, thereby suppressing the anti-cancer immune response. [00132] Immune checkpoint inhibitors expressed by the oncolytic virus and pharmaceutical combinations including the oncolytic vims, herein described can include any compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function as well as full blockade. In preferred embodiments, the immune checkpoint protein is a human checkpoint protein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of a human immune checkpoint . In some embodiments, the immune checkpoint inhibitor is a PD-1 antagoni st and/or an antagonist of its Ligand PD-L1 (programmed cell death receptor- 1 ligand)

[00133] In some embodiments, a PD-1 antagonist or PD-L1 antagonist expressed by the replicative oncolytic virus, or for use in combination with the oncolytic virus, is an antibody. The term "antibody" as used herein encompasses naturally occurring and engineered antibodies as well as full length antibodies or functional fragments or analogs thereof that are capable of binding e.g., the target immune checkpoint or epitope ( e.g., retaining the antigen-binding portion), including single chain Fv (scFv). The antibody for use according to the methods described herein may be from any origin including, without limitation, human, humanized, animal or chimeric and may be of any isotype. In some embodiments, the isotype is and IgGl, IgG2, IgG3, and IgG4. In some embodiments, the isotype is IgGl or IgG4. In some embodiments, the antibody may be glycosylated or non-glycosylated. The term antibody also includes bispecific or multispecific antibodies so long as they exhibit the binding specificity herein described. Humanized antibodies refer to non-human ( e.g., murine, rat, etc.) antibody whose protein sequence has been modified to increase similarity'- to a human antibody. Chimeric antibodies refer to antibodies comprising one or more element(s) of one species and one or more element(s) of another specifies, for example a non-human antibody comprising at least a portion of a constant region (Fc) of a human immunoglobulin. Another type of Ig domain of the heavy chain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CHI domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for IgG the antibody hinge is herein defined to include positions 221 (D221 in IgGl) to 236 (G236 in IgGl), wherein the numbering is according to the EU index as in Kabat. In some embodiments, for example in the context of an Fc region, the lower hinge is included, with the "lower hinge" generally referring to positions 226 or 230. In some embodiments, optional substitutions in the hinge region can be employed.

[00134] Many forms of antibody can be engineered for use in the combination of the invention, representative examples of which include an Fab fragment (monovalent fragment consisting of the VL, VH, CL and CHI domains), an F(ab')2 fragment (bivalent fragment comprising two Fab fragments linked by at least one disulfide bridge at the hinge region), a Fd fragment (consisting of the VH and CHI domains), a Fv fragment (consisting of the VL and VH domains of a single arm of an antibody), a dAb fragment (consisting of a single variable domain fragment (VH or VL domain), a single chain Fv (scFv) comprising the two domains of a Fv fragment, VL and VH, that are fused together, and in some embodiments, with a linker to make a single protein chain. In some preferred embodiments, the immune checkpoint inhibitor is an scFv against PD- 1, PD-L1, CTLA-4 or LAG3.

[00135] In some embodiments, the PD-1 antagonist or a PD-L.1 antagonist expressed by the replicative oncolytic virus, or for use in combination with the replicative oncolytic virus, is an antibody or fragment thereof. In some embodiments, the PD-1 antagonist or PD-L1 antagonist comprises a polypeptide comprising CDR1, CDR2, and CDR3 of the VL which is attached to a polypeptide comprising GDI, CDR2 and CDR3 of the VH. In some embodiments, the PD-1 antagonist or PD-L1 antagonist comprises a polypeptide comprising CDR.1 , CDR2, and CDR3 of the VL which is attached to a polypeptide comprising CD1, CDR2 and CDR3 of the VH via a linker. In some embodiments, the PD-1 antagonist or PD-L1 antagonist comprises a polypeptide comprising CDR1, CDR2, and CDR3 of the VL which is atached to a polypeptide comprising CD1, CDR2 and CDR3 of the VH without a linker. In some embodiments, the VL and VH are atached via a linker. In some embodiments, the PD-1 antagonist or PD-L.1 antagonist comprises the VL and VH attached together via a linker to form a scFv. In some embodiments, the VL and VH are not attached via a linker. In some embodiments, the VL and VH are noncovalently attached. In some embodiments, the VL and VH are attached via a linker.

[00136] In some embodiments, the oncolytic virus expresses and/or is co-administered with a PD-1 inhibitor (e.g., monoclonal antibody against PD-1). The complete nucleotide and amino acid sequences of human PD-1 can be found under GenBank Accession No. U64863 and NP__005009.2. Monoclonal antibodies against PD-1 include, without limitation, lambrolizumab (e.g., disclosed as KPD109A and its humanized derivatives I1409A11, h409A16 and h409A17 in U.S. Patent No. 8,354,509, incorporated herein by reference), Nivolumab (Opdivo®; Bristol- Myers Squibb; code name BMS-936558) disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference, Pembrolizumab (Keytruda®) and Pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)), Cemiplimab, or an antibody comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described in e.g., W02004/004771, W02004/056875, W02006/121168, WO2008/156712, W02009/014708, W02009/114335, WO2013/043569 and WO2014/047350. In a related embodiment, the checkpoint inhibitor is an anti-PD-1 fusion protein such as AMP -224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgGl).

{00137] In some embodiments, the oncolytic virus expresses and/or is co-administered with an inhibitor of CTLA-4 (cytotoxic T lymphocyte-associated molecule-4). In some aspects, the CTLA-4 inhibitor is an antibody (e.g., monoclonal antibody) against CTLA-4. Antibodies against CTLA-4 include ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca/Medlmmune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005/0201994, 2002/0039581, and 2002/086014, the contents of each of which are incorporated herein by reference, and antibodies disclosed in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281, and 8,491,895 the contents of each of which are incorporated herein by reference, or an antibody comprising the heavy and light chain variable regions of any of these antibodies or the CDR regions of any of these antibodies.

[00138] In some embodiments, the oncolytic virus expresses and/or is co-administered with a PD-L1 inhibitor (e.g., monoclonal antibody against PD-L1). Antibodies against PD-L1 include, without limitation, pembrolizumab (MK-3475, disclosed in W02009/114335)), BMS-936559 (MDX-1105), Atezolizumab (Genentech/Roche; MPDL33280A) disclosed in U.S. Patent No. 8,217,149, the contents of which are incorporated herein by reference, Durvalumab (AstraZeneca/Medlmmune; MEDI4736) disclosed in U.S. Patent No. 8,779,108, incorporated herein by reference, MIH1 (Asymetrix obtainable via eBioscience (16.5983.82)) and Avelumab (MSB0010718C; Merck KGaA) or an antibody comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-Ll fusion protein such as the PD-L2-Fc fusion protein known as AMP-224 (disclosed in Mkritchyan M., et al., J. Immunol., 189:2338-2347 (2010).

[00139] In other embodiments, the oncolytic virus expresses and/or is co-administered with an inhibitor of LAG3 such as soluble LAG.3 (IMP321, or LAG3-Ig disclosed in U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and in Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009)), IMP701 or other humanized antibodies blocking human LAG3 described in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Patent No. 5,773,578, incorporated herein by reference, or BMS- 986016 or other fully human antibodies blocking LAG3 described in U.S. Patent Application Publication No. 2011-0150892, incorporated herein by reference. Other LAG-3 inhibitors include LAG525 (IMP701), REGN3767 (R3767), BI 754,091, tebotelimab (MGD013), eftilagimod alpha (IMP321) and FS118.

[00140] In other embodiments, the oncolytic virus expresses and/or is co-administered with a BLTA inhibitor such as the antibody 4C7 disclosed in U.S. Patent No. 8,563,694, incorporated herein by reference.

[00141] In other embodiments, the oncolytic virus expresses and/or is co-administered with an inhibitor of B7H4 such as an antibody as disclosed in U.S. Patent Application Publication No. 2014/0294861, incorporated herein by reference or a soluble recombinant form of B7H4 e.g., as disclosed in U.S. Patent Application Publication No. 20120177645, incorporated herein by reference, or FPA150.

[00142] In other embodiments, the oncolytic virus expresses and/or is co-administered with an inhibitor of B7-H3 such as the antibody MGA271 disclosed as BRCA84D or a derivative as disclosed in U.S. Patent Application Publication No. 20120294796, incorporated herein by reference. [00143] In yet other embodiments, the oncolytic virus expresses and/or is co-administered with a TIM3 checkpoint inhibitor such as an antibody as disclosed in U.S. Patent No. 8,841,418, incorporated herein by reference or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-2779 (2008). Other TIM-3 inhibitors include MBG453, Sym023 and TSR-022.

[00144] In yet other embodiments, the oncolytic virus expresses and/or is co-administered with a KIR checkpoint inhibitor such as the antibody lirilumab (described in Romagne et al. , Blood, 114(13):2667-2677 (2009)).

[00145] In yet other embodiments, the oncolytic virus expresses and/or is co-administered with a TIGIT inhibitor. TIGIT checkpoint inhibitors preferably inhibit interaction of TIGIT with a poliovirus receptor (CD 155) and include, without limitation, antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication Nos. 20160355589 and 20160176963 (incorporated herein by reference) and poliovirus receptor variants such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

[00146] In yet other embodiments, In other embodiments, the oncolytic virus expresses and/or is co-administered with an IDO inhibitor. IDO is recognized as an immune checkpoint protein and its expression in tumor cells contributes to immune tolerance by shutting down effector T cells. IDO is thought, to contribute to resistance of anti-CLTA-4 therapies. Inhibitors of IDO for use according to the methods described herein include, without limitation, tryptophan mimetics such as D-1MT (D isoform of 1-methyl-DL-tryptophan (MT)), L-1MT (L isoform of MT), MTH-Trp (methylthiohydantoin-dl-tiyptophan; transcriptional suppressor of IDO), and [3- carbolines, indole mimetics such as napthoquinone-based agents, S-allyl-brassinin, S-benzyl- brassinin, 5-Bromo-brassinin, as well as phenylimidazole-based agents, 4-phenylimidazole, exiguamine A, epacadostat, rosmarinic acid, norharmane and NSC401366. Preferred IDO inhibitors include INCB 024360 (epacadostat; l,2,5-Oxadiazole-3-carboximidamide, 4-((2- ((AminosulfonyI)arnino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy~, (C(Z))-;

Incyte), indoximod (NLG2101; D-1MT; NewLink Genetics), IDO peptide vaccine (Copenhagen University) and NLG919 (NewLink Genetics). [00147] Treatments

[00148] In some aspects, a replication-competent recombinant oncolytic vaccinia virus as described herein is administered to a mammal as a monotherapy or as part of combined treatment regimen for the treatment and/or prevention of cancer. In some embodiments, the cancer includes but is not limited to a brain cancer, head & neck cancer, esophageal cancer, skin cancer, lung cancer, thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, renal cancer, testicular- cancer, rectal cancer, breast cancer, and pancreatic cancer. In some embodiments, the cancer selected from the group consisting of brain cancer, head & neck cancer, esophageal cancer, skin cancer, lung cancer, thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, renal cancer, testicular cancer, rectal cancer, breast cancer, and pancreatic cancer. In a preferred embodiment, the combination is used to treat and/or prevent a metastasis. In other preferred embodiments, the combination is used to treat a cancer including but not limited to hepatocellular- carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), stomach cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, cervical and liver cancer. In some embodiments, the combination is used to treat a cancer selected from the group consisting of hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non- small cell lung cancer), stomach cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, cervical and liver cancer. In some embodiments, the combination is used to treat colorectal cancer, particularly metastatic colorectal cancer. In some embodiments, the cancer is a solid cancer or solid tumor. In some embodiments, the tumor to be treated is a tumor with a high tumor mutational burden. In some embodiments, the mammal to be treated is a human.

[00149] In some preferred embodiments, the cancer is selected from breast cancer, colorectal cancer (e.g., colorectal carcinoma), bladder cancer, lung cancer (e.g., non-small cell lung cancer), hepatocellular carcinoma, renal cell carcinoma, and osteosarcoma. In particularly preferred embodiments, the cancer is microsatellite stable (MSS) colorectal cancer. [00150] In another preferred aspect, an oncolytic virus as herein described is used to treat a cancer that is resistant to one or more immune checkpoint inhibitors (e.g., the cancer is resistant to immunotherapy with PD-1, PD-L1, CTLA-4, LAG3, and/or TIGIT inhibitors). In such case, the oncolytic virus may encode one or more immune checkpoint inhibitors and/or may be coadministered with one or more immune checkpoint inhibitors to a human with a cancer that is refractory to the one or more immune checkpoint inhibitors.

[00151] In some embodiments, the oncolytic virus as described herein is sequentially or simultaneously co-administered with one or more immune checkpoint inhibitors to a mammal with cancer.

[00152] In some embodiments, the oncolytic virus is administered by intratumoral and/or intravascular (e.g., intravenous) administration. In other embodiments, the oncolytic virus is administered by intraperitoneal administration. In some embodiments, a replicative oncolytic vaccinia virus is administered by intratumoral and/or intravascular administration. In some embodiments, the replicative oncolytic vaccinia virus is administered by intravenous administration. In some embodiments, the replicative oncolytic vaccinia virus is administered by intra-arterial administration. In some embodiments, the replicative oncolytic vaccinia virus is administered only by intratumoral administration. In some embodiments, the replicative oncolytic vaccinia virus is administered only by intravenous administration.

[00153] The checkpoint inhibitor as disclosed herein can be administered by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbital ly, intracapsularly, intraperitoneally, intrarectally, intracistemally, intratumorally, intravasally, intradermally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively, In some embodiments, the checkpoint inhibitor is administered systemically. The checkpoint inhibitor also can be administered to the site of a pathologic condition, for example, intravenously or intra-arterially into a blood vessel supplying a tumor. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and/or TIM3. [00154] In some embodiments, the replicative oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicative oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicative oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicative oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically.

[00155] The total amount of an agent to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. One skilled in the art would know that the amount of the composition to treat a pathologic condition in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary. In general, the formulation of the composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.

[00156] Oncolytic vaccinia viruses as described herein may be administered in a single administration or multiple administrations ( e.g., 2, 3, 4, 5, 6, 7, 8 or more times). The virus may be administered at dosage of 1 x IO⁵ plaque forming units (PFU), 5 x 10⁵ PFU, 1 x 10⁶ PFU, at least 1 x 10⁶ PFU, 5 x 10⁶ or about 5 x 10⁶ PFU, 1 x 10', at least 1 x 10⁷ PFU, 1 x 10⁸ or about 1 x 10⁸ PFU, at least 1 x 10⁸ PFU, about or at least 5 x 10^s PFU, 1 x 10⁵ or at least 1 x 10⁹ PFU, 5 x 10⁹ or at least 5 x 10⁹ PFU, 1 x 10¹⁰ PFU or at least 1 x 10¹⁰ PFU, 5 x 10¹⁰ or at least 5 x 10¹⁰ PFU, 1 x 10ⁿ or at least 1 xlO¹¹, 1 x 10ⁱ² or at least 1 x 10¹², 1 x 10¹³ or at least 1 x 10ⁱ³. For example, the virus may be administered at a dosage of between about 10⁶-l 0¹³ pfu, between about 1O'-1O¹³ pfu, between about 10⁸-1O³³ pfu, between about 10⁹~l 0¹² pfu, between about 10⁸- 10¹² pfu, between about 10⁷-l()¹² pfu, between about 10⁶-10³²pfu, between about 10°-l 0⁹ pfu, between about 10⁶- 10^s pfu, between about 10⁷-10^!°pfu, between about 10⁷- 10⁹ pfu, between about 1 O⁸-1O¹⁰ pfu, or between about 10⁸-10⁹ pfu. Preferably, the virus is administered at a dosage of at least 10⁷ pfu, between 10⁷ and 10ⁱ⁰ pfu, between 10⁷-l 0⁹ pfu, between 10⁷~l 0⁸ pfo, between 10⁸-10³°pfu_; between 10⁸-10⁹ pfu or between 10⁹-10^{) 0}pfu.

[00157] It is con templated that a single dose of oncolytic virus refers to the amount administered to a subject or a tumor over a 0.1, 0.5, 1, 2, 5, 10, 15, 20, or 24-hour period, including all values there between. The dose may be spread over time or by separate injection. Typically, multiple doses are administered to the same general target region, such as in the proximity of a tumor. In certain aspects, the viral dose is delivered by injection apparatus comprising a syringe or single port needle or multiple ports in a single needle or multiple prongs coupled to a syringe, or a combination thereof. A single dose of the vaccinia virus may be administered or the multiple doses may be administered over a treatment period which may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. For example, the vaccinia virus may be administered every other day, weekly, every other w^reek, every third week for a period of 1 , 2, 3, 4, 5, 6 or more months.

[00158] In certain embodiments, a checkpoint inhibitor is sequentially or simultaneously coadministered with an oncolytic virus as herein described to a human with cancer. In some aspects, a checkpoint inhibitor is administered in 0.01-0.05 mg/kg, 0.05-0.1 mg/kg, 0.1-0.2 mg/kg, 0.2-0.3 mg/kg, 0.3-0.5 mg/kg, 0.5-0.7 mg/kg, 0.7-1 mg/kg, 1-2 mg/kg, 2-3 mg/kg, 3-4 mg/kg, 4-5 mg/kg, 5-6 mg/kg, 6-7 mg/kg, 7-8 mg/kg, 8-9 mg/kg, 9-10 mg/kg, at least 10 mg/kg, or any combination thereof doses. Suitable dosages of the checkpoint inhibitor range from about 0.5 mg/kg to 25 mg/kg, preferably from about 1 mg/kg to about 20 mg/kg, more preferably from about 2 mg/kg to about 15 mg/kg. In certain embodiments the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or multiple months. In certain embodiments, the checkpoint inhibitor is administered as a single dose, in two doses, in three doses, in four doses, in five doses, or in 6 or more doses. Preferably, the checkpoint inhibitor is administered intravenously (e.g. by intravenous infusion or injection) or intratumorally. By way of nonlimiting example, ipilimumab is preferably administered by intravenous infusion at a dose of 3mg/kg every' three weeks for a total of four doses. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and/or TIMS. [001S9] In some embodiments, an oncolytic virus as herein described is administered to a mammal with cancer in combination with one or more chemotherapeutic agents. In other embodiments, an oncolytic virus as herein described is administered to a mammal with cancer in combination with one or more radiation treatments. In other embodiments, an oncolytic virus as herein described is administered to a mammal with cancer in combination with a surgical procedure (e.g. tumor resection).

1001601 Pharmaceutical Compositions

[00161] In some aspects, a pharmaceutical composition is provided comprising a replication- competent recombinant oncolytic virus as herein described and a pharmaceutically acceptable carrier.

[00162] In some aspects, a replication-competent recombinant oncolytic virus as herein described is administered to treat cancer and/or directly to tumor cells and accordingly, the pharmaceutical compositions disclosed herein are formulated for the desired administration route (e.g. by intratumoral injection, intravenously, intra-arterially, and/or intraperitoneal administration). In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for administration by intratumoral, intravenously, intra-arterially, and/or intraperitoneal administration routes. In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for administration by intratumoral administration. In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for administration by intravenous administration. In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for intra-arterial administration. In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for administration by intraperitoneal administration. In some embodiments, the replicative oncolytic virus of the pharmaceutical combination is formulated for administration only by intratumoral administration or is formulated for administration only by intravenous administration.

[00163] Intratumoral injection of the oncolytic vims may be by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection. A novel needieless injection system has recently been described (U.S. Patent 5,846,233, incorporated herein by reference) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery. A syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Patent 5,846,225, incorporated herein by reference).

[00164] For intratumoral injection in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" .15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[00165] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar- as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[00166] The phrase "pharmaceutically-acceptable" or "pharmacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

[00167] In some embodiments, an oncolytic vaccinia virus as described herein may be administered in a single administration or multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8 or more times). In a preferred embodiment, the oncolytic vaccinia virus is administered as a single intradermal administration or two intradermal administrations. The virus may be administered at dosage of 1 x IO⁴ plaque forming units (PFU), 2.5 x IO⁴ PFU, 1 x 10' PFU, at least 1 x 10⁵ PFU, 2.5 x 10⁵ PFU or about 5 x 10³ PFU, 1 x 10⁶, at least 1 x 10⁶ PFU, 2.5 x 10⁶ PFU, 1 x 10⁷ or about 1 x 10⁷ PFU, at least 1 x 10⁷ PFU, about or at least 5 x 10⁷ PFU, 1 x 10⁸ or at least 1 x 10⁸ PFU, 5 x 10⁸ or at least 5 x 10⁸ PFU, 1 x 10⁹ PFU or at least 1 x 10⁹ PFU, 5 x IO⁹ or at least 5 x 10⁹ PFU, 1 x IO¹⁰ or at least 1 xlO^iO. 1 x 10^u or at least 1 x IO¹¹, 1 x 10ⁱ² or at least 1 x 10ⁱ². For example, the virus may be administered at a dosage of between about 10⁴-10¹² pfu, between about 10⁴-10^H pfu, between about 10⁴-10^l0pfu, between about 10⁴-10⁹pfu, between about 10⁴- 10⁸ pfu, between about 1 ()⁴-10⁷ pfu, between about 10⁵- 10¹⁰ pfu, between about ml 0⁵- 10⁹ pfu, between about 10⁶-l 0¹² pfu, between about 10^?-l O¹⁰ pfu, between about 10'-10⁹ pfu, or between about l()⁸-10⁵ pfu. Preferably, the virus is administered at a dosage of at least 1 x 10⁵.

[00168] The vaccinia virus vaccine of the pharmaceutical composition is administered intradermally or subcutaneously or by any route herein described and accordingly, the pharmaceutical compositions disclosed herein are formulated for intradermal administration, subcutaneous administration or the like.

[00169] Injection of the vaccinia virus vaccine (e.g. intradermal, subcutaneous) may be by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection. A novel needleless injection system has recently been described (U.S. Patent No. 5,846,233, incorporated herein by reference) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery⁷. A syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Patent No. 5,846,225, incorporated herein by reference). [00170] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity' may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions -can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00171] For injection in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15 ^,h Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[00172] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques wh ich yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.

[00173] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

[00174] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. [00175] The phrase ’’pharmaceutically-acceptable” or "phannacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar- untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared

EXAMPLES

[00176] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.

Materials and Methods - Examples 1-8

[00177] Plasmids

[00178] Plasmid containing hCD80-Fc, mIL-12, mIL-21, mIL-7, and mGM-CSF/LacZ were generated using gene synthesis techniques. All the sequences were codon optimized for human or mouse expression respectively, submitted to ThermoFisher Scientific for gene synthesis, and inserted into the pMA-RQ vector. hCD80-FL was generated by adding the c-terminal transmembrane domain replacing the Fc by PCR extension. The amino acid sequence of hCD80- Fc, hCD80-FL, mIL-12, mIL-21 and mIL-7 is annotated in SEQ ID NO:9, 10, 11, 13 and 15, respectively.

[00179] Viruses and cells

[00180] Wild-type poxvirus strain Western Reserve (WR) was used as an initial vector for further modifications. All the viruses were generated using a helper virus-mediated, restriction enzyme-guided, homologous recombination repair and rescue technique. Vaccinia virus (OVV- 001) with a deletion of J2R gene and a KI 5 IE mutation on A34 was constructed first using repair sequences with homologous amis to the left and right side of the modification site. The success of the modifications was evaluated initially by PCR and sanger sequencing covering the regions outside of the deletion of J2R and the KI 51 E substitution in the A34R gene and confirmed by whole genome sequencing (WGS). This vims was used as the parent to construct all our viruses expressing a single transgene as well as an intermediate virus expressing hCD80- Fc in the M2L region and mIL-7 in the B18R region (OVV-012). This later virus was then used to construct our virus with three or four transgene expressions.

[00181] Recombinant vaccinia vims control with the deletion of J2R and M2L (OVV-014) was constructed by recombination of a repair donor containing the homologous region outside of M2L of the parental virus OVV-OOl. Successful recombination of the repair donor to delete the M2L gene was verified by Sanger sequencing and WGS.

[00182] Recombinant vaccinia virus expressing human secreted CD80-Fc (OVV-004) and non-secreted CD80-FL (OVV-015) were constructed by recombination of the synthetized CD80- Fc or CD80-FL gene respectively under the control of the synthetic early-late promoter (pSEL), into the M2L region synthetic of the parental virus OVV-OOl. Successful insertion of the CD80 gene into the M2L region was verified by Sanger sequencing, western blotting and WGS.

[00183] Recombinant vaccinia virus expressing human CD80-Fc (OVV-016) w^ras constructed by recombination of the synthetized CD80-Fc gene under the control of the pSEL promoter into the J2R region of the parental virus OVV-OOl . Successful insertion of the CD80-Fc gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00184] Recombinant vaccinia virus expressing single mouse IL-12 (OVV-005, OVV-006 and OVV-013) were constructed by recombination of the synthetized IL-12 gene under the control of the late F17, A14 and pSEL promoter respectively into the B15-B17 region of the parental virus OVV-OOl. Successfill insertion of the IL-12 gene into the B15-B17 region was verified by Sanger sequencing, western blotting and WGS.

[00185] Recombinant vaccinia virus expressing mouse IL-7 (OVV-007) was constructed by recombination of the synthetized mIL-7 under the control of pSEL, promoter into the B18R region of the parental virus OVV-OOl. Successful insertion of the mIL-7 gene into the B18R region was verified by Sanger sequencing, western blotting and WGS.

[00186] Recombinant vaccinia virus expressing mouse IL-21 (OVV-003) was constructed by recombination of the synthetized mIL-21 under the controls of Fl 7 promoter into to J2R region of the parental virus OVV-OOl . Successful insertion of the mIL-21 gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00187] Recombinant vaccinia virus expressing human GM-CSF and LacZ (OVV-009) was constructed by recombination of the synthetized GM-CSF/LacZ genes under the control of the pSEL and p7.5 promoters respectively in opposite orientations, into the J2R region of the wild type (WT) WR virus. Successful insertion of the GM-CSF/LacZ genes into the J2R region was verified by Sanger sequencing and WGS.

[00188] Recombinant vaccinia virus expressing human CD80-Fc, mouse IL-21 and mouse IL- 7 (OVV-008) was constructed, by recombination of the synthetized IL-21 gene under the control of the late Fl 7 promoter into the J2R region, hCD80-Fc in the M2L region and mIL-7 in the B18R region of the parental virus OVV-OOl. Successful insertion of the mIL-21 gene into the J2R region, hCD80-Fc in the M2L region and mIL-7 in the B18R region was verified by Sanger sequencing, western blotting and WGS.

[00189] Recombinant vaccinia virus expressing human CD80-Fc, mouse IL-21, mouse IL- 12 and mouse IL-7 (OVV-010) was constructed by recombination of the synthetized IL- 12 gene under the control of the late Fl 7 promoter into the B15-B17 region, and mIL-21 gene into the J2R region of the parental virus OVV-012. Successful insertion of the mIL-12 gene into the B15- B17 region and mIL-21 gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00190] Recombinant vaccinia virus expressing human CD80-Fc, mouse IL-21, mouse IL-12 and mouse IL-7 (O VV-011) was constructed by recombination of the synthetized IL- 12 gene under the control of the late A14 promoter into the B15-B17 region, and mIL-21 gene into the J2R region of the parental virus OVV-012. Successful insertion of the mIL-12 gene into the B15- B 17 region, and mIL-21 gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00191] BSC40, A549, HCT-116 and U-2 OS cells were obtained from ATCC. HeLa cell line was obtained from GenTarget and Life Technologies. Colo 741 , and HT-29 were obtained from Sigma. MC-38 cells were kindly provided by Dr. Antoni Ribas at UCLA.

[00192] Virus amplification and purification

[00193] Monolayers of BSC40 cells infected with a serial dilution of the lysate from the transfection after viral recombination and covered by agarose overlay, were used to isolate and pick viral plaques. For each virus a total of 2 plaques were selected by 3 rounds of plaque isolation. After verification by Sanger sequencing of the correct insertion, deletion or mutation, a selected plaque was used for intermediate amplification. BSC-40 cells seeded in a T225 flask, were infected by adding virus and incubating for 1 hour. Following infection, the media was replaced with fresh media and incubated for 72 hours to allow for virus amplification. Following incubation, the cells were harvested and collected by centrifugation. The cell lysate was frozen/thawed three times to break down the cells and release the virus. The infected cell lysate was sonicated and used for large scale amplification in HeLa cells in a 10-layer cell factory. The virus was purified by sucrose gradient ultracentrifugation and thoroughly characterized in quality control assays, including full genome next generation sequencing.

[00194] Virus titering, and plaque assay

[00195] Virus titer was determined by ten-fold serial dilutions, with a final dilution of 10'⁸ of the stock concentrated, purified virus. The virus dilutions were used to infect U-2 OS cells to determine the number of plaque forming units per ml (PFU/mL). 0.7 ml., of each serial dilution was applied in duplicate to wells containing a confluent monolayer of U-2 OS cells in a standard 6-well microplate (Avantor). Cells were infected for 2 hours, viral inoculum removed, and overlaid with a solution of fresh media containing 1.5% carboxymethylcellulose (Teknova). Following 72 hours of incubation, the media was removed, and the cells were fixed and stained with a 20% methanol solution containing 0.1% crystal violet (Sigma). The stock titer was then determined by counting the number of plaques in each well, averaging between duplicate titers, and adjusting for the dilution factor. The titer was considered positive when the internal positive control was within the expected range.

[00196] Western blotting

[00197] HeLa cells were plated in 6-weil plates, after 18 hours post-seeding, the cells were confluent and infected with virus at. MOI of 3 for 18 hours. Cells were lysed in 200 pL. Laemmli buffer and lx NuPage LDS sample buffer was added prior to incubation at 95°C for 5 minutes and loading on a NuPage 4-12% Bis-Tris gel. Gel electrophoresis with IxMES running buffer was performed at 200V for 30 minutes. Proteins were transferred with PVDF membrane using an iBIot device and Western Blot was performed using an iBind device. For detection of hCD8-Fc and hCD80-FL we use anti-CD80 primary antibody (Nows, NBP-25255SS) at 1 :1000 dilution, mIL-12 was detected using anti-IL-12 primary antibody (Life Technologies, PA5-18741) at 1 :1000 dilution, mIL-21 was detected using anti-IL-21 primary' antibody (Life Technologies, PA5-46962) at 1 ;1000 dilution and mIL-7 was detected using anti-IL-7 primary antibody (Life Technologies, PA5-79509). As secondary antibodies Goat anti-mouse IgG-HRP (Southern Biotech, 1030-05) was used at a 1 :2000 dilution, Goat anti-rabbit IgG-HRP (Life Technologies, 31460) was used at a 1:10,000 dilution, and rabbit anti-goat IgG-HRP (Life Technologies. 31402) was used at a 1 : 10,000 dilution. TMB substrate w^ras subsequently added to the membrane to visualize bands and scanned.

[00198] ELISA

[00199] Coated ELISA Kits (Invitrogen), with plates ready to use were used to determine the concentration of expression of CD80 (BMS291INST), IL- 12 (BMS616), IL-21 (BMS6021) and IL-7 (EMIL 7). The concentration in the samples was determined by following the manufacturer instructions. Briefly, a monolayer of HeLa cells in a 6- well plate were infected with the recombinant vaccinia virus at an MOI of 3, after 24 hours the supernatant was collected. Serial dilutions of the supernatant were made and added to the coated plates. Absorbance was measured according to the manufactures instructions and concentrations were calculated based on the generated standard curve using a 4-parameter fit. [00200] IL-12 Functional Assay

[00201] Functional activity of the IL-12 containing viral supernatants was measured using an IL- 12 Bioassay (Promega, JA2601). Assay was run according to manufactures instructions, where cells were plated onto white 96-well plate (Life Technologies) Supernatants that were generated for ELIS As were serial diluted and added to the plate along with an IL- 12 protein standard (Life Technologies, 14-8121-80). After a 6-hour incubation, the kit supplied Bright-Gol regent was added and plate was read using a luminometer (Tecan). Luminescence was plotted versus dilution amount and fit using a 4-parameter logistic regression. ECso was calculated in GraphPad Prism 10.

[00202] Viral replication in tumor cells

[00203] Virus replication in the tumor cell lines A549, HCT-116, Colo 741, U-2 OS and HT- 29 was determined by infecting a monolayer of cells with virus at a multiplicity of infection (MOI) of 1 for 1 hour in triplicate. Following infection, the viral inoculum was replaced with fresh media. Cells were harvested and the lysate was frozen at -80°C at 24 and/or 48 hours postinfection. The viral titer for each sample was determined in duplicate via viral plaque assay.

[00204] C'ytptoxicity assay in tumor cells

[00205] Cell killing in the tumor cell line HCT-116 (NCI) was determined by infecting monolayers of cells with various MOI of virus in quadruplicate for 1 hour. Following infection, the viral inoculum was replaced with fresh media. At 72 hours post-infection, cytotoxicity was determined by counting cells alive with the colorimetric CCK-8 assay (Dojindo Laboratories) and measured using spectrophotometer (Tecan) at 450nm, following manufacturer instructions. Data analysis was performed with GraphPad Prism 10 software.

[00206] Animal model and tumor model preparation

[00207] C57BL/6J mice (The Jackson Laboratory) were housed in a temperature (68° - 79°F) and humidity' (30-70%) controlled facility. Animal rooms were maintained on 12-hour alternating light and dark cycles. Dry food (2018 18% protein diet (Envigo)) was made available ad libitum throughout acclimation and the biological phase of the study. MC38 cells were cultured and implanted (5x 10⁵ ceils in lOOpL) in the right rear flank of each mouse. Each animal was dosed intravenously with viral test agent (5x10⁷ PFU in 100 pL) when MC38 tumor volumes reached 50 to 100 mm³. Animals were monitored daily and tumor sizes measured twice per week.

Example 1,

[00208] Generation of recombinant vaccinia virus constructs,

[00209] Select features of the recombinant vaccinia virus constructs generated in connection with the examples provided are summarized in Table 1 : [00210] Each virus in table 1 has a deletion of the J2R gene and a KI 51 E mutation in A34 protein except OVV-009 that has an insertional inactivation of the J2R gene and not mutation in A34 protein. OVV-004 and OW-016 have the gene encoding a secreted human CD80 with a fused immunoglobin Fc fragment (hCD80-Fc) with M2L viral gene deletion or not respectively. OVV-0015 has the gene encoding a non-secreted full length human CD80 (hCD80-FL) and a M2L viral gene deletion. OVV-005, OVV-006 and OVV-013 have the gene encoding for mouse IL-12 under different promoters Fl 7, Al 4 and a synthetic early/late respectively. OVV-003 and OVV-007 have the gene encoding mouse IL-7 and mouse IL-21 respectively, the latter has a deletion of B18R viral gene. OVV-008, OVV-010 and OVV-011 express either the combination of three immunomodulatory transgenes (CD80-Fc, IL-21 and IL-7) or four immunomodulatory transgenes (CD80-Fc, IL-21, IL-7 and IL-12) with the only difference between the ones expressing four transgenes being the promoter driving the expression for IL- 12. All the transgene sequences were optimized for expression in either mouse or human cells respectively. FIG. LA. Provides schematic representation of full genomes for OVV-014, OW-004, OVV-015, OVV- 016, OVV-005, OVV-006 and OVV-013. FIG. IB. Provides schematic representation of full genomes for OVV-008, OVV-009, OVV-010 and OVV-011. FIG. 1C. Provides schematic representation of full genomes for OVV-007 and OVV-003.

Example 2 - Demonstration of transgene expression from recombinant vaccinia viruses in infected cells by Western blotting.

[00211] HeLa cells were infected with the respective recombinant aimed oncolytic vaccinia viruses and the total cell lysate was analyzed by western bloting, according to the procedure described in materials and methods. Results of human CD80 expression analysis following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 2A, as expected, a higher molecular weight is detected in viruses OVV-004 and OVV-016 expressing a human secreted CD80 fused with immunoglobulin Fc fragment (hCD80-Fc), where a lower molecular weight is detected from OVV-015 expressing a human non-secreted CD80 full length (hCD80-FL). Results of mouse IL- 12 expression analysis following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 2B, in which IL- 12 is observed in all the viruses tested. The level of expression changes depending on the strength of the promoter used for the expression of IL- 12 transgene. The level of expression of IL- 12 is comparable in the virus expressing this transgene under PSEL (OVV-013) and Fl 7 late promoter (OVV-05), and it is lower in the virus expressing IL-12 under A14 late promoter (OVV-006). In addition, the level of expression for IL- 12 also decreases when other transgenes are expressed like in OVV-Ol 1 and OVV-OIO. Results of mouse IL-7 expression analysis following infection of cells with recombinant oncolytic vaccinia viruses OW-007, OVV-008, OVV-OIO and OVV-Ol 1 are provided in FIG.2C, in which IL-7 is observed in all the viruses tested. Results of mouse IL-21 expression analysis following infection of cells with recombinant oncolytic vaccinia viruses OVV-003, OVV-008, OVV-OIO and OVV-Ol 1 are provided in FIG. 2D, in which IL-21 is observed in ah the viruses tested.

Example 3 - Quantification of secreted transgene expression from recombinant vaccinia viruses in supernatant of infected cells by ELISA.

[00212] Supernatant of infected HeLa cells with the respective recombinant armed oncolytic vaccina viruses was analyzed by ELISA according to the procedure described in materials and methods. Results of human CD80 concentration in supernatant following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 3 A, OVV-004 and OVV-Ol 6 which express only hCD80-Fc transgene with and without M2L viral gene deletion respectively, had higher concentration level in the supernatant when compared to OVV-008, OVV-IO and OVV- Ol 1 which express three or four transgenes respectively. OVV-Ol 5, which expresses the nonsecreted hCD80 full length was included in the study, however, the concentration measured was under the limit of detection for this assay, confirming that hCD80~FL expressed in OVV-015 is non-secreted. Results of mouse IL- 12 concentration in supernatant following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 3B. The concentration in the supernatant is similar in cells infected with virus expressing IL- 12 under the synthetic early late promoter (OVV-013) and virus expressing IL-12 under the late F17 promoter (OVV-005), however the concentration of IL- 12 detected in supernatant decreases with the virus expressing IL-12 under A14 late promoter, which is expected according to the different strength between the promoters. In addition, the concentration of IL-12 in supernatant is lower in infection with virus expressing a total of four transgenes, even when IL- 12 expression is driven by Fl 7 promoter (OVV-010) and even lower concentration when the IL- 12 expression is driven by Al 4 promoter (OVV-011). Results of mouse IL-7 concentration in supernatant following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 3C, infection with OW-007 which expresses a single IL-7 transgene has higher levels of this cytokine in the supernatant than the viruses expressing three (OVV-008) or four transgenes (OVV-010 and OVV-011) for winch the concentration of IL-7 was similar. Results of mouse IL-21 concentration in supernatant following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 3D, infection with OVV-003 which expresses a single IL-21 transgene has higher levels of this cytokine in the supernatant than the viruses expressing three (OVV-008) or four transgenes (OVV-010 and OVV-011). Altogether, the data suggest that all tire transgenes tested are expressed and secreted.

Example 4

[00213] The impact of hCD80-Fc, hCD80-FL arid IL-12 expressed under different promoters on oncolytic recombinant vaccinia virus was assessed in vitro using representative human cancer cell lines. f 00214] Recombinant oncolytic vaccinia virus not expressing any transgenes (OVV-014), a comparator virus similar to JX-594 expressing mGM-CSF and LacZ, a virus expressing the secreted hCD80~Fc with M2L viral gene deletion (OVV-004), a virus expressing the nonsecreted hCD80-FL with M2L viral gene deletion (OVV-015), a virus expressing the secreted hCD80-Fc without M2L viral gene deletion (OVV-016), a virus expressing mIL-12 under F17 promoter (OVV-005), a virus expressing mIL-12 under A14 promoter (OVV-006) and a virus expressing mlL- 12 under pSEL promoter (OVV-013), were generated and manufactured as described in materials and methods. A549 (lung adenocarcinoma) FIG. 4 A, HT-29 (colorectal adenocarcinoma) FIG. 4B, Colo 741 (colorectal adenocarcinoma) FIG. 4C, HCT1.16 (colorectal carcinoma) FIG. 4D, and U-2 OS (osteosarcoma) FIG. 4E cancer cells were infected with vaccinia viruses mentioned above. At 24 hours post-infection, virus replication was determined by viral plaque assays and plaques counted to determine the number of viral plaque-forming units produced per cell, as described in materials and methods. Fewer viral plaques per cell were produced by almost all recombinant viruses, except for OVV-004 and OVV-006 when compared to a virus not expressing any transgenes (OVV-014) in I IT-29 cells. However, no differences were observed against a comparator virus similar to J'X-594 in HT-29 cells. Fewer viral plaques per cell were produced by all recombinant viruses when compared to a virus not expressing any transgenes (OVV-014) in Colo 741 cells. However, no differences were observed against a comparator virus similar to JX-594 in Colo 741 cells. There were no major statistically significant differences in the other cancer cell lines tested. These data suggest that the expression of a single hCD80-Fc, hCD80-FL or IL-12 transgene does not affect the virus replication.

Example 5

[00215] The impact of hCD80-Fc, and IL- 12 expressed under different promoters on oncolytic recombinant vaccinia virus killing potency on HCT-116 colorectal cancer cell line.

[00216] HCT-116, colorectal carcinoma cell line was infected with recombinant oncolytic vaccinia virus not expressing any transgenes (OVV-014), a vims expressing the secreted hCD80- Fc with M2L viral gene deletion (OVV-004), a virus expressing mIL-12 under Fl 7 promoter (OVV-005), and a virus expressing mIL-12 under A14 promoter (OVV-006). At 72 hours postinfection, cell viability v/as determined using CCK-8 as described in materials and methods. Similar toxicity of the virus expressing hCD80-Fc (OVV-004), IL-12 expression driven under F17 promoter (OVV-005) and IL-12 expression driven under A14 promoter (OVV-006) when compared to a virus not expressing any transgenes (OVV-014) was observed. FIG. 5. At least in this cell line tested the transgene expression does not change the vaccinia virus specificity to replicate and kill this tumor cell line.

Example 6 - Demonstration of functional IL- 12 expressed under different promoters from recombinant vaccinia viruses in infected cells by functional assay.

[00217] Supernatant of infected HeLa cells with IL-12 driven under different promoters by recombinant oncolytic vaccina viruses was analyzed by an assay to detect concentration of functional mouse IL- 12 according to the procedure described in materials and methods. Results of EC50 of functional mouse IL-12 concentration in supernatant follo wing infection of cells with recombinant oncolytic vaccinia viruses OVV-013 (mIL-12 expression under pSEL promoter), OVV-005 (mIL-12 expression under Fl 7 promoter), OVV-006 (mIL-12 expression under A14 promoter), OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression under F17 promoter) and OVV-011 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expression under A14 promoter) are provided in FIG. 6. The EC50 of the concentration of functional mouse IL- 12 in the supernatant is higher in supernatants of cells infected with virus expressing IL- 12 under the synthetic early- late promoter (OV V -013), followed by a virus expressing IL- 12 under the late F 17 promoter (OVV-005) and lower with a virus expressing IL-12 under the late A14 promoter (OVV-006), this is expected according to the different strength between the viral promoters. In addition, the EC50 for the concentration of functional mIL-12 in the supernatant from infected cells with OVV-010 and OVV-11 that express four different transgenes (hCD80-Fc, mIL-21, mIL-7 and mIL-12 under different promoters) is overall lower than the ones expressing the single mIL-12 transgene under the same promoter.

Example 7

[00218] The impact of recombinant oncolytic vaccinia virus expressing human CD80-Fc activity in MC38 tumor-bearing C57BL/6 mice

[00219] Western Reserve (WR) recombinant vaccinia virus unarmed lacking J2R (viral thymidine kinase) and M2L viral gene (OVV-014), a virus expressing secreted human CD80 fused to an immunoglobulin Fc fragment (hCD80-Fc) with M2L viral gene deleted (OVV-004), a virus expressing hCD80-Fc with M2L viral gene intact (OVV-016) and a virus expressing nonsecreted full length CD80 (hCD80-FL) with M2L viral gene deleted (OVV-015) were generated and manufactured as described in materials and methods. In this study, female C57BL/6J mice (8-10 weeks old) were implanted subcutaneously in the rear flank with MC38 cancer cells as described in materials and methods. After the tumors reached a volume range between 50 and 100 mm³, the animals were randomized in five groups (n^lO). On day 11 after tumor implant, mice were intravenously dosed with vehicle (30 mM Tris, 10% sucrose, pH 8.0), or 100 pL vehicle containing 5E7 plaque forming units (PFU) of OVV-014 (control virus not expressing transgenes), OVV-004 (secreted hCD80-Fc - M2L), OVV-016 (secreted hCD80-Fc +M2L), and OVV-015 (non-secreted hCD80-FL). Tumor volumes were measured twice per week until endpoint (tumor volume exceeding 2000 mm³). [00220] Comparisons between tumor growth profiles of groups (FIG. 7 A, 7C-G) revealed that treatment with the virus expressing secreted hCD80~Fc with M2L deletion (OVV-004) vims produced a statistically significant inhibitory effect on tumor growth over multiple consecutive days when compared to the group dosed with vehicle. Treatment with virus expressing nonsecreted hCD80-FL with M2L deletion (OVV-015) had a negative impact tumor growth inhibition. Treatment with unarmed virus (OVV-014) and vims expressing secreted hCD80-Fc with M2L viral gene intact (OVV-016), had a partial effect but it was not statistically significant (FIG. 7 A).

[00221] The body weight of all the animals included in this study and treated as described above was measured twice per week (FIG. 7B). Overall, all the animals in the groups dosed with recombinant vaccinia virus tended to lose weight during the first 8 days after viral treatment but they recovered after that. In addition, none of the groups treated with virus lost any significant weight in this study.

[00222] Survival of animals in each treatment group was also assessed up through day 46 posttumor implantation (FIG. 7H). The unarmed vaccinia virus control (O VV-014) or virus armed with non-secreted hCD80-FL (OVV-015) did not significantly improve survival over vehicle control. However, mice treated with armed vimses expressing secreted hCD80-Fc ± M2L viral gene (OVV-004 and OVV-0016) showed an improved survival with a median survival of 46 days. These data suggest that overall, the virus armed with hCD80-Fc and M2L gene deletion (OVV-004) had a positive effect on tumor growth inhibition and prolonging survival without toxicity by body weight.

Example 8

[00223] The impact of recombinant oncolytic vaccinia virus expressing multiple transgenes human CD80-Fc, mIL-21, mIL-7 and mIL-12 activity' in MC38 tumor-bearing C57BL/6 mice.

[00224] Western Reserve (WR) recombinant vaccinia virus unarmed Sacking J2R (viral thymidine kinase) and M2L viral gene (OVV-014), a comparator virus similar to JX-594 (OVV- 009), an armed virus expressing hCD80-Fc, mIL-21 and mIL-7 (OVV-008), an armed virus expressing hCD80-Fc, mIL-21, mIL-7 and mIL-12 under F17 promoter (OOV-OIO) and an armed virus expressing hCD80-Fc, mIL-21, mIL-7 and mIL-12 under A14 promoter (OOV-Ol 1) were generated and manufactured as described in materials and methods. In this study, female C57BL/6J mice (8-10 weeks old) were implanted subcutaneously in the rear flank with MC38 cancer cells as described in materials and methods. After the tumors reached a volume range between 50 and 100 mm³, the animals were randomized in six groups (n i t)). On day 11 after tumor implant, mice were intravenously dosed with vehicle (30 mM Tris, 10% sucrose, pH 8.0), or 100 pL vehicle containing 5E7 plaque forming units (PFU) of OVV-014 (control unarmed virus), OVV-009 (comparator virus similar to JX-594), OVV-008 (armed with hCD80-Fc, mlL- 21 and mIL-7), OVV-010 (armed with hCD80-Fc, mIL-21, mIL-7 and mIL-12 under F17 promoter) and OVV-011 (armed with hCD80-Fc, mIL-21, mIL-7 and mIL-12 under A14 promoter). Tumor volumes were measured twice per week until endpoint (tumor volume exceeding 2000 mm³).

[00225] Comparisons between tumor growth profiles of groups (FIG. 8A, 8C-H) revealed that treatment with the virus armed with four transgenes OVV-010 (hCD80-FC, mIL-21, mIL-7 and mIL-12 under Fl 7 promoter) produced a statistically significant inhibitory effect on tumor growth over multiple consecutive days when compared to the group dosed with vehicle. All the other viruses including the comparator virus mimetic to JX-594 (OVV-009) did not have any effect in tumor growth in this model. The unarmed virus (OVV-014) had a partial effect as the armed virus expressing four transgenes OVV-011 (hCD80-FC, mIL-21, mIL-7 and mIL-12 under A14 promoter), however the difference was not statistically significant (FIG. 8A)

[00226] The body weight of all the animals included in this study and treated as described above was measured twice per week (FIG. 8B). Overall, all the animals in the groups dosed with recombinant vaccinia virus tended to lose weight during the first 8 days after viral treatment but they recovered after that. In addition, none of the groups treated with virus lost any significan t weight in this study. This is an important observation as it is known that the treatment with IL- 12 in other modalities can cause animal death.

[00227] The survival of animals in each treatment group was also assessed up through day 46 post-tumor implantation (FIG. 81). The treatment with armed virus expressing four transgenes OW-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expressed under F17 promoter) showed an improved survival with a median survival of 46 days. None of the other viruses has a significant effect on survival when compared to the vehicle or the unarmed virus. These data suggest that the virus armed with four transgenes OVV-010 (hCD80-Fc, mIL-21, mIL-7 and mIL-12 expressed under Fl 7 promoter) has a positive effect on tumor growth inhibition and prolonging survival without toxicity measured by body weight.

[00228] CONCLUSIONS

[00229] The present inventors, for the first time, have engineered 4 different transgenes - CD80-Fc. IL-21, and IL-12 - in recombinant oncolytic vaccinia virus. Further, the present inventors have, for the first time, demonstrated detrimental effects when combining specific transgenes due to, e.g., a negative impact in virus replication.

[00230] The present inventors have, for the first time, shown that vaccinia virus requires a deletion of the M2L viral gene in order to observe efficacy in a syngeneic animal model of cancer such as MC38 when the virus expresses secretory CD80. Further, it is shown that vaccinia virus expressing full length (non-secreted CD80) does not have efficacy. It was also determined that expressing CD80-Fc in certain locations from the vaccinia virus has an impact in the level of expression of this transgene.

[00231] The present inventors, for the first time, have demonstrated that the combination of M2L viral gene deletion and expression of CD80-Fc, IL-21, and IL- 12 from vaccinia virus results exhibits potent anticancer effects while avoiding toxicity.

[00232] It was also determined that expressing IL- 12 from vaccinia virus using a late promoter decreases toxicity that correlates with the transgene level of expression and the time in which the transgene is expressed during the viral replication cycle.

[00233] Further, it was determined that specific combination of secreted CD80-Fc, IL-21, IL-7 and IL- 12 under Fl 7 (native late) promoter has a stronger effect on efficacy in vivo, whereas the same combination but expressing IL-12 under A14 (native late) promoter does not have any effect in MC38 in vivo model. This suggests that when combining different transgenes, it is important to note the level and timing in which each transgene is combined, the promoter for each transgene. Even when a good effect is observed in vivo, viral replication may suffer a negative impact with the combination.

Materials and Methods - Examples 9-16

[00234] Plasmidg

[00235] Plasmid containing mIL-12, hIL-12, hIL-21, mIL-21, hCD80-Fc, mIL-2v, hIL-2gv, HSV-TK.007, A33/A34 substitutions and B5 substitutions were generated using gene synthesis techniques. All the sequences were codon optimized for human or mouse expression respectively, submitted to ThermoFisher Scientific for gene synthesis, and inserted into the pMA-RQ vector. The amino acid sequence of hCD80-Fc, mIL-12, hIL-12, mIL-21, hIL-21, HSV-TK.007, mIL-2v, hIL-2gv, A33/A34 substitutions and B5 substitutions are set forth in SEQ ID NO: 9, 11, 12, 13, 14, 1, 36, 41, 3, 5 and 7, respectively.

[00236] Viruses and cells

[00237] Wild-type poxvirus strain Western Reserve (WR) and Copenhagen (Cop) were used as initial vectors for further modifications. All the viruses were generated using a helper virus- mediated, restriction enzyme-guided, homologous recombination repair and rescue technique. Vaccinia vims (OVV-029) with a deletion of J2R gene, a K151E mutation in A34 and expression of Luc-2 A-GFP reporter was constructed using Copenhagen with repair sequences with homologous arms to the left and right side of the modification site. The success of the modifications was evaluated initially by PCR and sanger sequencing covering the regions outside of the deletion of J2R, M2L and the KI 5 IE substitution in the A34R gene and confirmed by whole genome sequencing (WGS). This virus was used as the parent to construct viruses OVV- 030 to OW-033 expressing a combination of transgenes.

[00238] Recombinant cop vaccinia virus expressing the transgenes combination of human secreted CD80-Fc and human IL -12 with the deletion of J2R and M2L (OVV-030) was constructed by recombination of two repair donors containi ng the homologous region outside of M2L and Luc-2A-GFP of the parental virus OVV-029. Successful recombination of the repair donors to delete the M2L and Luc-2A-GFP genes was verified by Stinger sequencing and WGS.

[00239] Recombinant cop vaccinia virus expressing human secreted CD80-Fc. hIL-12 and HSV-TK.007 (OVV-31) was constructed by recombination of the synthetized HSV-TK.007 gene under the control of the pF 17 late promoter, into the B16 region of the parental virus OVV-030. Successful insertion of the HSV-TK.007 gene into the B 16 region was verified by Sanger sequencing, western blotting and WGS.

[00240] Recombinant cop vaccinia vims expressing human secreted CD80-Fc, human IL-21 and human IL-12 (OVV-032) was constructed by recombination of the synthetized human IL-21 under the control of the pF 17 later promoter, into the J2R region and human IL-12 gene under the control of pF17 later promoter into the B16 region of the parental virus OVV-030. Successful insertion of the human IL-21 gene into the J2R region and human IL- 12 into the Bl 6 region was verified by Sanger sequencing, western blotting and WGS.

[00241] Recombinant cop vaccinia virus expressing IL-2gv and HSV-TK.007 (OVV-033) was constructed by recombination of the synthetized IL-2gv gene under the control of the synthetic late promoter (pSEL) and HSV-TK.007 under the pF 17 late promoter into the J2R and B16 region respectively of the parental virus OVV-029. Successful insertion of the IL-2gv and HS V- TK.007 genes into the J2R and B16 region was verified by Sanger sequencing, western blotting and WGS.

[00242] Recombinant WR vaccinia virus including J2R and M2L deletions, A34 substitution K151E and expressing HSV-TK.007 (OVV-034) was constructed by recombination of the synthetized HSV-TK.007 under the control of F17 promoter into the B15-B17 region of the parental virus OVV-017. Successful insertion of the HSV-TK.007 gene into the Bl 5-B17 region was verified by Sanger sequencing, western blotting and WGS.

[00243] Recombinant WR vaccinia virus expressing human secreted CD80-Fc and HSV- TK.007 (OVV-035) was constructed by recombination of the synthetized hCD80-Fc under the control of pSEL promoter into the M2L region of the parental virus OVV-017. Successful insertion of the hCD80~Fc gene into the M2L region was verified by Sanger sequencing, western blotting and WGS.

[00244] Recombinant WR vaccinia virus expressing mouse IL-2v and HSV-TK.007 (OVV- 036) was constructed by recombination of the synthetized mIL-2v under the control of synthetic early-late promoter into the J2R region of the parental virus OVV-034. Successful insertion of the mIL-2v gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00245] Recombinant WR vaccinia virus expressing human secreted CD80-Fc, mouse IL-2v and HSV-TK.007 (OVV-037) was constructed by recombination of the synthetized mIL-2v under the control of synthetic early-late promoter into the J2R region of the parental virus OVV- 035. Successful insertion of the mIL-2v gene into the J2R region was verified by Sanger sequencing, western blotting and WGS.

[00246] Recombinant WR vaccinia virus expressing mouse IL-21, mouse IL-2v and HSV- TK.007 (OVV-038) was constructed by recombination of the synthetized mIL-21 under the control of Fl 7 late promoter into the M2L region of the parental virus OVV-036. Successful insertion of the mIL-21 gene into the M2L region was verified by Sanger sequencing, western bloting and WGS.

[00247] Recombinant WR vaccinia virus expressing human secreted CD80-Fc, mouse IL-2v, HSV-TK.007 and substitutions in A33 and A34 proteins (OVV-039) was constructed by recombination of the synthetized substitutions in A33 and A34 into the A33 and A34 region of the parental virus OVV-037. Successful insertion of the A33R and A34R gene mutations into the A33R-A34R region were verified by Sanger sequencing, western blotting and WGS.

[00248] Recombinant WR vaccinia virus expressing human secreted CD80-Fc, mouse IL-2v, HSV-TK.007 and substitutions in B5 protein (OVV-040) was constructed by recombination of the synthetized substitutions in B5 into the B5 region of the parental virus OVV-037. Successful insertion of the B5R gene mutations into the B5R region were verified by Sanger sequencing, western blotting and WGS.

[00249] BSC40, A549, HCT-116 and U-2 OS cells were obtained from ATCC. Colo 741, and HT-29 were obtained from Sigma. MC-38 cell were kindly provided by Dr. Antoni Ribas at UCLA

[00250] Virus amplification and twificatioti

[00251] Monolayers of BSC40 cells infected with a serial dilution of the lysate from the transfection after viral recombination and covered by agarose overlay, were used to isolate and pick viral plaques. For each virus, 2 plaques were selected by 3 rounds of plaque isolation. After verification by Sanger sequencing of the correct insertion, deletion or mutation, a selected plaque was used for intermediate amplification. BSC-40 cells seeded in a T225 flask, were infected by adding virus and incubating for 1 hour. Following infection, the media was replaced with fresh media and incubated for 72 hours to allow for virus amplification. Following incubation, the cells were harvested and collected by centrifugation. The cell lysate was frozen/thawed three times to break down the cells and release the virus. The infected cell lysate was sonicated and used for large scale amplification in HeLa cells in a 10-layer cell factory. The virus was purified by sucrose gradient ultracentrifugation and thoroughly characterized in quality control assays, including full genome next generation sequencing.

[00252] Virus titering and plaque assay

[00253] Virus titer was determined by ten-fold serial dilutions, with a final dilution of 10'* of the stock concentrated, purified virus. The vims dilutions were used to infect U-2 OS cells to determine the number of plaque forming units per mL (PFU/mL). 0.7 mL of each serial dilution was applied in duplicate to wells containing a confluent monolayer of U-2 OS cells in a standard 6-well microplate (Avantor). Cells were infected for 2 hours, viral inoculum removed, and overlaid with a solution of fresh media containing 1.5% carboxymethylcellulose (Teknova). Following 72 hours of incubation for WR, 48 hours for Cop, the media was removed, and the cells were fixed and stained with a 20% methanol solution containing 0.1% crystal violet (Sigma). The stock titer was then determined by counting the number of plaques in each well, averaging between duplicate titers, and adjusting for the dilution factor. The titer was considered positive when the internal positive control was within the expected range.

[00254] Western blotting

[00255] HeLa cells were plated in 6-well plates, after 18 hours post-seeding, the cells were confluent and infected with virus at MOI of 3 for 22 hours. Cells were lysed in 200 uL Laemmli buffer and lx NuPage LDS sample buffer was added prior to incubation at 95°C for 5 minutes and loading on a NuPage 4-12% Bis-Tris gel. Gel electrophoresis with IxMES running buffer was performed at 200V for 30 minutes. Proteins were transferred with PVDF membrane using an iBlot device and Western Blot was performed using an iBind device. For detection of mIL-2v we use anti-IL2 primary antibody (ThermoFisher Scientific, 26156-1-AP) at 1 :500 dilution, hCD80- Fc w^ras detected using anti-CD80 primary antibody (Novus, NBP-25255SS) at 1 : 1000 dilution, mIL-21 was detected using anti-IL-21 primary antibody (Life Technologies, PA5-46962) at 1 :1000 dilution. As secondary antibodies, goat anti-mouse IgG-HRP (Southern Biotech, 1030- 05) was used at a 1:5,000 dilution, goat anti-rabbit IgG-HRP (Life Technologies, 31460) was used at a 1:5,000 dilution, and rabbit anti-goat IgG-HRP (Life Technologies, 31402) was used at a 1:5,000 dilution. TMB substrate was subsequently added to the membrane to visualize bands and scanned.

[00256] ELISA

[00257] Coated ELISA Kits (Invitrogen), with plates ready to use were used to determine the concentration of expression of CD80 (BMS291INST). The concentration in the samples was determined by following the manufacturer instructions. Briefly, 1:10 serial dilutions of lOpL of each mouse serum were made and added to the coated plates. Absorbance was measured according to the manufactures instructions and concentrations were calculated based on the generated standard curve using a 4-parameter fit.

[00258] Viral replication in tumor cells, [00259] Virus replication in the tumor cell lines MC38, A549, Colo741, and HT-29 was determined by infecting a monolayer of cells with virus at a multiplicity of infection (MOI) of 1 for 1 hour in triplicate. Following infection, the viral inoculum was replaced with fresh media. Cells were harvested and the lysate was frozen at -80°C at 48 hour's post-infection. The viral titer for each sample was determined via viral plaque assay.

[00260] Cytotoxicity assay in tumor cells

[00261] Cell killing in the tumor cell lines MC38, HCT-116 (NCI), A549 and Colo741 was determined by infecting monolayers of cells with various MOI of virus in quadruplicate for 1 hour. At 72 hours post-infection for HCT-1 16 and MC38 cells, or 96 hours post-infection for A549 and Colo741 cells, cytotoxicity' was determined by counting cells alive with the colorimetric CCK-8 assay (Dojindo Laboratories) and measured using spectrophotometer (Tecan) at 450nm, following manufacturer instructions. Data analysis was performed with GraphPad Prism 10 software.

[00262] Comet assay

[00263] Monolayers of BSC-40 cells seeded in 6-well plates were infected with 0.7 mL of 10- fold serial dilutions of virus for 1 hour. The infected cells were washed 2 times, repleni shed with fresh media and incubated for 72 hours at a 20 angle at 37°C. Cells monolayers were washed then stained with a 20% methanol solution containing 0.1% crystal violet (VWR) for 1 hour to visualize comets.

[00264] Animal model and tumor model ptoparatinn

[00265] C57BL/6J mice (The Jackson Laboratory) were housed in a temperature (68° - 79°F) and humidity (30-70%) controlled facility'. Animal rooms were maintained on 12 -hour alternating light and dark cycles. Dry food (2018 18% protein diet (Envigo)) was made available ad libitum throughout acclimation and the biological phase of the study. MC38 cells were cultured and implanted (5xlO⁵ cells in lOOpL) in the right rear flank of each mouse. Each animal was dosed intravenously with viral test agent (5x10⁷ PFU in 100 pL) when MC38 tumor volumes reached 50 to 100 mm³. Animals were monitored daily, and tumor sizes measured twice per week.

Example 9

[G0266] Generation of recombinant vaccinia virus constructs

[00267] Selected features of the recombinant vaccinia virus constructs generated in connection with examples 9-16 are provided are summarized in Table 2:

Table 2

[00268] Virus engineered using Copenhagen (Cop) as a base s train have a deletion of the J2R gene and a KI 5 IE mutation in A34 protein. OVV-029 has a gene encoding for Luc-2A-GFP reporter. OVV-030 has a combination of the genes encoding a secreted human CD80 with a fused imrnunoglobin Fc fragment (hCD80-Fc) with M2L viral gene deletion and the gene encoding a human IL-12. OVV-031 encodes genes for hCD80-Fc, hIL-12 and HSV-TK.007 with a M2L deletion. OVV-032 encodes genes for hCD80-Fc, hIL-21 and hIL-12 with a M2L deletion and OVV-033 encodes genes for hIL-2gv and HSV-TK.007. Viruses engineered using Western Reserve (WR) as base strain have a deletion of the J2R and M2L viral genes, gene encoding a variant of herpes simplex virus 1 thymidine kinase 00.7 (HSV-TK.007) inserted in the B16R region and a K151E mutation in A34 protein (OVV-034). OVV-035 encodes the gene for hCD80-Fc. OVV-036 encodes the gene for mIL-2v. OVV-037 encodes genes for hCD80-Fc and mIL-2v. OW-038 encodes genes for mIL-21 and mIL-2v. OVV-039 encodes genes for hCD80- Fc, mIL-2v, M63R mutation in A33 protein and M66T mutation in A34 protein. OVV-040 encodes genes for hCD80-Fc, mIL-2v, N241T, E243V, V247D, G250R and A276F mutations in B5 protein. A schematic representation of full genomes based on Copenhagen strain OVV-029, OVV-030, OVV-031, OVV-032 and O VV-033 is provided by Fig. 9. A schematic representation of full genomes based on Western Reserve strain OVV-034, OVV-035. OVV-036, OVV-037, OVV-038, OVV-039 and OVV-040 are provided by Fig. 12.

Example 10

[00269] The impact of hCD80-Fc, IL-12, IL-21, IL-2gv and HSV-TK.007 combinations expressed on oncolytic recombinant vaccinia virus was assessed in vitro using representative human cancer cell lines HT-29 (colorectal adenocarcinoma), A549 (lung adenocarcinoma), and Colo 741 (colorectal adenocarcinoma).

[00270] Recombinant Cop oncolytic vaccinia virus expressing the reporter transgenes Luc-2A- GFP was used as a control for the experiment. A virus expressing the secreted hCD80-Fc and hIL-12 with M2L viral gene deletion (OVV-030), a virus expressing the secreted hCD80-Fc, hIL-12 and HSV-TK.007 with M2L deletion (OVV-031), a virus expressing the secreted hCD80- Fc, ML12 and hIL-21 with M2L deletion (OVV-032) and a virus expressing hIL-2gv and HSV- TK.007 (OVV-033), were generated and manufactured as described in materials and methods. HT-29 (FIG. 10A), A549 (FIG. 10B) and Colo 741 (FIG. 10C) human cancer cells were infected with Cop vaccinia viruses mentioned above. At 48 hours post-infection, virus replication was determined by viral plaque assays to determine the number of viral plaque-forming units (PFU) produced per cell, as described in materials find methods. No statistically significant differences were observed in any of the human cell lines when comparing all recombinant viruses against OVV-029, the reference virus expressing reporter genes (Luc-2A-GFP). This data indicates that expression of the hCD80-Fc, hIL-21, hIL-12, hlL-2gv and HSV-TK..007 transgenes in different combinations, expressed in Cop vaccinia recombinant virus, does not affect viral replication in human cancer cell lines.

Example 11

[00271] The impact of expression of hCD8()-Fc, IL- 12, IL-21 , IL-2gv and HSV-TK.007 from oncolytic recombinant vaccinia virus on cancer cell killing potency was assessed in vitro using representative human cancer cell line

[00272] Recombinant Cop oncolytic vaccinia vims expressing the reporter transgenes Luc-2A- GFP was used as a control for the experiment. A virus expressing the secreted hCD80-Fc and hIL-12 with M2L viral gene deletion (OVV-030), a vims expressing the secreted hCD80-Fc, hIL-12 and HSV-TK.007 with M2L deletion (OVV-031), a virus expressing the secreted 11CD80- Fc, hIL12 and hIL-21 with M2L deletion (OVV-032) and a virus expressing hIL-2gv and HSV- TK.007 (OVV-033), were generated and manufactured as described in materials and methods. HCT-116 (FIG. 11A), Colo 741 (FIG. 1 IB) and A549 (FIG. 11C) human cancer cells were infected with Cop vaccinia recombinant viruses described above. After 72 hours post-infection for HCT-116 cells and 96 hours post-infection for Colo 741 and A549 cells, cell viability was determined using CCK-8 as described in materials and methods. Similar toxicity of the virus expressing different therapeutic transgenes was observed. Viability in the cell lines tested was not significantly different from any the virus tested when compared with the control (OV V -029) expressing reporter transgenes (Lnc-2A-GFPL therefore the data suggest that the different combinations of transgenes expressed in the Cop recombinant vaccinia virus as a backbone does not change the potency for the armed recombinant vaccinia virus to replicate and kill the human cancer cells evaluated.

Example 12

[00273] Transgene expression from recombinant vaccinia viruses in infected cells was confirmed by Western blotting. [00274] HeLa cells were infected with the respective recombinant armed oncolytic vaccinia viruses and the total cell lysate was analyzed by western blotting, according to the procedure described in materials and methods. Results of the mouse IL-2v expression analysis is provided in FIG. 13 A, in which mIL-2v is observed in virus expressing such transgene (OW-036, OVV- 037, OVV-038, OVV-039 and OVV-040). The expression in OVV-035 is contamination from OVV-036 sample loaded in the gel to the right. Results of human CD80 expression analysis following infection of cells with recombinant oncolytic vaccinia viruses are provided in FIG. 13B. As expected a high molecular weight is detected in virus expressing the human secreted CD80 fused with immunoglobulin Fc fragment (hCD80-Fc) OV V-035, OV V-037, OVV-039 and OVV-040. Results of mouse IL-21 expression analysis following infection of cells with recombinant oncolytic vaccinia virus OVV-038 are provided in FIG. 13C, in which mIL-21 is observed in OVV-038 as expected.

Example 13

[00275] The impact of hCD80-Fc, mIL-2v and mIL-21, alone or in combinations, on viral replication was assessed in vitro using representative human and mouse cancer cell lines.

[00276] Recombinant oncolytic vaccinia virus with M2L and J2R viral gene deletions, expressing HSV-TK.007 transgene and KI 5 IE substitution in A34 protein (OVV-034 backbone) was used as a control for the experiment. All viruses evaluated had the same deletions, substitutions and expressed HSV-TK.007. A virus expressing hCD80-Fc (OVV-035), a virus expressing mIL-2v (OVV-036), a virus expressing hCD80-Fc and mIL-2v combination (OVV- 037) and a virus expressing mIL-21 and mIL-2v combination (OVV-038), were generated and manufactured as described in materials and methods. MC38 (murine colorectal carcinoma; FIG. 14A), A549 (human lung adenocarcinoma; FIG. 14B) and HT29 (human colorectal adenocarcinoma; FIG. 14C) cells were infected with WR vaccinia viruses. At 48 hours postinfection, virus replication was determined by viral plaque assays to determine the number of plaque forming units (PFU) produced per cell, as described in materials and methods. OVV-035 recombinant virus expressing single hCD80-Fc and OVV-038 recombinant virus expressing mIL-21 and mIL-2v combination decrease their replication (PFU/cell) in MC38 with a statistically significant difference of p=0.007 and p=0.014 respectively. All the recombinant vaccinia virus evaluated decreased viral replication when compared against the backbone virus (OVV-034) not expressing any cytokine transgenes. However, no differences in viral replication (PFU/cells) were observed in A549 cells when compared against the backbone virus not expressing cytokine transgenes. Therefore, no specific trends in virus replication differences for the recombinant viruses tested were observed in vitro in mouse or human cancer cell lines.

Example 14

[00277] The impact of hCD80~Fc, mIL-2v and mIL-21, alone or in combinations, expressed on oncolytic recombinant vaccinia virus cancer cell killing potency was assessed in vitro using representative human and mouse cancer cell lines.

[00278] Recombinant oncolytic vaccinia virus with M2L and J2R viral gene deletions, expressing HSV-TK.007 transgene and K151E substitution in A34 protein (OVV-034 backbone) was used as a control for the experiment. All viruses evaluated had the same deletions, substitutions and expressed HSV-TK.007. A virus expressing hCD80-Fc (OVV-035), a virus expressing mIL-2v (OVV-036), a virus expressing hCD80-Fc and mIL-2v combination (OVV- 037) and a virus expressing mIL-21 and mIL-2v combination (OW-038), were generated and manufactured as described in materials and methods. MC38 (murine colorectal carcinoma; FIG. 15 A) and A549 (human king adenocarcinoma: FIG. 15B) cells were infected with WR vaccinia viruses. After 72 hours post-infection for MC38 cells and 96 hours post-infection for A549 cells, cell viability was determined using CCK-8 as described in materials and methods. Similar toxicity of the virus expressing different therapeutic transgenes was observed. Therefore, viability in the cell lines tested was not significantly different from any of the virus tested when compared with the backbone control (OVV-034) not expressing cytokine transgenes. The data indicate that cytokine transgenes expressed in WR recombinant vaccinia virus, alone or in combinations, does not alter the potency to replicate and kill the mouse and human cancer cells evaluated. Example 15

[00279] The tumor cell-killing potential of recombinant oncolytic vaccinia virus expressing transgenes hCD80-Fc, mIL-21, and mIL-2v activity (alone or in combinations) in MC38 tumorbearing C57BL/6 mice was evaluated.

[00280] Western Reserve (WR) recombinant oncolytic vaccinia virus lacking M2L and J2R (viral thymidine kinase), expressing HSV-TK.007 transgene and KI 5 IE substitution in A34 protein (OVV-034 backbone) was used as a control for the study. All the viruses evaluated had the same viral deletions, substitutions and expressed HSV-TK.007. An armed virus expressing hCD80-Fc (OVV-035), an armed virus expressing m!L-2v (OVV-036), an armed vims expressing hCD80-Fc and mIL-2v combination (OVV-037), an armed virus expressing mIL-21 and mIL-2v combination (OVV-038), an armed virus expressing hCD80-Fc and mIL-2v combination with a M63R substitution in A33 and M66T substitution in A34, and an armed virus expressing hCD80-Fc and mIL-2v combination with N241T, E243V, V247S, G250R and A276F substitutions in B5 were generated and manufactured as described in materials and methods. In this study, female C57BL/6J mice (8-10 weeks old) were implanted subcutaneously in the rear flank with MC38 cancer cells as described in materials and methods. After the tumors reached a volume range between 50 and 100 mm³, the animals were randomized in eight groups (n=10). On day 11 after tumor implant, mice were intravenously (IV) dosed with vehicle (30 mM Tris, 10% sucrose, pH 8.0), or 100 pL vehicle containing 5E7 plaque forming units (PFU) of OVV- 034 (control backbone unarmed virus), OVV-035 (armed with hCD80-Fc), OVV-036 (armed with mIL-2v), OVV-037 (armed with hCD80-Fc and mIL-2v), OVV-038 (armed with mIL-21 and mIL-2v), OVV-039 (armed with hCD80-Fc, mIL-2v, M63R substitution in A33 and M66T substitution in A34) and OW-040 (armed with hCD80-Fc, mIL-2v, N421T, E243V, V247S, G250R and A276F substitutions in B5. Tumor volumes were measured twice per week until endpoint (tumor volume exceeding 2000 mm^J).

[00281] Comparisons between tumor growth profiles of treatment groups (FIG. 16A, 16B-I) revealed that treatment with the virus armed with hCD80-Fc and mIL-2v (OVV-037), produced 70% complete tumor regressions, with a statistically significant inhibitor)' effect on tumor growth over multiple consecutive days when compared to the group dosed with vehicle or control virus backbone (OVV-034). The virus armed with hCD80-Fc and mIL-2v and the substitutions of N241T, E243V, V247S, G250R and A276F in B5 (for better intratumoral virus spreading) (OVV-040) produced 60% complete tumor regressions. In contrast, viruses armed with a single transgene - hCD80-Fc (OVV-035) or mIL-2v (OVV-036) - produced 10% and 50% complete tumor regressions respectively. The virus armed with mIL-21 and mIL-2v (OVV-038) produced 10% of complete tumor regressions. All armed viruses either with single or combination transgenes hCD80-Fc (OVV-035), mIL-2v (OVV-036), hCD80-Fc + mIL-2v (OVV-037), mIL-2 + mIL-2v (OVV-038, hCD80 + mIL-2v + substitutions in A33 and A34 (OVV-039) and hCD80-Fc + mIL~2v + substitutions in B5 (OVV-040) produced a statistically significant inhibitory’ effect on tumor growth over multiple consecutive days when compared to the group dosed with vehicle (FIG. 16A)

[00282] The body weight of all the animals included in this study as described above was measured twice per week (FIG. 16J). Overall, all the animals in the groups dosed with recombinant vaccinia virus tended to lose weight during the first 8 days after viral treatment but they recovered after that. In addition, none of the groups treated with virus lost any significant weight in this study.

[00283] The survival of animals in each treatment group due to tumor burden was also assessed through day 56 post-tumor implantation (FIG. 16K). The treatment with armed virus expressing either single or transgene combinations mentioned above showed an improved survival with an undefined median survival at 56 days, significantly improving on survival when compared to the vehicle or the unarmed (backbone OVV-034) virus. In addition, in the group receiving IV treatment with an armed virus expressing hCD80-Fc and mIL-2v (OVV-037), none of the animals reached tumor burden within the 56 days of the study.

[00284] Expression of hCD80 transgene was measured in the serum of the animals from groups dosed with virus expressing this transgene either alone (OVV-035) or in combination with mIL-2v (OVV -037, OVV-039 and OVV-040) after 72 hours post-treatment (see FIG. 16L). hCD80 was detected in the serum of ah the animals treated with viruses expressing CD80 as a transgene. These data indicate that transgenes are being expressed from replicating viruses and detected in the serum of the animals after 72 hours post intravenous treatment. Armed viruses produced complete tumor regressions and prolonged survival (measured by tumor burden) without toxicity measured by body weight.

Example 16

[00285] Recombinant oncolytic vaccinia vims engineered with additional substitutions in A33, A34 and B5 to enhance virus spreading in vitro were assessed.

[00286] The impact of the substitution variants on viral spreading and extracellular enveloped virus (EEV) production was assessed in vitro using BSC-40 (African green monkey kidney) cell line. Western Reserve (WR) vaccinia viruses containing the wild-type A33, A34 and B5 sequences in a comparator to JX-594 virus expressing mGM-CSF and LacZ (OVV-009), a KI 51 E substitution in A34 (a known mutation to increase spreading and EEV production) expressing hCDSO and mIL-2v (OVV-037), a M63R substitution in A33, a M66T and K151E substitutions in A34 expressing hCD80 and mIL-2v (OVV-039) and a KI 5 IE substitution in A34, aN241T, E243V, V247S, G250R and A276F substitutions in B5 expressing hCD80 and mIL-2v (OVV-040) were generated and manufactured as described in materials and methods. The comet assay was performed as described in materials and methods. The presence of longer comet, tails and appearance of more satellite plaques for the virus with substitutions in A34 (OVV-037), substitutions in A33 and A34 (OVV-039) and substitutions in A34 and B5 (OVV- 040) compared to WR containing the wild-type A33, A34 and B5 sequences (OVV-009), indicates enhanced virus spreading and therefore more EEV being produced by the recombinant vaccinia viruses containing mutations in A34R, A33R and B5R viral genes. These data illustrate that the incorporation of substitutions to A33, A34 and B5 in particular at the M63 location in A33, M66 location in A34 in combination with KI 5 IE substitution in A34, and N241, E243, V247, G250 and A276 locations in B5 in combination with K151E substitution in A34 leads to enhanced virus spreading of recombinant armed virus with hCD80-Fc and mIL-2v in vitro.

Claims

1 . A replication competent recombinant oncolytic vaccinia virus comprising, in its genome, a nucleotide sequence encoding a soluble human CD80, said nucleotide sequence operably linked to an expression control sequence, wherein said virus comprises a mutation in the M2L gene that results in a negative M2L phenotype and comprises a mutation in the J2R gene that results in a negative J2R phenotype.

2. The replication competent oncolytic vaccinia virus vector according to claim 2, wherein the nucleotide sequence encoding a soluble human CD80 is a human CD80-Fc fusion protein comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

3. The replication competent recombinant oncolytic vaccinia virus according to claim 1 or 2, wherein the nucleotide sequence encoding a soluble human CD80 encodes a human CD80-Fc fusion polypeptide of SEQ ID NO:9 or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

4. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1 to 3, wherein the negative M2L phenotype results from an insertion of the nucleotide sequence encoding a soluble human CD80 into the M2L gene.

5. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1 to 3, wherein the negative J2R phenotype results .from an insertion of the nucleotide sequence encoding a soluble human CD80 into the J2R gene.

6. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1 to 5, wherein the vaccinia virus comprises a K151E amino acid substitution in the A34R gene.

7. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1 to 6, wherein the expression control sequence comprises an SEL promoter.

8. The replication competent recombinant oncolytic vaccinia virus according to claim 7, wherein the SEL promoter comprises the nucleotide sequence set forth in SEQ ID NO:25 or a sequence at least 80%, at least 85%, at least 90% or at least 95% identical thereto.

9. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1-8, wherein the vaccinia virus is a Copenhagen or Western Reserve strain.

10. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 1-9, wherein the vaccinia virus further comprises, in its genome, (i) nucleotide sequence encoding IL-7 operably linked to an expression control sequence, (ii) nucleotide sequence encoding IL- 12 operably linked to an expression control sequence and (iii) nucleotide sequence encoding IL-21 operably linked to an expression control sequence.

11. The replication competent recombinant oncolytic vaccinia virus according to claim 10, wherein the nucleotide sequence encoding IL-7 comprises the nucleotide sequence of SEQ ID NO:23 or SEQ ID NO:24 or a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

12. The replication competent recombinant oncolytic vaccinia virus according to claim 10 or 11, wherein the nucleotide sequence encoding IL-7 encodes a polypeptide of SEQ ID NO: 15 or 16 or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

13. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10 to 12, wherein the nucleotide sequence encoding IL- 12 comprises the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO:20 or a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

14. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10 to 13, wherein the nucleotide sequence encoding IL- 12 encodes a polypeptide of SEQ ID NO:11 or SEQ ID NO: 12 or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

15. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10 to 14, wherein the nucleotide sequence encoding IL-21 comprises the nucleotide sequence of SEQ ID NO:21 or SEQ ID NO:22 or a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

16. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10 to 15, wherein the nucleotide sequence encoding IL-21 encodes a polypeptide of SEQ ID NO:13 or SEQ ID NO:14 or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

17. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-16, wherein the vaccinia virus comprises a mutation in the A45R gene that results in a negative A45R phenotype and comprises a mutation in the B 13R gene that results in a negative B13R phenotype and comprises a mutation in the A53R gene that results in a negative A53R phenotype.

18. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10 to 17, wherein the vaccinia virus is a Western Reserve strain and the nucleotide sequence encoding IL-12 is inserted into the B15R-B17L intergenic region, or wherein the vaccinia virus is Copenhagen strain and the nucleotide sequence encoding IL- 12 is inserted into the B16 gene, preferably wherein the remaining portion of the Bl 6 gene is deleted.

19. The replication competent recombinant oncolytic vaccinia virus according to claim 17 or 18, wherein the negative Bl 8R phenotype results from an insertion of the nucleotide sequence encoding IL-7 into the B18R.

20. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-19, wherein the nucleotide encoding IL- 12 is operably linked to an F17 or Al 4 promoter and is preferably operably linked to an F17 promoter.

21. The replication competent recombinant oncolytic vaccinia virus according to claim 20, wherein the A14 promoter comprises the nucleotide sequence set forth as SEQ ID NO:27 or a sequence at least 80%, at least 85%, at least 90% or at least 95% identical thereto and/or wherein the Fl 7 promoter comprises the nucleotide sequence set forth as SEQ ID NO:25 or a sequence at least 80%, at least 85%, at least 90% or at least 95% identical thereto.

22. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-21, wherein the nucleotide encoding IL-21 is operably linked to an Fl 7 promoter.

23. The replication competent recombinant oncolytic vaccinia virus according to claim 22, wherein the Fl 7 promoter comprises the nucleotide sequence set forth as SEQ ID NO:25 or a sequence at least 80%, at least 85%, at least 90% or at least 95% identical thereto.

24. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-23, wherein the nucleotide encoding IL-7 is operably linked to an SEL promoter

25. The replication competent recombinant oncolytic vaccinia virus according to claim 24, wherein the SEL promoter comprises the nucleotide sequence set forth as SEQ ID NO:25 (or a sequence at least 80%, at least 85%, at least 90% or at least 95% identical thereto.

26. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-25. wherein the vaccinia virus comprises a KI 5 IE amino acid substitution in the A34R gene and/or wherein the vaccinia virus comprises a nucleotide sequence encoding a herpes simplex virus (HSV) thymidine kinase polypeptide or variant thereof and/or wherein the vaccinia virus comprises a mutation, preferably a deletion, in the A45R, B13R and A53R genes and optionally further comprises a mutation, preferably a deletion, in one or more of the N I R, A44L- A46R and A49R genes.

27. The replication competent recombinant oncolytic vaccinia virus according to any one of claims 10-26, wherein the vaccinia virus is a Copenhagen or Western Reserve strain.

28. A pharmaceutical composition comprising the replication competent vaccinia virus according to any one of claims 1 -27 and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition according to claim 28, comprising between about 10⁴- 1 O' ² plaque forming units (pfu), between about 10⁴-10¹¹pfu, between about. 10⁴-10¹⁰pfu, between about 10⁴-10⁹pfu, or between about 10⁴~10⁸ pfo.

30. The pharmaceutical composition according to claim 28 or 29 for use as a medicament.

31 The pharmaceutical composition according to claim 28 or 29 for use in the treatment of cancer.

32. The pharmaceutical composition for use according to claim 31, wherein the pharmaceutical composition is administered by intravascular and/or intratumoral injection.

33. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject one or more doses of a pharmaceutical composition according to claim 28 or 29.

34. The method according to claim 33, wherein the pharmaceutical composition is administered to the subject by intravascular and/or intratumoral administration.

35. The method according to any one of claims 32 to 34, wherein the cancer is selected from brain cancer, head & neck cancer, esophageal cancer, skin cancer, lung cancer (including nonsmall cell lung cancer), thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, renal cancer, testicular cancer, rectal cancer, breast cancer, pancreatic cancer, hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, sarcoma, mesothelioma, melanoma, cervical and liver cancer, preferably wherein the cancer is selected from breast cancer, colorectal cancer (e.g., colorectal carcinoma), bladder cancer, lung cancer (e.g., non-small cell lung cancer), hepatocellular carcinoma, renal cell carcinoma, and osteosarcoma, more preferably wherein the cancer is microsatellite stable (MSS) colorectal cancer.

36. A method for treating and/or preventing a metastasis in a subject in need thereof, comprising administering to the subject one or more doses of a pharmaceutical composition according to claim 28 or 29.

37. The method according to claim 36, wherein the metastasis is a liver or lung metastasis and/or wherein the metastasis comprises metastatic melanoma.

38. The method according to claim 36 or 37, wherein the pharmaceutical composition is administered to the subject by intravascular and/or intratumoral administration.

39. The method according to any one of claims 33-38, wherein the pharmaceutical composition is administered to the subject in combination with one or more immune checkpoint inhibitors.

40. The method according to claim 39, wherein the pharmaceutical composition and the one or more immune checkpoint inhibitors are administered sequentially.

41. The method according to claim 39, wherein the pharmaceutical composition and the one or more immune checkpoint inhibitors are administered simultaneously or concurrently.

42. The method according to any one of claims 39 to 41, wherein the one or more immune checkpoint, inhibitors comprises a PD-1 inhibitor and/or a CTLA4 inhibitor.

43. The method according to any one of claims 33 to 42, wherein the subject is a primate.

44. The method according to claim 43, wherein the primate is a human.

45. The method according to any one of claims 33 to 44, wherein the pharmaceutical composition comprises a Western Reserve or Copenhagen strain vaccinia virus comprising: -a KI 51 E amino acid substitution in the A34R gene,

-a nucleotide sequence encoding a soluble human CD80 polypeptide operably linked to a promoter, preferably an SEI.; promoter, said nucleotide sequence inserted into the M2L gene, thereby rendering the M2L gene non-functional,

-a nucleotide sequence encoding a murine or human IL -21 polypeptide operably linked to a promoter, preferably an Fl 7 promoter, said nucleotide sequence inserted into the J2R gene, thereby rendering the J2R gene non-functional,

-a nucleotide sequence encoding a murine or human IL- 12 polypeptide operably linked to a promoter, preferably an Fl 7 promoter, said nucleotide sequence inserted into the B15R-B17L intergenic region in a Western Reserve strain or is inserted into the B16 gene in a Copenhagen strain, and

-a nucleotide sequence encoding a murine or human IL-7 polypeptide operably linked to a promoter, preferably an SEL promoter, inserted into the B18R gene, thereby rendering the B18R gene non-functional.

46. A nucleic acid comprising a recombinant vaccinia virus genome, said genome derived from the Vaccinia Copenhagen strain genome, wherein said recombinant vaccinia virus genome comprises (i) nonfunctional J2R and M2L vaccinia genes; (ii) a nucleotide sequence encoding a soluble CD-80 protein (iii) a nucleotide sequence encoding an IL-2 gene or variant thereof.

47. The nucleic acid according to claim 46, wherein said recombinant vaccinia virus genome further comprises a heterologous nucleotide sequence encoding a thymidine kinase.

48. The nucleic acid according to claim 46 or 47, wherein the nucleotide sequence encoding a soluble CD-80 protein encodes a fusion protein comprising a soluble human CD-80 fused to the Fc region of immunoglobulin class G (IgG).

49. The nucleic acid according to claim 48, wherein the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:9 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto, preferably wherein the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:9.

50. The nucleic acid according to claim 49, wherein the nucleotide sequence comprises the nucleotide sequence set forth as SEQ ID NO: 17 or a nucleotide sequence at least 90%, at least 95% or at least 98% identical thereto.

51. The nucleic acid according to any one of claims 46-50, wherein the nucleotide sequence encoding an IL-2 gene or variant thereof encodes a human IL-2 variant.

52. The nucleic acid according to claim 51 , wherein the human IL-2 variant comprises the amino acid set forth as SEQ ID NO:38 or 41 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

53. The nucleic acid according to claim 52, wherein the nucleotide sequence encoding the human IL-2 variant comprises the nucleotide sequence of SEQ ID NO:39 or SEQ ID NO:40 or a nucleotide sequence at least 90%, at least 95% or at least 98% identical thereto.

54. The nucleic acid according to any one of claims 46-53, wherein the nucleic acid comprises a deletion of the entire J2R and/or M2L vaccinia genes.

55. The nucleic acid according to any one of claims 46-53, wherein the nucleic acid comprises a deletion of a portion of the J2R and/or M2L vaccinia genes and wherein said deletion renders the J2R and/or M2L nonfunctional upon introduction into a host cell.

56. The nucleic acid according to any one of claims 46-55. wherein the nucleotide sequence encoding the soluble CD80 polypeptide is inserted into the M2L gene region, thereby rendering the M2L gene non-functional.

57. The nucleic acid according to any one of claims 46-56, wherein the nucleotide sequence encoding the soluble CD 80 polypeptide is operably linked to a promoter, preferably a synthetic late promoter (pSEL).

58. The nucleic acid according to any one of claims 46-57, wherein the nucleotide sequence encoding the IL-2 polypeptide is inserted into the J2R gene region, thereby rendering the J2R gene non-functional.

59. The nucleic acid according to any one of claims 46-58, wherein the nucleotide sequence encoding the IL-2 polypeptide is operably linked to a promoter, preferably a synthetic late promoter (pSEL).

60. The nucleic acid according to any one of claims 47-59, wherein the heterologous nucleotide sequence encoding a thymidine kinase encodes a herpes simplex virus thymidine kinase and is inserted into the Bl 6 gene region, thereby rendering the B16 gene nonfunctional, preferably wherein the nucleotide sequence encodes an HSV-TK polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1.

61. The nucleic acid according to any one of claims 47-60, wherein the heterologous nucleotide sequence encoding a thymidine kinase is operably linked to a promoter, preferably a pF 17 late promoter.

62. The nucleic acid according to any one of claims 46-61, wherein said recombinant vaccinia virus genome further comprises one or more mutations in the A34R, A33R and/or B5R viral genes, wherein said one or more mutations confer enhanced viral spreading to a recombinant vaccinia virus comprising the nucleic acid relative to a recombinant vaccinia virus comprising an otherwise identical nucleic acid but lacking the one or more mutations in the A34R, A33R and/or B5R viral genes.

63. The nucleic acid according to claim 62, wherein said recombinant vaccinia virus genome further comprises a K151E amino acid substation in the A34R gene.

64. The nucleic acid according to claim 63, wherein said recombinant vaccinia virus genome further comprises an amino acid substitution at M63 of the A33R gene and/or an amino acid substit ution at M66 of the A34R gene, and/or an amino acid substitution at one or more of N241 , E243. V247, G250 and A276 of the B5R gene.

65. The nucleic acid according to claim 63, wherein the A33R gene comprises an M63R amino acid substitution, the A34R gene comprises an M66T amino acid substitution and the B5R gene comprises N241T, E243V, V247S, G250R and A276F amino acid substitutions.

66. The nucleic acid according to any one of claims 46-65, wherein said recombinant vaccinia virus genome further comprises a heterologous nucleotide sequence encoding IL-21 , preferably human IL-21, optionally wherein said IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical thereto.

67. A recombinant vaccinia virus encoded by the vaccinia virus genome of any one of claims 1- 34.

68. A pharmaceutical composition comprising the recombinant vaccinia virus according to claim 67 and a pharmaceutically acceptable carrier.

69. A method of treating cancer in a mammalian patient in need thereof, said method comprising administering a therapeutically effective amount of the pharmaceutical composition claim 68 to said patient.

70. The method of claim 69, wherein said mammalian patient is a human patient.

71. The method of claim 69 or 70, wherein said cancer is selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer.

72. The method of claim 69 or 70, wherein said cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS- related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ ceil tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, 'fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wihns tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobuiinemia.