Interleukin 2 and interleukin 12 fusion proteins and methods of using

Abstract

Provided herein is a recombinant oncolytic virus comprising an oncolytic virus genome comprising a nucleic acid encoding an IL-2 / IL-12 fusion protein. The fusion protein may be membrane-anchored. Also provided are cells expressing the recombinant oncolytic virus as well as method of using the recombinant oncolytic virus and cells described herein, for example for the treatment of cancer. In a preferred embodiment, the oncolytic virus is a vaccinia virus.

Description

INTERLEUKIN 2 AND INTERLEUKIN 12 FUSION PROTEINS AND METHODS OF USING CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 63 / 557,788, filed February 26, 2024, which is hereby incorporated by reference in its entirety. REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on February 24, 2025, is named 106641_09202_SL.xml and is 183,946 bytes in size. FIELD

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating cancer. BACKGROUND

[0004] Conventional cancer therapies generally target the proliferation, survival, or metabolic activity of tumor cells directly. In contrast, newly emerging cancer immunotherapies seek to restore anticancer immunity by modulating the tumor microenvironment (TME), tipping the equilibrium between factors that stimulate or inhibit anticancer immunity. Modern cancer immunotherapies, including immune checkpoint blockade, adoptive cell transfer, and cancer vaccines are ultimately dependent on immune cells (especially T cells) for their antitumor effects. While dramatic, durable and therapeutic responses are observed after checkpoint inhibitor therapy or chimeric antigen receptor (CAR)-T cell therapy, these successes are still limited to a small percentage of solid tumors, owing to the fact that the majority of solid tumors are characterized by a paucity of intratumoral T cell infiltrate, and defined as non-T cell-inflamed or “cold” tumors.

[0005] As such, new approaches that can improve intratumoral T cell infiltrate and transform “cold” tumors into “hot” or T cell-inflamed tumors are urgently needed to improve the efficacy of cancer immunotherapy. 168734056.1SUMMARY

[0006] Provided herein is a viral vector comprising a viral genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein. In one embodiment, provided is a recombinant oncolytic virus comprising an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a soluble IL-2 / IL-12 fusion protein. In one embodiment, provided is a recombinant oncolytic virus comprising an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a membrane-anchored IL-2 / IL-12 fusion protein. Also provided are cells comprising the viral vector and / or the viral genome as well as method of using the viral vectors and cells described herein, for example for the treatment of cancer. In a preferred embodiment, the viral vector is a recombinant oncolytic virus, even more preferably a recombinant vaccinia virus.

[0007] Provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding: (a) a fusion protein comprising: a first polypeptide comprising an interleukin 2 (IL-2) or an IL- 2 variant and a second polypeptide comprising (i) an interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant, (ii) an interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant, and optionally (iii) a membrane-anchoring polypeptide; (b) a heterodimeric fusion protein comprising a first polypeptide comprising an IL-12p35 or an IL-12p35 variant and a second polypeptide comprising: (i) an IL-2 or an IL-2 variant; (ii) an IL-12p40 or an IL-12p40 variant, and optionally (iii) a membrane-anchoring polypeptide; or (c) a heterodimeric fusion protein comprising first polypeptide comprising an IL-12p40 or an IL-12p40 variant and a second polypeptide comprising: (i) IL-2 or an IL-2 variant; (ii) IL- 12p35 or an IL-12p35 variant, and optionally (iii) a membrane-anchoring polypeptide.

[0008] In one aspect provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding a fusion protein comprising: (a) a first polypeptide comprising interleukin 2 (IL-2) or an IL-2 variant; (b) a second polypeptide comprising (i) interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant and (ii) interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant; and (c) optionally a membrane-anchoring polypeptide.

[0009] In one aspect provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding: (a) IL-12p35 or an IL- 12p35 variant; and (b) a fusion protein comprising: (i) a first polypeptide comprising IL-2 or an IL-2 variant; (ii) a second polypeptide comprising IL-12p40 or an IL-12p40 variant; and 2 168734056.1(iii) optionally a membrane-anchoring polypeptide. In one aspect provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding: (a) IL-12p40 or an IL-12p40 variant; and (b) a fusion protein comprising: (i) a first polypeptide comprising IL-2 or an IL-2 variant; (ii) a second polypeptide comprising IL- 12p35 or an IL-12p35 variant; and (iii) optionally a membrane-anchoring polypeptide. In some embodiments, the IL-12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently linked. In one embodiment, the IL-12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently by a disulfide bond.

[0010] In one embodiment, the nucleic acid encoding the fusion protein is operatively linked to a promoter. In some embodiments, the promoter is a p7.5 or a pSe / l promoter or is derived from a p7.5 or a pSe / l promoter.

[0011] In some embodiments, the fusion protein comprises human or murine IL-2. In some embodiments, the first polypeptide of the fusion protein comprises a sequence that is at least 90% identical to SEQ ID NO:39 or SEQ ID NO:40. In some embodiments, the first polypeptide of the fusion protein comprises SEQ ID NO:39 or SEQ ID NO:40. In one embodiment, the first polypeptide comprises SEQ ID NO:40.

[0012] In some embodiments, the fusion protein comprises human or murine IL-12p35. In some embodiments, the second polypeptide of the fusion protein comprises a sequence that is at least 90% identical to SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, the second polypeptide of the fusion protein comprises SEQ ID NO:45 or SEQ ID NO:46. In one embodiment, the second polypeptide comprises SEQ ID NO:46.

[0013] In some embodiments, the fusion protein comprises human or murine IL-12p40. In some embodiments, the second polypeptide of the fusion protein comprises a sequence that is at least 90% identical to SEQ ID NO:47 or SEQ ID NO:48. In some embodiments, the second polypeptide of the fusion protein comprises SEQ ID NO:47 or SEQ ID NO:48. In one embodiment, the second polypeptide SEQ ID NO:48.

[0014] In some embodiments, the fusion protein comprises a first linker connecting (i) the first and (ii) the second polypeptide. In one embodiment, the fusion protein comprises a second linker connecting (i) the membrane-anchoring polypeptide and (ii) the first or the second polypeptide. In one embodiment, the IL-12p35 or IL-12p35 variant is located N-terminally of the IL-12p40 or IL-12p40 variant. In one embodiment, the IL-12p35 or IL-12p35 variant is located C-terminally of the IL-12p40 or IL-12p40 variant. 3 168734056.1

[0015] Provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding a fusion protein comprising from N-terminus to C- terminus: (a) a polypeptide comprising interleukin 2 (IL-2) or an IL-2 variant; (b) a first linker; (c) a polypeptide comprising (i) interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant and (ii) interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant; (d) a second linker; and (e) optionally a membrane-anchoring polypeptide. Provided is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding a fusion protein comprising from N-terminus to C-terminus: (a) a polypeptide comprising (i) interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant and (ii) interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant; (b) a first linker; (c) a polypeptide comprising interleukin 2 (IL-2) or an IL-2 variant; (d) a second linker; and (e) optionally a membrane-anchoring polypeptide.

[0016] In some embodiments, the IL-12p35 or IL-12p35 variant and the IL-12p40 or IL- 12p40 variant are linked by a third linker.

[0017] In some embodiments, the first linker, the second linker, and / or the third linker is a polypeptide linker. In one embodiment, the first linker, the second linker, and / or the third linker is a flexible polypeptide linker. In some embodiments, the flexible polypeptide linker predominantly comprises glycines and serines. In some embodiments, the flexible polypeptide linker comprises SEQ ID NO:53 (GGGGS) or one or more repeats of SEQ ID NO:53 (GGGGS). In some embodiments, the flexible polypeptide linker comprises a sequence selected from the group consisting of GGS, SEQ ID NO:54 (GGSGGGS), SEQ ID NO:55 ((GGGGS)2), SEQ ID NO:56 ((GGGGS)3), and SEQ ID NO:57 ((GGGGS)4). In one embodiment, the flexible polypeptide linker comprises SEQ ID NO:56 ((GGGGS)3).

[0018] In some embodiments, the first linker and / or the second linker is a rigid polypeptide linker. In some embodiments, the rigid polypeptide linker predominantly comprises alanines. In some embodiments, the rigid polypeptide linker comprises any one of SEQ ID NO:58 (A(EA3K)1AAA), SEQ ID NO:59 (A(EA3K)2AAA), SEQ ID NO:60 (A(EA3K)3AAA), SEQ ID NO:61 (A(EA3K)4AAA), or SEQ ID NO:62 (A(EA3K)5AAA). In some embodiments, the rigid polypeptide linker SEQ ID NO:61 (A(EA3K)4AAA).

[0019] In some embodiments, the fusion protein comprises a membrane-anchoring polypeptide, wherein the membrane-anchoring polypeptide is between about 10 and about 50 amino acids in length. In one embodiment, the membrane-anchoring polypeptide comprises a glycosylphosphatidylinositol (GPI)-anchor acceptor peptide or a variant thereof. In some embodiments, the membrane-anchoring polypeptide comprises a sequence that is at least 90% 4 168734056.1identical to a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104. In some embodiments, the membrane-anchoring polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104. In some embodiments, the membrane- anchoring polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:63. In one embodiment, the membrane-anchoring polypeptide comprises SEQ ID NO:63.

[0020] Provided herein is a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding a fusion protein comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:11-34 In some embodiments, the fusion protein comprises any one of SEQ ID NOs: 11-34.

[0021] In some embodiments, the vaccinia virus genome has a deletion of one or more of the thymidine kinase (TK) gene, the vaccinia growth factor (VGF gene), and / or the A56R gene. In some embodiments, the vaccinia virus genome has a deletion of one or more of the viral genes for A41L, A44L, A46R, A49, A52R, A53R, B5R, B8R, B13R (SPI-2), B15R, B18R, C3L (VCP), C6, C7L, C12L, E3L, F1L, K1L, K3L, K7R, M1L, and N1L or combinations thereof.

[0022] Provided herein is a cell comprising a viral vector, a nucleic acid, or a recombinant vaccinia virus disclosed herein. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. The cell may be a cancer cell. The cell may be a stromal cell in a tumor microenvironment.

[0023] Provided herein is a pharmaceutical composition comprising a viral vector, a nucleic acid, or a recombinant vaccinia virus disclosed herein and one or more pharmaceutically acceptable excipients.

[0024] Provided herein is a method for treating cancer in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, gastric cancer, sarcoma, mesothelioma, ovarian cancer, endometrial cancer, or cervical cancer.

[0025] Provided herein is a method for reducing tumor growth, the method comprising administering to a subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein. 5 168734056.1

[0026] Provided herein is a method for reducing tumor metastasis in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0027] Provided herein is a method for increasing cytokine production in the tumor microenvironment in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0028] Provided herein is a method for increasing anti-tumor immunity in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0029] Provided herein is a method for increasing infiltration of a tumor with immune cells in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0030] Provided herein is a method for reducing T cell tolerance in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0031] Provided herein is a method for enhancing T cell expansion in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0032] Provided herein is a method for increasing the number of memory T cells in a subject in thereof, the method comprising administering to the subject in need thereof a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein.

[0033] In some embodiments, in the methods disclosed herein, the step of administering comprises intraperitoneal administration. In some embodiments, in the methods disclosed herein, the step of administering comprises systemic administration. In some embodiments, the method further comprises administering to the subject an additional antineoplastic agent. In one embodiment, the antineoplastic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group of a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a CTLA4 inhibitor, or combinations thereof. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof. In some 6 168734056.1embodiments, the antineoplastic agent is a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR natural killer cell, or a tumor infiltrating lymphocyte.

[0034] Provided herein is a method of generating tumor-infiltrating vaccinia virus-induced T lymphocytes, the method comprising: (a) administering to a subject having cancer a viral vector or a recombinant vaccinia virus disclosed herein or a pharmaceutical composition disclosed herein; (b) isolating tumor-infiltrating T lymphocytes from the subject; and (c) expanding the tumor-infiltrating T lymphocytes ex vivo. In some embodiments, the method further comprises expanding the tumor-infiltrating T lymphocytes ex vivo in the presence of IL-2, IL-7, GSK3b, or a combination thereof.

[0035] In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. BRIEF DESCRIPTION OF THE FIGURES

[0036] Figs. 1A, 1B, and 1C provide schematic diagrams of illustrative viral vectors. The indicated vectors were generated by homologous recombination of related genes of interest into the tk locus of the vaccinia viral genome of VSC20. TKR = thymidine kinase right arm; TKL = thymidine kinase left arm; YFP = yellow fluorescent protein; p7.5 = vaccinia early and late p7.5 promoter; pSEe / l = synthetic early and late promoter (pSE / L); IRES = Internal Ribosome Entry Sites. “RG” presents a rigid linker fused to a membrane anchor. “FG” presents a flexible linker fused to a membrane anchor. “R” presents a rigid liker. “F” presents a flexible liker. P2A represents a self-cleaving site. “S” presents an IL-12p40 signal peptide. Membrane anchored constructs are shown in Figs. 1A and 1B. Soluble constructs are show in Fig. 1C. See Table 1 for viral vector nomenclature and Tables 2-4 for sequences.

[0037] Fig.2 illustrates viral vector replication and cytokine expression. Tumor cell MC38- luc (3×105cells), CT26-luc (2×105cells) or B16 (2×105cells), were mock-infected or infected with vvDD or vvDD expressing indicated cytokines or fusion proteins, respectively, at an MOI of 1. The cell pellets were harvested to measure A34R, IL-2, or IL-12p40 expression 24 hours after infection using RT-qPCR. Cytokine expression is presented relative to hypoxanthine phosphoribosyl transferase 1 (HPRT1) expression. HPRT1 served as a housekeeping gene. Vaccinia virus outer envelope protein A34 served as a control. For each gene, data points are shown from left to right: PBS, vvDD, vvDD-IL-2-RG, vvDD-scIL-12-FG, vvDD-scIL-12-R- IL-2-FG. See Table 1 for viral vector nomenclature. 7 168734056.1

[0038] Fig. 3 illustrates the in vitro viral cytotoxicity of membrane-anchored cytokines. Tumor cell MC38-luc (1×104cells), CT26-luc (1×104cells), or B16 (8×103cells), respectively, were mock-infected or infected with vvDD or vvDD expressing indicated cytokine(s) at different MOIs. The viable cells were measured 48 hours after infection using Cell Counting Kit-8. See Table 1 for viral vector nomenclature. MC38-luc cells (traces from top to bottom): vvDD, vvDD-IL-2-RG, vvDD-scIL-12-FG, vvDD-scIL-12-R-IL-2-RG. CT26-luc cells (traces from top to bottom): vvDD, vvDD-scIL-12-FG, vvDD-IL-2-RG, vvDD-scIL-12-R-IL-2-RG. B16 cells (traces from top to bottom): vvDD, vvDD-IL-2-RG, vvDD-scIL-12-R-IL-2-RG / vvDD-scIL-12-FG.

[0039] Figs. 4A and 4B illustrate that a vvDD vector expressing a membrane-anchored fusion protein elicits potent therapeutic effects in a murine colon cancer model. Fig. 4A. C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells and treated with PBS, vvDD, or vvDD expressing the indicated cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. Traces from lowest % survival to highest % survival (as determined by endpoint): PBS, vvDD, vvDD-IL-2-RG, vvDD-scIL-12-FG, vvDD-scIL-R-IL-2-RG. Fig. 4B. BALB / c mice were i.p. inoculated with 5×105Renca-luc cells and treated with PBS, vvDD, or vvDD expressing the indicated cytokine at 1×108PFU / mouse five days after tumor inoculation. Traces from lowest % survival to highest % survival (as determined by endpoint): PBS, vvDD, vvDD-scIL-12-FG, vvDD-IL-2-RG, vvDD-scIL12-R-IL-2-RG. Figs.4A and 4B. The Kaplan Meier survival curve is shown. A log-rank (Mantel-Cox) test was used to compare survivalrates. ** P 0.01; and **** P 0.0001. See Table 1 for viral vector nomenclature.

[0040] Fig. 5 illustrates that vaccinia viruses expressing membrane-anchored IL-2 / IL-12 fusion proteins elicit potent therapeutic effects in a murine colon cancer model. C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells and treated with PBS, vvDD, or vvDD expressing indicated cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. Traces from lowest % survival to highest % survival (as determined by endpoint): PBS, vvDD-IL- 12p35-P2A-IL-12p40-R-IL-2-RG, vvDD-IL-12p35-IRES-IL-12p40-R-IL-2-RG, vvDD-IL- 12p35-IRES-IL-12p40-F-IL-2-RG, vvDD-IL-12p35-P2A-IL-12p40-F-IL-2-RG, vvDD-scIL- 12-F-IL-2-RG, vvDD-scIL-12-FG-IRES-IL-2-RG, vvDD-scIL-12-R-IL-2-RG. The Kaplan Meier survival curve is shown. A log-rank (Mantel-Cox) test was used to compare survivalrates. ** P 0.01; *** P 0.001 and **** P 0.0001. ns: not significant. See Table 1 for viralvector nomenclature. 8 168734056.1

[0041] Figs.6A and 6B illustrate that vaccinia viruses expressing membrane-anchored IL- 2 / IL-12 fusion proteins elicit potent, long-term tumor-specific antitumor effects. MC38-luc- intraperitoneal-bearing C57BL / 6 mice were treated with vvDD expressing the indicated cytokines. The mice that survived more than 150 days were s.c. injected with 1×106MC38 cells in the left flanks (Fig.6A) and 6×105B16 cells in the right flanks (Fig.6B), respectively. A two-way ANOVA test was used to compare tumor growth cures. Naïve (PBS treated) group represented by top trace.

[0042] Figs. 7A, 7B, and 7C illustrate the toxicity and therapeutic efficacy of vaccinia viruses expressing different secreted IL-2 / IL-12 fusion proteins. Viral toxicity was assessed by determining mouse body weight loss (increased toxicity resulting in less body weight loss) and survival (longer survival indicating decreased toxicity) in a murine colon cancer model. C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells and treated with vvDD expressing the indicated cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. The body weight variation curves are shown. A log-rank (Mantel-Cox) test was used to compare survival rates after viral treatment. Fig. 7A shows results for vaccinia vectors expressing IL-12; IL-12 fused to IL-2 via a flexible linker; and IL-12 fused to IL-2 via a rigid linker. Genes encoding the different subunits of IL-12 were separated by an IRES site. Lower right figure (traces from highest survival to lowest): vvDD-IL-12p35-IRES-IL-12p40, vvDD- IL-12p35-IRES-IL-12p40-F-IL-2, vvDD-IL-12p35-IRES-IL-12p40-R-IL-2. Fig. 7B shows results for vaccinia vectors expressing single chain IL-12; single chain IL-12 fused to IL-2 with a flexible linker; and single chain IL-12 fused to IL-2 with a rigid linker. Lower right figure (traces from highest survival to lowest): vvDD-scIL-12-F-IL-2, vvDD-scIL-12, vvDD-scIL- 12-R-IL-2. Fig. 7C shows results for vaccinia vectors expressing IL-12; IL-12 fused to IL-2 via a flexible linker; and IL-12 fused to IL-2 via a rigid linker. Genes encoding the different subunits of IL-12 were separated by a P2A site. Lower right figure (traces from highest survival to lowest): vvDD-IL-12p35-P2A-IL12p40-F-IL-2, vvDD-IL12p35-P2A-IL-12p40, vvDD-IL- 12p35-P2A-IL12p40-R-IL-2. Traces from lowest % toxicity to highest % toxicity (as determined by survival after viral treatment) for Figs. 7A, 7B, and 7C overall: vvDD-IL- 12p35-P2A-IL-12p40-F-IL-2, vvDD-scIL-12-F-IL-2, vvDD-IL-12p35-P2A-IL-12p40-R-IL- 2, vvDD-IL-12p35-IRES-IL-12p40-R-IL-2, vvDD-IL-12p35-IRES-IL-12p40-F-IL-2, vvDD- scIL-12-R-IL-2. See Table 1 for viral vector nomenclature.

[0043] Fig. 8 illustrates that vaccinia viruses expressing the indicated secreted IL-2 / IL-12 fusion proteins elicit potent therapeutic effects in a murine colon cancer model. C57BL / 6 mice 9 168734056.1were i.p. inoculated with 5×105MC38-luc cells and treated with PBS, or vvDD expressing the indicated cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. Traces from lowest % survival to highest % survival (as determined by endpoint): PBS, Group 1 (vvDD- scIL-12-R-IL-2, vvDD-IL-12p35-IRES-IL-12p40-F-IL-2, vvDD-IL-12p35-IRES-IL-12p40- R-IL-2), Group 2 (vvDD-IL-12p35-P2A-IL-12p40, vvDD-scIL-12, vvDD-IL-12p35-IRES-IL- 12p40,), Group 3 (vvDD-IL-12p35-P2A-IL-12p40-R-IL-2, vvDD-scIL-12-F-IL-2, vvDD-IL- 12p35-P2A-IL-12p40-F-IL-2). The Kaplan Meier survival curve is shown. A log-rank (Mantel-Cox) test was used to compare survival rates. ** P 0.01; *** P 0.001 and **** P 0.0001. ns:not significant. See Table 1 for viral vector nomenclature.

[0044] Figs.9A and 9B show that a lentiviral vector expressing a membrane-anchored IL- 2 / IL-12 fusion protein promotes human T cell proliferation and viability. Activated T cells (2.5×104) were transduced with lentiviruses expressing various IL-2 / IL-12 proteins, respectively. The dead and alive cells were counted on Day 3 and 5 after transduction. Fig.9A. Absolute alive T cell number. Fig.9B. Cell viability percentage. DETAILED DESCRIPTION

[0045] Provided herein is a viral vector, including, but not limited to, a recombinant oncolytic virus comprising an oncolytic virus genome comprising a nucleic acid encoding a fusion protein comprising: (a) interleukin 2 (IL-2) or an IL-2 variant; (b) interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant, and (iii) interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant. Provided herein is a viral vector, including, but not limited to, a recombinant oncolytic virus comprising an oncolytic virus genome comprising a nucleic acid encoding a fusion protein comprising: (a) IL-2 or an IL-2 variant; (b) an IL-12p35 or an IL-12p35 variant, (c) IL-12p40 or an IL-12p40 variant; and (d) a membrane-anchoring polypeptide. Also provided are cells expressing the viral vector, including, but not limited to, the recombinant oncolytic virus as well as method of using the viral vector, including, but not limited to, the recombinant oncolytic virus and cells described herein, for example, for the treatment of cancer. In a preferred embodiment, the oncolytic virus is a vaccinia virus.

[0046] Cytokines such as interleukins IL-2 and IL-12 play a pivotal role in immunotherapy and have shown efficacy in multiple preclinical animal cancer models. However, these cytokine’s roles in human cancer patients have been largely limited due to toxicity induced by high-dose administration. Indeed, both IL-2 and IL-12 are exceedingly toxic when administered as a monotherapy, let alone as a combination therapy. 10 168734056.1

[0047] In contrast, the oncolytic viruses provided herein allow delivery of genetic information encoding a membrane-anchored IL-2 / IL-12 fusion protein directly to tumor cells or stromal cells in tumors, allowing for the expression of the membrane-anchored IL-2 / IL-12 fusion protein on the surface of the target cell. The presence of IL-2 and IL-12 on the target cells in turn allows for an effective retention of the cytokines within the tumor microenvironment (TME) and the elicitation of potent antitumor effects in tumor models. Similarly, the oncolytic viruses provided herein allow delivery of genetic information encoding a soluble IL-2 / IL-12 fusion protein directly to tumor cells or stromal cells in tumors, allowing for the expression of the membrane-anchored IL-2 / IL-12 fusion protein in the microenvironment of the tumor.

[0048] Moreover, delivery of genes encoding the soluble or membrane-anchored IL-2 / IL- 12 fusion proteins disclosed herein provides additional, powerful benefits. Oncolytic viruses can selectively replicate in and lyse tumor cells and stromal cells in tumors, induce immunogenic cell death with the release of tumor antigens and damage- and pathogen- associated molecular patterns, and upregulate chemokines and cytokines that induce an inflammatory infiltrate in the TME. Thus, oncolytic viruses can cause immune cell-mediated clearance of tumor cells. Disclosed herein is a tumor-selective, oncolytic vaccinia virus (vvDD), generated by inactivating the thymidine kinase and vaccinia growth factor genes from the Western Reserve (WR) strain of vaccinia. This strain demonstrates superior potency against multiple cancer cell lines and is safe in clinical trials as an intratumoral or intravenous injection.

[0049] In sum, the compositions and methods disclosed herein can improve intra-tumoral T cell infiltration, transform the TME from immune-suppressive to immune-favorable, and convert ‘cold’ tumors into ‘hot’ or T cell-inflamed tumors. Importantly, these effects can be achieved without the toxic effects of systemic IL-2 and / or IL-12 exposure. Thus, the compositions and methods disclosed herein provide urgently needed improvements in cancer immunotherapy.

[0050] Fusion proteins

[0051] Cytokines

[0052] IL-2, also referred to as T cell growth factor (TCGF), is small, 15.5-kDa, four α- helical bundle cytokine. This pleiotropic cytokine is produced predominately by antigen- simulated CD4+T cells, but can also be produced by CD8+cells, natural killer (NK) cells, as well as activated dendritic cells (DC). IL-2 plays a critical role in regulating the adaptive immune system by controlling the survival and proliferation of regulatory T cells, which are 11 168734056.1required for the maintenance of immune tolerance. IL-2 plays a critical role in the differentiation of CD4+T cells into T helper-1 (Th1) and T helper-2 (Th2) cells while inhibiting T helper-17 (Th17) differentiation and promotes CD8+T cell and NK cell cytotoxicity activity. While high-dose (HD) IL-2 has shown promising results in treating cancer, a major drawback of IL-2 monotherapy is the severe toxicity of HD IL-2 therapy, which can result in vascular leak syndrome (VLS), pulmonary edema, hypotension, and heart toxicities. See, e.g., Jiang et al., Role of IL-2 in cancer immunotherapy, Oncoimmunology. 2016 Apr 25;5(6):e1163462, incorporated herein by reference in its entirety.

[0053] As used herein, the term “IL-2 variant” refers to a modified IL-2 protein that comprises one or more alterations when compared to the parental protein, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the IL-2 variant retains at least 10% of the immune-activating activity of the parental protein. In embodiments, the “IL-2 variant” retains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the immune-activating activity of the parental protein. The IL-2 variant may have a higher immune-activating activity as compared to the parent protein. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The IL-2 variant may be a portion of the parental protein that comprises an immune-activating portion of the parental protein. In some embodiments, the IL-2 variant comprises one or more mutations as compared to its parental counterpart. In some embodiments, the IL-2 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to its parental counterpart. In some embodiments, the IL-2 variant comprises one or more conservative mutations as compared to its parental counterpart. In some embodiments, the IL-2 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative mutations as compared to its parental counterpart.

[0054] As used herein, the terms “conservative amino acid substitutions” and “conservative modifications” refer to amino acid modifications that do not significantly affect or alter the function and / or activity of the presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0055] Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, 12 168734056.1amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0056] IL-12 is a heterodimeric cytokine encoded by two genes, IL-12A and IL-12B. IL-12 subunit alpha (IL-12A), also referred to as cytotoxic lymphocyte maturation factor 35 kDa subunit (CLMF p35), IL-12 subunit p35 (IL-12p35), or NK cell stimulatory factor chain 1 (NKSF1), consists of a bundle of four alpha helices. IL-12 subunit beta (IL-12B), also referred to as cytotoxic lymphocyte maturation factor 40 kDa subunit (CLMF p40), IL-12 subunit p40 (IL-12p40), or NK cell stimulatory factor chain 2 (NKSF2) has three beta sheet domains. The two subunits of IL-12 are linked by a disulfide bond to form the p70 heterodimer. IL-12 enhances activation as well as the cytotoxic capacities of T cells and NK cells, induces differentiation of TH1 cells, and inhibits immunosuppressive cells including tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). IL-12 also induces the production IFNγ, which in turn is cytostatic / cytotoxic, anti-angiogenic, and can cause upregulation of MHC I and II expression on tumor cells, leading to enhanced recognition and lysis. Although IL-12 has shown antitumor activity in preclinical studies, systemic administration of IL-12 has been shown to be exceedingly toxic.

[0057] As used herein, the term “IL-12p35 variant” refers to a modified IL-12p35 protein that comprises one or more alterations when compared to the parental protein, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the IL-12p35 variant retains at least 10% of the immune-activating activity of the parental protein. In embodiments, the “IL-12p35 variant” retains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the immune-activating activity of the parental protein. The IL-12p35 variant may have a higher immune-activating activity as compared to the parent protein. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The IL- 12p35 variant may be a portion of the parental protein that comprises an immune-activating 13 168734056.1portion of the parental protein. In some embodiments, the IL-12 variant comprises one or more mutations as compared to its parental counterpart. In some embodiments, the IL-12p35 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to its parental counterpart. In some embodiments, the IL-12 variant comprises one or more conservative mutations as compared to its parental counterpart. In some embodiments, the IL-12p35 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative mutations as compared to its parental counterpart.

[0058] As used herein, the term “IL-12p40 variant” refers to a modified IL-12p40 protein that comprises one or more alterations when compared to the parental protein, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the IL-12p40 variant retains at least 10% of the immune-activating activity of the parental protein. In embodiments, the “IL-12p40 variant” retains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the immune-activating activity of the parental protein. The IL-12p40 variant may have a higher immune-activating activity as compared to the parent protein. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The IL- 12p40 variant may be a portion of the parental protein that comprises an immune-activating portion of the parental protein. In some embodiments, the IL-12 variant comprises one or more mutations as compared to its parental counterpart. In some embodiments, the IL-12p40 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to its parental counterpart. In some embodiments, the IL-12 variant comprises one or more conservative mutations as compared to its parental counterpart. In some embodiments, the IL-12p40 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative mutations as compared to its parental counterpart.

[0059] IL-2 / IL-12 fusion proteins

[0060] Provided herein are protein complexes comprising: (a) IL-2 or an IL-2 variant, (b) IL-12p35 or an IL-12p35 variant, (c) IL-12p40 or an IL-12p40 variant, and (d) optionally a membrane-anchoring polypeptide. The term “IL-2 / IL-12 fusion protein” or “IL-12 / IL-12 fusion protein” encompasses a fusion protein in which all fusion protein components are part of a continuous amino acid chain. The term “IL-2 / IL-12 fusion protein” or “IL-12 / IL-12 fusion protein” also encompasses heterodimeric fusion proteins as discussed herein. The IL-12p35 / IL- 12p40 portion of the membrane-anchored IL-2 / IL-12 fusion protein is also referred to herein as an IL-12 portion.

[0061] The IL-2 / IL-12 fusion protein may or may not comprise a membrane-anchor. Fusion proteins comprising a membrane-anchoring polypeptide are also referred to herein as 14 168734056.1“membrane-anchored IL-2 / IL-12 fusion proteins” or “membrane-anchored IL-12 / IL-2 fusion proteins.”

[0062] In some embodiments, the components of IL-2 / IL-12 fusion protein are provided in the form of a single polypeptide chain. In one embodiment, the IL-12p35 subunit (or variant thereof) is located N-terminally of the IL-12p40 subunit (or variant thereof). In one embodiment, the IL-12p35 subunit (or variant thereof) is located C-terminally of the IL-12p40 subunit (or variant thereof). In one embodiment, the two IL-12 subunits are connected by a linker, including, but not limited, to a linker disclosed herein. In one embodiment, the IL-2 (or variant thereof) is located N-terminally of the IL-12p35 subunit (or variant thereof) and / or the IL-12p40 subunit (or variant thereof). In one embodiment, the IL-2 (or variant thereof) is located C-terminally of the IL-12p35 subunit (or variant thereof) and / or the IL-12p40 subunit (or variant thereof).

[0063] In some embodiments, the fusion protein comprises IL-12p35 and IL-12p40, wherein the two subunits are covalently bound. Such a covalent bond may, for example, be a peptide bond or a disulfide bond.

[0064] In some embodiments, the IL-2 / IL-12 fusion protein is a heterodimeric fusion protein in which the two IL-12 subunits are not part of a single polypeptide chain. In some embodiments, the IL-2 / IL-12 fusion protein is a heterodimer comprising: (a) (i) a first polypeptide comprising an IL-2 or an IL-2 variant and an IL-12p35 or an IL- 12p35 variant and (ii) a second polypeptide comprising an IL-12p40 or an IL-12p40 variant; or (b) (i) a first polypeptide comprising an IL-2 or an IL-2 variant and an IL-12p40 or an IL- 12p40 variant and (ii) a second polypeptide comprising an IL-12p35 or an IL-12p35 variant.

[0065] The first and / or the second polypeptide may be linked to a membrane-anchoring polypeptide.

[0066] In some embodiments, the two IL-12 subunits are expressed separately, for example, by including an IRES site or a sequence encoding a self-cleaving peptide (including, but not limited to, P2A) between the genes encoding each subunit. An IRES sequence may be used to produce more than one polypeptide from a single gene transcript. A P2A sequence can induce ribosomal skipping during translation of a protein in a cell. As such, a P2A sequence may be used to produce more than one polypeptide from a single gene transcript. Self-cleaving sequences are known in the art. See, e.g., Liu et al., Systematic comparison of 2A peptides for 15 168734056.1cloning multi-genes in a polycistronic vector, Sci Rep. 2017 May 19;7(1):2193, incorporated herein by reference in its entirety. In one embodiment, provided is a single polypeptide chain comprising IL-2 and IL-12p35, wherein the IL-12p35 subunit is covalent linked to a (separately expressed) IL-12p40 subunit. In one embodiment, provided is a single polypeptide chain comprising IL-2 and IL-12p40, wherein the IL-12p40 subunit is covalent linked to a (separately expressed) IL-12p35 subunit. In one embodiment, provided is a single polypeptide chain comprising IL-2, IL-12p35, and a membrane-anchoring polypeptide, wherein the IL-12p35 subunit is covalent linked to a (separately expressed) IL-12p40 subunit.

[0067] In one embodiment, provided is a single polypeptide chain comprising IL-2, IL- 12p40, and a membrane-anchoring polypeptide, wherein the IL-12p40 subunit is covalent linked to a (separately expressed) IL-12p35 subunit.

[0068] In some embodiments, the IL-2 is a mammalian or a primate IL-2. In some embodiments, the IL-2 is a human, mouse, rat, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat IL-2. In some embodiments, the IL-2 is human IL-2. In some embodiments, the IL-2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 In one embodiment, the IL-2 comprises sequence SEQ ID NO:39. In some embodiments, the IL-2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:40. In one embodiment, the IL-2 comprises sequence SEQ ID NO:40.

[0069] In some embodiments, the IL-12p35 is a mammalian or a primate IL-12p35. In some embodiments, the IL-12p35 is a human, mouse, rat, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat IL-12p35. In some embodiments, the IL-12p35 is human IL-12p35. In some embodiments, the IL-12p35 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45. In one embodiment, the IL-12p35 comprises sequence SEQ ID NO:45. In some embodiments, the IL-12p35 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46. In one embodiment, the IL-12p35 comprises sequence SEQ ID NO:46.

[0070] In some embodiments, the IL-12p40 is a mammalian or a primate IL-12p40. In some embodiments, the IL-12p40 is a human, mouse, rat, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat IL-12p40. In some embodiments, the IL-12p40 is human IL-12p40. In some embodiments, the IL-12p40 comprises a sequence that is at least 80%, at least 85%, at 16 168734056.1least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47. In one embodiment, the IL-12p40 comprises sequence SEQ ID NO:47. In some embodiments, the IL-12p40 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48. In one embodiment, the IL-12p40 comprises sequence SEQ ID NO:48.

[0071] In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47. In one embodiment, the fusion protein comprises an IL-12 comprising: (a) SEQ ID NO:45 and (b) SEQ ID NO:47. In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48. In one embodiment, the fusion protein comprises an IL-12 comprising: (a) SEQ ID NO:46 and (b) SEQ ID NO:48.

[0072] In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45; (b) a linker; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47. In one embodiment, the fusion protein comprises an IL- 12 comprising: (a) SEQ ID NO:45; (b) a linker; and (c) SEQ ID NO:47. In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46; (b) a linker; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48. In one embodiment, the fusion protein comprises an IL-12 comprising: (a) SEQ ID NO:46; (b) a linker; and (c) SEQ ID NO:48. The linker may be a flexible linker, including a flexible linker disclosed herein.

[0073] In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45; (b) a sequence comprising any one of SEQ 17 168734056.1ID NOs:53-57; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47. In one embodiment, the fusion protein comprises an IL-12 comprising: (a) SEQ ID NO:45; (b) a sequence comprising any one of SEQ ID NOs: 53-57; and (c) SEQ ID NO:47. In embodiments, the fusion protein comprises an IL-12 comprising: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46; (b) a sequence comprising any one of SEQ ID NOs:41-45; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48. In one embodiment, the fusion protein comprises an IL-12 comprising: (a) SEQ ID NO:46; (b) a sequence comprising any one of SEQ ID NOs: 53-57; and (c) SEQ ID NO:48.

[0074] In embodiments, the fusion protein comprises an IL-12 comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:85. In one embodiment, the fusion protein comprises an IL-12 comprising SEQ ID NO:85. In embodiments, the fusion protein comprises an IL-12 comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:86. In one embodiment, the fusion protein comprises an IL-12 comprising SEQ ID NO:86.

[0075] For the avoidance of doubt, if multiple components of a fusion protein are provided, the order of the individual components is not limited by the specific order of the components provided in the list of components (unless, for example, it is stated explicitly that a fusion protein comprises several components “from N-terminus to C-terminus” or that a polypeptide is “located C-terminally” of another polypeptide).

[0076] Provided is an IL-2 / IL-12 fusion protein, in which the IL-2 protein or IL-2 variant is located N-terminally of the IL-12 protein or IL-12 variant. Provided is an IL-2 / IL-12 fusion protein, in which the IL-2 protein or IL-2 variant is located C-terminally of the IL-12 protein or IL-12 variant. Provided is an IL-2 / IL-12 fusion protein, in which the IL-12p35 subunit or IL-12p35 subunit variant is located N-terminally of the IL-12p40 subunit or IL-12p40 subunit variant. Provided is an IL-2 / IL-12 fusion protein, in which the IL-12p35 subunit or IL-12p35 subunit variant is located C-terminally of the IL-12p40 subunit or IL-12p40 subunit variant.

[0077] Provided are IL-2 / IL-12 fusion proteins comprising one or more IL-2 proteins or IL- 2 variants and / or one or more IL-12 proteins or IL-12 variants.

[0078] In some embodiments, the fusion protein comprises: 18 168734056.1(a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47; and (d) optionally a membrane-anchoring polypeptide.

[0079] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:39; (b) SEQ ID NO:45; (c) SEQ ID NO:47; and (d) optionally a membrane-anchoring polypeptide.

[0080] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:40; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48; and (d) optionally a membrane-anchoring polypeptide.

[0081] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:40; (b) SEQ ID NO:46; (c) SEQ ID NO:48; and (d) optionally a membrane-anchoring polypeptide.

[0082] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; and (d) optionally a membrane-anchoring polypeptide.

[0083] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; 19 168734056.1(c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; and (d) optionally a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:63 or 97-104.

[0084] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:39 or SEQ ID NO:40; (b) SEQ ID NO:45 or SEQ ID NO:34; (c) SEQ ID NO:47 or SEQ ID NO:48; and (d) optionally a membrane-anchoring polypeptide.

[0085] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:39 or SEQ ID NO:40; (b) SEQ ID NO:45 or SEQ ID NO:46; (c) SEQ ID NO:47 or SEQ ID NO:48; and (d) optionally any one of SEQ ID NOs:63 or 97-104.

[0086] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (f) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (g) optionally a membrane-anchoring polypeptide.

[0087] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) any one of SEQ ID NOs:53-62; (c) SEQ ID NO:47 or SEQ ID NO:48; (d) any one of SEQ ID NOs:53-62; (e) SEQ ID NO:39 or SEQ ID NO:40; (f) any one of SEQ ID NOs:53-62; and 20 168734056.1(g) optionally a membrane-anchoring polypeptide.

[0088] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (f) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; and (g) optionally a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:63 or 97-104.

[0089] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) any one of SEQ ID NOs:53-62; (c) SEQ ID NO:47 or SEQ ID NO:48; (d) any one of SEQ ID NOs:53-62; (e) SEQ ID NO:39 or SEQ ID NO:40; (f) any one of SEQ ID NOs:53-62; and (g) optionally any one of SEQ ID NOs:63 or 97-104.

[0090] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-57; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; 21 168734056.1(d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-62; (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (f) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:58-62; and (g) optionally a membrane-anchoring polypeptide.

[0091] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) any one of SEQ ID NOs:53-57; (c) SEQ ID NO:47 or SEQ ID NO:48; (d) any one of SEQ ID NOs:53-62; (e) SEQ ID NO:39 or SEQ ID NO:40; (f) any one of SEQ ID NOs:58-62; and (g) optionally a membrane-anchoring polypeptide.

[0092] In some embodiments, the fusion protein comprises: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56 or SEQ ID NO:61; (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (f) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61; and (g) optionally a membrane-anchoring polypeptide.

[0093] In some embodiments, the fusion protein comprises: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) SEQ ID NO:56; (c) SEQ ID NO:47 or SEQ ID NO:48; 22 168734056.1(d) SEQ ID NO:56 or SEQ ID NO:61; (e) SEQ ID NO:39 or SEQ ID NO:40; (f) SEQ ID NO:61; and (g) optionally a membrane-anchoring polypeptide.

[0094] In some embodiments, the fusion protein comprises from N- to C-terminus: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56 or SEQ ID NO:61; (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (f) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61; and (g) optionally a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:63 or 97-104.

[0095] In some embodiments, the fusion protein comprises from N- to C-terminus: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) SEQ ID NO:56; (c) SEQ ID NO:47 or SEQ ID NO:48; (d) SEQ ID NO:56 or SEQ ID NO:61; (e) SEQ ID NO:39 or SEQ ID NO:40; (f) SEQ ID NO:61; and (g) optionally any one of SEQ ID NOs:63 or 97-104.

[0096] Provided herein is a fusion protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:64 or SEQ ID NO:65. Provided herein is a fusion protein comprising SEQ ID NO:64 or SEQ ID NO:65. 23 168734056.1

[0097] Provided herein is a heterodimeric fusion protein comprising (1) an IL-12p35 covalently linked to (2) a fusion protein comprising (a) IL-12p40; (b) a linker; (c) IL-2; (d) a linker; and (e) optionally a membrane-anchoring polypeptide.

[0098] Provided herein is a heterodimeric fusion protein comprising (1) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46, covalently linked to (2) a fusion protein comprising (a) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (b) a linker; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (d) a linker; and (e) optionally a membrane-anchoring polypeptide. Provided herein is a heterodimeric fusion protein comprising (1) SEQ ID NO:45 or SEQ ID NO:46, covalently linked to (2) a fusion protein comprising (a) SEQ ID NO:47 or SEQ ID NO:48; (b) a linker; (c) SEQ ID NO:39 or SEQ ID NO:40; (d) a linker; and (e) optionally a membrane-anchoring polypeptide.

[0099] Provided herein is a heterodimeric fusion protein comprising (1) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46, covalently linked to (2) a fusion protein comprising (a) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (b) a flexible linker; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (d) a rigid linker; and (e) optionally a membrane-anchoring polypeptide. Provided herein is a heterodimeric fusion protein comprising (1) SEQ ID NO:45 or SEQ ID NO:46, covalently linked to (2) a fusion protein comprising (a) SEQ ID NO:47 or SEQ ID NO:48; (b) a flexible linker; (c) SEQ ID NO:39 or SEQ ID NO:40; (d) a rigid linker; and (e) optionally a membrane-anchoring polypeptide.

[0100] Provided herein is a heterodimeric fusion protein comprising (1) an IL-12p40 covalently linked to (2) a fusion protein comprising (a) IL-12p35; (b) a linker; (c) IL-2; (d) a linker and (e) optionally a membrane-anchoring polypeptide.

[0101] Provided herein is a heterodimeric fusion protein comprising (1) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48, covalently linked to (2) a fusion 24 168734056.1protein comprising (a) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a linker; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (d) a linker; and (e) optionally a membrane-anchoring polypeptide. Provided herein is a heterodimeric fusion protein comprising (1) SEQ ID NO:47 or SEQ ID NO:48, covalently linked to (2) a fusion protein comprising (a) SEQ ID NO:45 or SEQ ID NO:46; (b) a linker; (c) SEQ ID NO:39 or SEQ ID NO:40; (d) a linker; and (e) optionally a membrane-anchoring polypeptide.

[0102] Provided herein is a heterodimeric fusion protein comprising (1) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48, covalently linked to (2) a fusion protein comprising (a) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a flexible linker; (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (d) a rigid linker; and (e) optionally a membrane-anchoring polypeptide. Provided herein is a heterodimeric fusion protein comprising (1) SEQ ID NO:47 or SEQ ID NO:48, covalently linked to (2) a fusion protein comprising (a) SEQ ID NO:45 or SEQ ID NO:46; (b) a flexible linker; (c) SEQ ID NO:39 or SEQ ID NO:40; (d) a rigid linker; and (e) optionally a membrane-anchoring polypeptide.

[0103] Provided herein is a fusion protein comprising (1) IL-12p35, (2) a first flexible linker, (3) IL-12p40, (4) a second flexible linker, and (5) IL-2. Provided herein is a fusion protein comprising from N-to C-terminus: (1) IL-12p40, (2) a first flexible linker, (3) IL-12p35, (4) a second flexible linker, and (5) IL-2. Provided herein is a fusion protein comprising from N-to C-terminus: (1) IL-12p35, (2) a first flexible linker, (3) IL-12p40, (4) a second flexible linker, and (5) IL-2.

[0104] In some embodiments, the fusion protein comprises (optionally in this order from N- to C-terminus): (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-57; 25 168734056.1(c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-57; and (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40.

[0105] In some embodiments, the fusion protein comprises (optionally in this order from N- to C-terminus): (a) SEQ ID NO:45 or SEQ ID NO:46; (b) any one of SEQ ID NOs:53-57; (c) SEQ ID NO:47 or SEQ ID NO:48; (d) any one of SEQ ID NOs:53-57; and (e) SEQ ID NO:39 or SEQ ID NO:40.

[0106] In some embodiments, the fusion protein comprises (optionally in this order from N- to C-terminus): (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56; (c) sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48; (d) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56; and (e) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:40.

[0107] In some embodiments, the fusion protein comprises (optionally in this order from N- to C-terminus): (a) SEQ ID NO:46; (b) SEQ ID NO:56; (c) SEQ ID NO:48; (d) SEQ ID NO:56; and (e) SEQ ID NO:40. 26 168734056.1

[0108] Provided herein is a fusion protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Tables 3 or 4.

[0109] Soluble fusion proteins

[0110] In some embodiments, the IL-2 / IL-12 fusion protein is secreted by a cell. In someprotein is soluble.

[0111] Membrane-anchoring polypeptides

[0112] In embodiments, provided herein are fusion proteins comprising: (a) IL-2 or an IL- 2 variant; (b) an IL-12p35 or an IL-12p35 variant, (c) an IL-12p40 or an IL-12p40 variant; and (d) a membrane-anchoring polypeptide.

[0113] As used herein, a membrane-anchoring polypeptide is a polypeptide that anchors the IL-2 / IL-12 fusion protein to a cell membrane. A membrane-anchoring polypeptide is also referred to as a membrane anchor. In some embodiments, the membrane-anchoring polypeptide comprises one or more transmembrane domains. In some embodiments, the membrane- anchoring polypeptide can comprise sequences derived from one or more naturally occurring and / or from one or more artificial proteins, including transmembrane proteins. In some embodiments, membrane-anchoring polypeptide comprises a lipid anchor. As a non-limiting example, a glycosylphosphatidylinositol (GPI) is a lipid anchor that can be used to anchor a polypeptide, including an IL-2 / IL-12 fusion protein, to a cell surface. A GPI anchor is a posttranslational modification of a polypeptide with a glycolipid that allows embedding of the glycolipid in the cell membrane (and hence anchoring of the polypeptide to the cell membrane). As illustrated by GPI, the anchoring function of the membrane-anchoring polypeptide can be achieved by a modification of an underlying peptide or protein that contains non-peptide elements, including but not limited to carbohydrate, lipid, etc.

[0114] In some embodiments, the membrane-anchoring polypeptide is between about 10 and about 50 amino acids, or between about 15 and about 30, or about 20 amino acids in length. In some embodiments, the membrane-anchoring polypeptide is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acids in length. 27 168734056.1

[0115] In some embodiments, the membrane-anchoring polypeptide is a GPI-anchor peptide. In some embodiments, the anchoring peptide comprises a GPI-anchor sequence of the human CD16b anchor peptide. In some embodiments, the anchor is a GPI anchor peptide from a protein as set forth in Ferguson et al., “Chapter 11: Glycosylphosphatidyl Anchors” in Glycobiology, 2nd Edition, Varki et al., editors, Cold Spring Harbor Press, 2009, the contents of which is incorporated by reference in its entirety herein. In some embodiments, the anchor is a glycosylphosphatidylinositol (GPI)-anchor derived from alkaline phosphatase, CD58, CD14, NCAM-120 and TAG-1. In some embodiments, the membrane-anchoring polypeptide comprises a sequence that is at least is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63. In one embodiment, the membrane-anchoring polypeptide comprises SEQ ID NO:63. In some embodiments, the IL-2 / IL-12 fusion protein is linked to the membrane-anchoring polypeptide via a rigid linker, including, but not limited to, a rigid linker disclosed herein. In some embodiments, the Il-2 / IL-12 fusion protein is linked to the membrane-anchoring polypeptide via a flexible linker, including, but not limited to, a flexible linker disclosed herein.

[0116] In some embodiments, the GPI anchor peptide comprises a signal peptide portion (“SPP”), which functions during GPI addition and is cleaved in the process. In certain embodiments, a SPP comprises three domains: (1) a first domain comprising three relatively small amino acids (for example, but not limited to, Gly (G), Ala (A), Ser (S), Asn (N), Asp (D), or Cys (C) or any combination thereof), ω, (ω+1), and (ω+2), where ω is attached to the GPI anchor and (ω+1) and (ω+2) are the first 2 residues of the cleaved peptide; (2) a relatively polar domain spacer of about 5-10 amino acid residues and (3) a hydrophobic domain of about 15-20 amino acids. This signal peptide portion, extending from the protein C terminus, can be diagrammed as: ω−(ω+1)−(ω+2)−polar spacer region−hydrophobic domain. See, e.g., Galian e tal., Efficient(GPI) modification of membrane proteins requires a C-terminal anchoring signal of marginal hydrophobicity, J Biol Chem. 2012 May 11;287(20):16399-409. In certain non-limiting embodiments, the ω−(ω+1)−(ω+2)−polar spacer region can include a sequence of about ten amino acids of which at least about 2 or at least about 3 residues are G and at least about 2 or at least about 3 or at least about 4 or at least about 5 residues are S. In some embodiments, the hydrophobic domain can include between about 15 and about 20 amino acids comprising at least about 10 residues selected from the group of A, Leu (L), Val (V), Phe (F) and combinations thereof. 28 168734056.1

[0117] In some embodiments, the membrane-anchoring polypeptide comprises at least about 10, at least about 15, or at least about 20 amino acids of any one of SEQ ID NOs:63 or 97-104. In some embodiments, the membrane-anchoring polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:63 or 97-104. In some embodiments, the membrane-anchoring polypeptide comprises any one of SEQ ID NOs:63 or 97-104. In some embodiments, the membrane-anchoring polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63. In one embodiment, the membrane-anchoring polypeptide comprises SEQ ID NO:63. In some embodiments, the membrane-anchoring polypeptide comprises a sequence that contains about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, insertions or deletions, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 1conservative amino acid substitutions as compared to a membrane-anchoring polypeptide sequence disclosed herein.

[0118] Linkers

[0119] In some embodiments, the IL-2 protein or IL-2 variant is directly fused to the IL-12 protein or IL-12 variant. In some embodiments, the IL-2 protein or IL-2 variant is covalently linked to the IL- IL-12 protein or IL-12 variant through a linker. In some embodiments, the IL- 12p39 subunit or IL-12p39 subunit variant is directly fused to the IL-12p40 subunit or IL- 12p40 subunit. In some embodiments, the IL-12p39 subunit or IL-12p39 subunit variant is covalently linked to the IL-12p40 subunit or IL-12p40 subunit through a linker. In some embodiments, the IL-2 protein or IL-2 variant is directly fused to the membrane-anchoring polypeptide. In some embodiments, the IL-2 protein or IL-2 variant is covalently linked to membrane-anchoring polypeptide through a linker. In some embodiments, the IL-12p39 subunit or IL-12p39 variant is directly fused to the membrane-anchoring polypeptide. In some embodiments, the IL-12p39 subunit or IL-12p39 variant is covalently linked to membrane- anchoring polypeptide through a linker. In some embodiments, the IL-12p40 subunit or IL- 12p40 variant is directly fused to the membrane-anchoring polypeptide. In some embodiments, the IL-12p40 subunit or IL-12p40 variant is covalently linked to membrane-anchoring polypeptide through a linker.

[0120] In one embodiment, the linker is a flexible linker. In one embodiment, the linker is a rigid linker. In one embodiment, the linker is a cleavable linker. In one embodiment, the 29 168734056.1linker is a non-cleavable linker. In one embodiment, the linker is a helical linker. In one embodiment, the linker is a non-helical linker. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker is between 3 and 30 amino acids long. In certain embodiments, the peptide linker can, for example and not by way of limitation, be between about 1 and about 25, between about 5 and about 20, between about 5 and about 15, or between about 5 and about 10 amino acids in length. Non-limiting examples of linkers for use in the presently disclosure subject matter are disclosed in International Patent Publication WO 2017 / 165464 (e.g., SEQ ID NOs:42, 44, 45, 75, 76, 77 and 78), the contents of which are incorporated by reference herein in its entirety.

[0121] In some embodiments, the flexible linker predominantly comprises glycines and serines. In some embodiments, the flexible linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the following sequences: GGS, SEQ ID NO:53 (GGGGS), SEQ ID NO:54 (GGSGGGS), SEQ ID NO:55 (GGGGSGGGGS), SEQ ID NO:56 (GGGGSGGGGSGGGGS), and SEQ ID NO:57 (GGGGSGGGGSGGGGSGGGGS). In embodiments, the linker comprises GGS, SEQ ID NO:53 (GGGGS), SEQ ID NO:54 (GGSGGGS), SEQ ID NO:55 (GGGGSGGGGS), SEQ ID NO:56 (GGGGSGGGGSGGGGS), or SEQ ID NO:57 (GGGGSGGGGSGGGGSGGGGS). In embodiments, the flexible linker comprises repeats of one or more flexible linkers disclosed herein.

[0122] In some embodiments, the rigid linker predominantly comprises alanines. In some embodiments, the rigid linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the following sequences: SEQ ID NO:58 (A(EA3K)1AAA), SEQ ID NO:59 (A(EA3K)2AAA), SEQ ID NO:60 (A(EA3K)3AAA), SEQ ID NO:61 (A(EA3K)4AAA), or SEQ ID NO:62 (A(EA3K)5AAA). In some embodiments, the rigid linker comprises SEQ ID NO:58 (A(EA3K)1AAA), SEQ ID NO:59 (A(EA3K)2AAA), SEQ ID NO:60 (A(EA3K)3AAA), SEQ ID NO:61 (A(EA3K)4AAA), or SEQ ID NO:62 (A(EA3K)5AAA). In embodiments, the rigid linker comprises repeats one or more rigid linkers disclosed herein.

[0123] In embodiments, provided is a linker that comprises flexible and rigid portions.

[0124] Signal sequences

[0125] In some embodiments, the IL-2 protein (or variant) or the IL-12 protein (or variant) is fused to a signal peptide. The terms “signal peptide” and “signal sequence” are used interchangeably herein. In some embodiments, the IL-12p35 protein (or variant) or the IL- 30 168734056.112p40 protein (or variant) is fused to a signal peptide. In some embodiments, the signal sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs:49-52. In some embodiments, the signal sequence comprises a sequence selected from any one of SEQ ID NOs: 49-52. Sequences of suitable signal peptides are known in the art. A person skilled in the art may employ any signal sequence compatible with the compositions and methods disclosed herein. See, e.g., Owji et al., A comprehensive review of signal peptides: Structure, roles, and applications, Eur J Cell Biol. 2018 Aug;97(6):422-441, which is incorporated herein by reference in its entirety.

[0126] Provided herein is a fusion protein comprising sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs:41-44. Provided herein is a fusion protein comprising any one of SEQ ID NOs:41-44.

[0127] Fusions with functional moieties

[0128] In some embodiments, the IL-2 / IL-12 fusion protein, which may or may not be membrane-anchored, is conjugated to a functional moiety, including, but not limited to, a diagnostic moiety, a detectable moiety, a therapeutic moiety, or a moiety useful for purification.

[0129] Examples of moieties useful for purification include, but are not limited to, Albumin-binding protein (ABP), Alkaline Phosphatase (AP), AU1 epitope, AU5 epitope, Bacteriophage T7 epitope (T7-tag), Bacteriophage V5 epitope (V5-tag), Biotin-carboxy carrier protein (BCCP), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), Chloramphenicol Acetyl Transferase (CAT), Cellulose binding domain (CBP), Chitin binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase (DHFR), E2 epitope, FLAG epitope, Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glutathione S-transferase (GST), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag (HAT), Horseradish Peroxidase (HRP), HSV epitope, Ketosteroid isomerase (KSI), KT3 epitope, LacZ, Luciferase, Maltose-binding protein (MBP), Myc epitope, NusA, PDZ domain, PDZ ligand, Polyarginine (Arg-tag), Polyaspartate (Asp-tag), Polycysteine (Cys-tag), Polyhistidine (His-tag), Polyphenylalanine (Phe-tag), Profinity eXact, Protein C, S1-tag, S-tag, Streptavadin-binding peptide (SBP), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep-tag, Streptavadin, Small Ubiquitin- like Modifier (SUMO), Tandem Affinity Purification (TAP), T7 epitope, Thioredoxin (Trx), TrpE, Ubiquitin, Universal, and VSV-G. 31 168734056.1

[0130] Examples of detectable moieties include, but are not limited to, fluorescent moieties or labels, imaging agents, radioisotopic moieties, radiopaque moieties, and the like, e.g., detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Non-limiting examples of fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, and the like) and other luminescent molecules (e.g., luminal). A fluorophore may be environmentally-sensitive such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g., dansyl probes). Non-limiting examples of radiolabels include small molecules containing atoms with one or more low sensitivity nuclei (13C,15N,2H,125I,123I,99Tc,43K,52Fe,67Ga,68Ga,111In and the like). Other useful moieties are known in the art.

[0131] Examples of diagnostic moieties include, but are not limited to, detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for determining the presence, absence, or level of a molecule, for example, a target peptide, protein, or proteins, that is associated with a disease or disorder. Such diagnostics are also suitable for prognosing and / or diagnosing a disease or disorder and its progression.

[0132] Examples of therapeutic moieties include, but are not limited to, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents, or generally a therapeutic moiety. The functional moiety may also have one or more of the herein-mentioned functions.

[0133] Non-limiting examples of therapeutic moieties include radionuclides with high- energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer cell). Non-limiting examples of high-energy radionuclides include:90Y,125I,131I,123I,111In,105Rh,153Sm,67Cu,67Ga,166Ho,177Lu,186Re and188Re. These isotopes typically produce high-energy α- or β-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0134] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as cytostatics (e.g., alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenesis agents, and the like. 32 168734056.1

[0135] Non-limiting examples of therapeutic moieties also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Non-limiting examples of DNA- intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0136] Non-limiting examples of therapeutic moieties also include transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other non-limiting examples of cytostatic agents that are compatible with the embodiments disclosed herein include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0137] Non-limiting examples of therapeutic moieties also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; tubulin binding agents such as taxoids (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g., Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatins (e.g., Combretastatin A- 4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenesis compounds such as Angiostatin K1-3, DL-α-difluoromethyl- ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0138] Non-limiting examples of therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens, (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminogluthetimide), 17-(allylamino)- 17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, 33 168734056.1dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0139] Non-limiting examples of therapeutic moieties also include enzyme inhibitors such as, S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D- ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

[0140] Non-limiting examples of therapeutic moieties also include gene regulators such as 5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0141] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.

[0142] Still other cytotoxins that are compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0143] A moiety may have more than one function.

[0144] Nucleic acids, genomes, and vectors

[0145] Also provided herein are nucleic acids, genomes, and vectors comprising nucleic acids encoding the IL-2 / IL-12 fusion proteins, which may or may not be membrane-anchored, disclosed herein. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0146] Provided herein are nucleic acids comprising (i) a promoter and (ii) a transgene encoding an IL-2 / IL-12 fusion protein disclosed herein (or a portion thereof), wherein the transgene is operably linked to the promoter. 34 168734056.1

[0147] Provided herein is a nucleic acid comprising (a) a promoter and (b) a sequence encoding a fusion protein comprising: (i) IL-2 or an IL-2 variant, (ii) IL-12p35 or an IL-12p35 variant, (iii) IL-12p40 or an IL-12p40 variant, and (iv) optionally a membrane-anchoring polypeptide, wherein the sequence encoding a fusion protein is operatively linked to the promoter.

[0148] In the case of heterodimeric fusion proteins, the two subunits may be expressed from two separate nucleic acids. Alternatively, the two subunits may be expressed may be expressed from the same nucleic acid. For example, the nucleic acid sequences encoding for the two subunits of a heterodimeric fusion protein may be located on the same nucleic acid molecule, but may be separated by an including a IRES site or a sequence encoding a self-cleaving peptide (including, but not limited to, P2A).

[0149] Provided herein is a nucleic acid or a set of nucleic acid comprising (a) a first nucleic acid encoding a first polypeptide comprising an IL-2 or an IL-2 variant and an IL-12p35 or an IL-12p35 variant, wherein the first nucleic acid is operatively linked to a promoter and (b) a second nucleic acid encoding a second polypeptide comprising an IL-12p40 or an IL-12p40 variant, wherein the second nucleic acid is operatively linked to a promoter.

[0150] Provided herein is a nucleic acid or a set of nucleic acid comprising (a) a first nucleic acid encoding a first polypeptide comprising an IL-2 or an IL-2 variant and an IL-12p40 or an IL-12p40 variant, wherein the first nucleic acid is operatively linked to a promoter and (b) a second nucleic acid encoding a second polypeptide comprising an IL-12p35 or an IL-12p35 variant, wherein the second nucleic acid is operatively linked to a promoter.

[0151] As used herein, “operably linked” refers to both expression control sequences that are contiguous with the transgene and expression control sequences that act in trans or at a distance to control the expression of the transgene. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion.

[0152] In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a vaccinia virus promoter. Non-limiting examples of vaccinia virus promoters include p7.5 (E / L), psyn (L), pSe (L) (also referred to as Pse / l), or pH5 (E / L) (E / L = early and late promoter; L = late promoter). In one embodiment, the transgene encoding the IL-2 / IL-12 35 168734056.1fusion protein is operably linked to a p7.5 promoter. In one embodiment, the transgene encoding the IL-2 / IL-12 fusion protein is operably linked to a Pse / l promoter. In some embodiments, the nucleic acid comprises (i) a promoter, (ii) a transgene encoding an IL-2 / IL- 12 fusion protein (or a portion thereof) disclosed herein, wherein the transgene is operatively linked to the promoter, and two tk gene flanking regions.

[0153] Provided herein is an oncolytic virus genome having a deletion of or an inactivating mutation in one or more of the following non-limiting examples of genes: ribonucleotide reductase-large subunit, ribonucleotide reductase-small subunit, DNA ligase, dUTPase, tk, vgf, or A56R gene. Provided herein is an oncolytic virus genome having a deletion of or an inactivating mutation in one or more of the viral genes for A41L, A44L, A46R, A49, A52R, A53R, B5R, B8R, B13R (SPI-2), B15R, B18R, C3L (VCP), C6, C7L, C12L, E3L, F1L, K1L, K3L, K7R, M1L, and N1L. In some embodiments, the oncolytic virus genome is a vaccinia virus genome. Provided herein is an oncolytic virus genome comprising (i) a promoter and (ii) a transgene encoding an IL-2 / IL-12 fusion protein (or a portion thereof) disclosed herein, wherein the transgene is operably linked to the promoter. Provided herein is an oncolytic virus genome comprising (i) a promoter, (ii) a transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof) disclosed herein, wherein the transgene is operatively linked to the promoter, and two tk gene flanking regions. In one embodiment, the transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof) is operably linked to a p7.5 promoter. In one embodiment, the transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof) is operably linked to a Pse / l promoter.

[0154] Provided herein is a vector comprising a nucleic acid comprising (i) a promoter and (ii) a transgene encoding an IL-2 / IL-12 fusion protein (or a portion thereof) disclosed herein, wherein the transgene is operably linked to the promoter. In one embodiment, the transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof) is operably linked to a p7.5 promoter. In one embodiment, the transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof) is operably linked to a Pse / l promoter.

[0155] “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini- circle, or a virus (including virus-derived sequences). A recombinant vaccinia virus is a vector as used herein. A vector may also refer to a viral particle comprising a nucleic acid to be delivered into a host cell, either in vitro, in vivo or ex vivo. 36 168734056.1

[0156] Provided is a vector comprising a nucleic acid or set of nucleic acids disclosed herein.

[0157] In one embodiment, the vector is a viral vector.

[0158] In one embodiment, the vector is a lentiviral vector. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome providing the potential for very long-term gene expression.

[0159] In one embodiment, the vector is a vaccinia virus vector.

[0160] In some embodiments, the vector comprises a nucleic acid encoding an additional therapeutic agent. In some embodiments, the therapeutic agent is, for example, a cytokine, a chemokine, a tumor antigen, or a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD1 / L1 minibody.

[0161] The nucleic acids, oncolytic virus genome, or vectors disclosed herein may comprise nucleic acids comprising other regulatory sequences known in the art. In some embodiments, nucleic acids, oncolytic virus genome, or vectors disclosed herein comprise an IRES sequence. In some embodiments, nucleic acids, oncolytic virus genome, or vectors disclosed herein comprise a sequence encoding a self-cleaving peptide, including, but not limited to a P2A sequence.

[0162] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising a nucleic acid encoding (1) IL-12 p35 or a variant thereof, (2) IL-12 p40 or a variant thereof, (3) IL-2 or a variant thereof, and optionally (4) a membrane-anchoring polypeptide.

[0163] In some embodiments, provided is a nucleic acid (or set of nucleic acids), oncolytic virus genome, or vector comprising a nucleic acid encoding: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40; (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48.

[0164] In some embodiments, provided is a nucleic acid (or set of nucleic acids), oncolytic virus genome, or vector comprising a nucleic acid encoding: (a) SEQ ID NO:39 or SEQ ID NO:40; (b) SEQ ID NO:45 or SEQ ID NO:46; and 37 168734056.1(c) SEQ ID NO:47 or SEQ ID NO:48.

[0165] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising (1) a nucleic acid encoding IL-12 p35, (2) a P2A sequence, (3) a nucleic acid encoding: IL-12 p40 fused to IL-2 via a flexible linker.

[0166] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (b) a flexible linker, optionally, wherein the flexible linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-57; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40.

[0167] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding SEQ ID NO:45 or SEQ ID NO:46; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) SEQ ID NO:47 or SEQ ID NO:48; (b) a flexible linker, optionally, wherein the flexible linker any one of SEQ ID NOs:53- 57; and (c) SEQ ID NO:39 or SEQ ID NO:40.

[0168] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising (1) a nucleic acid encoding IL-12 p35, (2) a P2A sequence, (3) a nucleic acid encoding IL-12 p40 fused to IL-2 via a rigid linker.

[0169] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: 38 168734056.1(1) a nucleic acid encoding a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to NO:45 or SEQ ID NO:46; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (b) a rigid linker, optionally, wherein the rigid linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:58-62; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40.

[0170] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding SEQ ID NO:45 or SEQ ID NO:46; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) SEQ ID NO:47 or SEQ ID NO:48; (b) a rigid linker, optionally, wherein the rigid linker comprises any one of SEQ ID NOs:58-62; and (c) SEQ ID NO:39 or SEQ ID NO:40.

[0171] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising (1) a nucleic acid encoding IL-12 p40 (2) a P2A sequence, (3) a nucleic acid encoding: IL-12 p35 fused to IL-2 via a flexible linker.

[0172] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or SEQ ID NO:48; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; 39 168734056.1(b) a flexible linker, optionally, wherein the flexible linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:53-57; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40.

[0173] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding SEQ ID NO:47 or SEQ ID NO:48; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) a flexible linker, optionally, wherein the flexible linker any one of SEQ ID NOs:53- 57; and (c) SEQ ID NO:39 or SEQ ID NO:40.

[0174] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising (1) a nucleic acid encoding IL-12 p40, (2) a P2A sequence, (3) a nucleic acid encoding IL-12 p35 fused to IL-2 via a rigid linker.

[0175] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: (1) a nucleic acid encoding a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to NO:47 or SEQ ID NO:48; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:45 or SEQ ID NO:46; (b) a rigid linker, optionally, wherein the rigid linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:58-62; and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39 or SEQ ID NO:40.

[0176] In some embodiments, provided is a nucleic acid, oncolytic virus genome, or vector comprising: 40 168734056.1(1) a nucleic acid encoding SEQ ID NO:47 or SEQ ID NO:48; (2) a P2A sequence; and (3) a nucleic acid encoding: (a) SEQ ID NO:45 or SEQ ID NO:46; (b) a rigid linker, optionally, wherein the rigid linker comprises any one of SEQ ID NOs:58-62; and (c) SEQ ID NO:39 or SEQ ID NO:40.

[0177] Provided herein is a nucleic acid sequence that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:1-38. Provided herein is a nucleic acid sequence that comprises any one of SEQ ID NOs:1-38.

[0178] Provided herein is a nucleic acid sequence that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Table 2. Provided herein is a nucleic acid sequence that comprises any one of the sequences in Table 2.

[0179] Provided herein is a nucleic acid sequence that comprises a sequence encoding a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:39-106. Provided herein is a nucleic acid sequence that comprises a sequence encoding any one of SEQ ID NOs:39- 106.

[0180] Provided herein is a nucleic acid sequence that comprises a sequence encoding a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Table 3. Provided herein is a nucleic acid sequence that comprises a sequence encoding any one of the sequences in Table 3.

[0181] A person skilled in the art will appreciate that the constructs presented in the Tables herein may be constructed using IL-2 and IL-12 proteins from species other than human and mouse as well.

[0182] In one embodiment, the vector is an oncolytic viral vector.

[0183] In one aspect, the transgene encoding the IL-2 / IL-12 fusion protein (or a portion thereof), which may or may not be membrane-anchored, disclosed herein is delivered to a cancer cell using an oncolytic virus (OV). As such, provided herein are oncolytic viral vectors comprising a nucleic acid encoding an IL-2 / IL-12 fusion protein disclosed herein. As used 41 168734056.1herein, an “oncolytic virus” is a virus that exhibits increased replication in, and lysis of, cancer cells relative to comparable non-cancer cells. In certain embodiments, the oncolytic virus exhibits selective replication in cancer cells and less or essentially no replication in non-cancer cells. In certain embodiments, less replication means that replication in cancer cells versus comparable non-cancer cells is at least about 30 % greater, or at least about 50 % greater, or at least about 80 % greater. Non-limiting examples of oncolytic viruses include types of (i) adenovirus (“Ad”), for example hTERT-Ad; (ii) herpes simplex virus (“HSV”), for example G207, HSV-1716, T-VEC, and HSV-2 APK mutant; (iii) poxvirus, for example vaccinia virus, for example vSP and vvDD (tk− / vgf−); (iv) arbovirus; (v) paramyxovirus, for example, measles virus, mumps and Newcastle disease virus; (vi) rhabdovirus, for example, vesicular stomatitis virus; (vii) picornavirus, for example Coxsackie virus, Seneca Valley Virus, and polio virus; (viii) reovirus; (ix) parvovirus; and (x) recombinant / engineered versions of any of the oncolytic viruses disclosed herein.

[0184] OVs can directly kill tumor cells like conventional cancer therapies. This killing further provides a natural repertoire of tumor-specific antigens / tumor-associated antigens (TSAs / TAAs), danger signals (including damage-associated molecular pattern (DAMP) and OV-derived pathogen-associated molecular pattern (PAMP) molecules), and inflammatory cytokines to trigger innate and adaptive anti-tumor immunity. This anti-tumor immune reactivity in turn results in the infiltration of diverse immune cells, including T lymphocytes, into the TME, transforming “cold” tumors. Provided herein are oncolytic viruses that are further armed with transgenes coding for IL2 / IL-12 fusion proteins that can help transform the TME so as to harness the immune system to attack and treat tumors.

[0185] In some embodiments, the oncolytic virus is an oncolytic virus that has been approved by the U.S. Food and Drug Administration (FDA) or is undergoing clinical trials. For example, but not by way of limitation, the oncolytic virus can be Talimogene laherparepvec (Imlygic™; Amgen, Inc.), also referred to as T-VEC. In certain embodiments, the oncolytic virus can be pelareorep (Reolysin®; Oncolytics Biotech, Inc.). In certain embodiments, the oncolytic virus can be DNX-2401 (DNAtrix Therapeutics). In certain embodiments, the oncolytic virus can be H101 (Oncorine®; Shanghai Sunway Biotech Co., Ltd.). In certain embodiments, the oncolytic virus can be pexastimogene devacirepvec (JX-594; SillaJen Inc.). In certain embodiments, the oncolytic virus can be CG0070 (Cold Genesys, Inc.). In certain embodiments, the oncolytic virus can be G47Δ (Daiichi-Sankyo Company, Limited). 42 168734056.1

[0186] In a preferred embodiment, the oncolytic virus is a vaccinia virus. In certain non- limiting embodiments, the oncolytic virus is an engineered (also referred to as “recombinant”) vaccinia virus. In certain non-limiting embodiments, the virus is a recombinant vaccinia virus based on the Western Reserve (“WR”) strain of vaccinia, for example, the WR strain commercially available from the American Type Culture Collection as ATCC No. VR1354. Other vaccinia virus strains suitable for engineering include, but are not limited, to the Wyeth strain (ATCC VR-1536), the Lederle-Chorioallantoic strain (ATCC VR-325), and the CL strain (ATCC VR-117).

[0187] In some embodiments, the oncolytic virus is an engineered double-deleted vaccinia virus (vvDD) vaccinia viral construct comprising, for example, a modified version of a virus described in U.S. Pat. No. 8,506,947, McCart et al., 2001, Cancer Research 61:8751-8757, and / or Thorne S et al., 2007, J. Clin. Invest. 117:3350-3358, all of which are incorporated by reference herein in their entireties. For example, but not by way of limitation, the vaccinia virus can have deletions of the thymidine kinase (tk) and / or vaccinia growth factor (vgf) genes.

[0188] In some embodiments, a vaccinia virus has an inactivating mutation or deletion in one or more genes where the product of said gene or genes functions in viral replication. For example, but not by way of limitation, one or more of the following genes can bear an inactivating mutation or deletion: ribonucleotide reductase-large subunit, ribonucleotide reductase-small subunit, DNA ligase, dUTPase, tk, vgf, or A56R (coding for hemagglutinin) gene. In certain embodiments, an inactivating mutation is a mutation that either reduces or eliminates activity of the gene product. In certain embodiments, gene activation can be achieved by mutagenesis, e.g., site-directed mutagenesis or PCR-mediated mutagenesis. Alternatively, or additionally, in some embodiments, a nucleic acid can be inserted into one or more of the foregoing genes to achieve inactivation. In some embodiments, a nucleic acid encoding a protein can be inserted into one or more of the foregoing genes to achieve inactivation and to further achieve expression of the nucleic acid. In some embodiments, a nucleic acid encoding an IL-2 / IL-12 fusion protein can be inserted within one of the foregoing genes to achieve inactivation.

[0189] In some embodiments, the oncolytic virus is a vaccinia virus having an inactivating mutation (a mutation that either reduces or eliminates activity of the gene product) in the tk gene. For example, but not limited to, the inactivation of the thymidine kinase gene can be generated by the insertion of a cytosine deaminase (fcy1) gene within the thymidine kinase gene locus of the vaccinia viral genome, resulting in the expression of the fcy1 gene rather than 43 168734056.1the tk gene. In another non-limiting embodiment, a nucleic acid encoding a detectable protein, for example a fluorescent protein, for example yellow fluorescent protein (“yfp”), can be inserted into the tk gene, thereby inactivating it. In certain embodiments, a nucleic acid encoding an IL-2 / IL-12 fusion protein can be inserted into the tk gene.

[0190] In additional or alternative embodiments, the recombinant vaccinia virus can have an inactivating mutation in the vaccinia growth factor gene. For example, but not by way of limitation, an insertion of a lacZ gene within the vgf gene locus results in the expression of the lacZ gene rather than vgf. In certain embodiments, a nucleic acid encoding an IL-2 / IL-12 fusion protein can be inserted into the vgf gene.

[0191] In some embodiments, the vaccinia virus is modified to reduce immunogenicity in a mammal, including, but not limited to in a human. In some embodiments, the vaccinia virus has a deletion of or an inactivating mutation in one or more of the viral genes for A41L, A44L, A46R, A49, A52R, A53R, B5R, B8R, B13R (SPI-2), B15R, B18R, C3L (VCP), C6, C7L, C12L, E3L, F1L, K1L, K3L, K7R, M1L, and N1L.

[0192] Methods for generating recombinant vectors, including viral vectors for the delivery of transgenes are known in the art. Likewise, methods for generating recombinant oncolytic viral vectors (including vaccinia virus vectors) that comprise transgenes are known in the art.

[0193] For example, a vaccinia viral particle comprising a nucleic acid comprising a transgene may be generated by transfecting a shuttle vector or plasmid containing transgene operatively linked to a vaccinia virus promoter into a cell that has been infected with vaccinia virus and introducing the exogenous sequence by homologous recombination. Alternative methods may be used, employing two viruses, one defective for some genes and one wild-type acting as a helper. In both cases, the recombinant viruses are produced by homologous recombination in the infected cell. Selection techniques may be employed to select the resulting, recombinant viral particles. In some embodiments, the promoter and the transgene operatively linked to the promoter are flanked by tk gene flanking sequences, so that they replace a portion or all of the genomic tk gene during the homologous recombination process.

[0194] In another variation, the bacteriophage T7 RNA polymerase gene can be integrated into the genome of vaccinia so that a transgene controlled by a T7 promoter, either in a transfected plasmid or a recombinant vaccinia virus, is expressed.

[0195] Cells expressing IL-2 / IL-12 fusion proteins 44 168734056.1

[0196] In one aspect, provided is a cell comprising a transgene encoding an IL-2 / IL-12 fusion protein disclosed herein. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the cell is a human cell.

[0197] Provided herein is also a cell comprising any nucleic acid disclosed herein. Provided herein is also a cell comprising a nucleic (or pair of nucleic acids) encoding any polypeptide disclosed herein (including any fusion protein disclosed herein). Provided is a cell comprising a vector disclosed herein.

[0198] If the IL-2 / IL-12 fusion protein is a membrane-anchored fusion protein, a portion of the fusion protein may be presented on the surface of the cell.

[0199] In one embodiment, the IL-2 / IL-12 fusion protein is expressed by a cancer cell or a stromal cell in a tumor.

[0200] In one embodiment, the IL-2 / IL-12 fusion protein is expressed by an immune cell. In some embodiments, the immune cell is a lymphocyte, a dendritic cell, a natural killer cell, or a macrophage. In one embodiment, the immune cell is a T lymphocyte. In one embodiment, the immune cell is a B lymphocyte. In some embodiments, the cell is a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR NK, a T cell receptor (TCR) T lymphocyte, or a tumor infiltrating lymphocyte (TIL). In some embodiments, the IL-2 / IL-21 fusion protein is expressed on the surface of the immune cell.

[0201] In some embodiments, the immune cell further expresses on its surface a moiety that targets the immune cell to a cancer cell. In some embodiments, the targeting moiety recognizes a cancer antigen. Non-limiting examples of cancer antigen include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, Folate Receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGE A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, midkin, BAGE, CASP-8, β-catenin, CA-125, CDK-1, ESO-1, gp75, gp100 , MART-1, MUC- 1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orf112, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orf153, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, ART1, ART4, B-cycline, Gli1, Cav-1, Cathepsin B, CD74, E- Cadherin, EphA2 / Eck, Fra-1 / Fosl 1, GAGE-1, Ganglioside / GD2, GnT-V, β1, 6-Ν, Ki67, Ku70 / 80, PROX1, PSCA, SOX10, SOX11, Survivin, βhCG, WT1, mesothelin, melan-A, NY- BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the targeting moiety recognizes a neoantigen. In some 45 168734056.1embodiments, the targeting moiety is a chimeric antigen receptor (CAR). In some embodiments, the targeting moiety is a T-cell receptor (TCR).

[0202] The cell may be isolated.

[0203] Pharmaceutical compositions

[0204] Provided herein are pharmaceutically acceptable compositions comprising any of the compositions disclosed herein. For example, provided herein are pharmaceutically acceptable compositions that comprise a nucleic acid (or set of nucleic acids) encoding an IL- 2 / IL-12 fusion protein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, formulated together with one or more pharmaceutically acceptable excipients.

[0205] The active agent and excipient(s) may be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions disclosed herein may be specially formulated in solid or liquid form, including those adapted for parenteral administration, for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.

[0206] The therapeutic compositions disclosed herein may formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and / or a preservative. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non- toxic compatible substances employed in pharmaceutical formulations. 46 168734056.1

[0207] A bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present disclosure include mannitol, glycine, polyethylene glycol and sorbitol.

[0208] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particulates in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. Suitable surfactants according to the present disclosure include polysorbates (e.g., polysorbates 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.).

[0209] Preservatives may be used in formulations disclosed herein. Suitable preservatives for use in the formulations disclosed herein include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl- dimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g., in “Remington’s Pharmaceutical Sciences”, The Science and Practice of Pharmacy, 19thEd. Mack Publishing Company, Easton, Pa., (1995).

[0210] Methods of treatment

[0211] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a composition provided herein. Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus comprising a 47 168734056.1nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein or an IL-2 / IL-12 fusion protein disclosed herein. Provided herein is a method of treating cancer, the method comprising administering to a subject in need thereof a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein.

[0212] By “subject” is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline, etc. Individuals and patients are also subjects herein. In embodiments, a therapeutically effective amount of the nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein, the vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or the IL-2 / IL-12 fusion protein disclosed herein is administered. In one embodiment, therapeutically effective amount of a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein is administered. “Therapeutically effective amount” means an amount that, when administered to a subject, is effective in producing the desired therapeutic effect. For example, the term “therapeutically effective” amount can refer to the amount of a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL- 12 fusion protein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL- 2 / IL-12 fusion protein disclosed herein, an oncolytic virus comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or an IL-2 / IL-12 fusion protein disclosed herein that is sufficient to elicit the desired biological or medical response of a cell, tissue, system, animal, or human.

[0213] The term “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term “cancer” as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject that has a cancer is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location 48 168734056.1and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[0214] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder, or slow its course of development.

[0215] Provided herein is an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein for use in the treatment of cancer.

[0216] Provided herein is the use of an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL- 12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein in the manufacture of a medicament for the treatment of cancer.

[0217] Also provided herein is a method of reducing tumor growth in subject in need thereof, the method comprising administering to a subject in need thereof an IL-2 / IL-12 fusion 49 168734056.1protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein. “Reducing” includes inhibiting and / or reversing and can refer to, for example, the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or the size of the dormant tumor.

[0218] The embodiments disclosed herein may be used for treating metastasis, which relates to the spreading of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life- threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.

[0219] Accordingly, provided herein is a method of reducing metastasis, the method comprising administering to a subject in need thereof an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein.

[0220] Also contemplated are methods of reducing cancer stemness by administering to a subject in need thereof an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus 50 168734056.1genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL- 12 fusion protein disclosed herein. Cancer stemness may refer to the ability of a cell to self- renew and to generate an additional, phenotypically distinct cell type. Cancer stem cells (CSCs) are cancer cells that exhibit stem-cell like properties. CSCs often exhibit at least one hallmark of cancer, and are capable of generating at least one additional, phenotypically distinct cell type. Furthermore, cancer stem cells are capable of both asymmetric and symmetric replication. It is appreciated that a cancer stem cell may result from differentiated cancer cells that acquire stemness traits and / or stem cells that acquire phenotypes associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within a tumor.

[0221] Provided herein are methods of increasing cytokine production in the tumor microenvironment by administering to a subject in need thereof an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein.

[0222] Provided herein are methods of increasing anti-tumor immunity in a subject by administering to a subject in need thereof an IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL- 2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding (or set of nucleic acids) an IL-2 / IL-12 fusion protein disclosed herein.

[0223] Provided herein are methods of increasing infiltration of a tumor with immune cells by administering to a subject in need thereof a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL- 12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) 51 168734056.1encoding an IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein.

[0224] Provided herein are methods of reducing T cell tolerance by administering to a subject in need thereof a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein.

[0225] Provided herein are methods of enhancing T cell expansion by administering to a subject in need thereof a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein.

[0226] Provided herein are methods of increasing the number of memory T cells (or of increasing the ratio of memory T cell to non-memory T cells) by administering to a subject in need thereof a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein.

[0227] Also contemplated are methods of inducing cell death of a dividing cancer cell, the method comprising contacting the cancer cell with a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic 52 168734056.1acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein.

[0228] Examples of cancer that can be treated with the compositions and methods disclosed herein include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; mesothelioma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; vulvar sarcomas; as well as other carcinomas and sarcomas.

[0229] The efficacy of the treatment methods for cancer comprising administering a IL- 2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein can be measured by various endpoints commonly used in evaluating cancer treatments, including but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, and quality of life. The treatment with a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic 53 168734056.1acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the disorder. In cases where a patient has more than one type of cancer, the method of treatment disclosed herein can be effective in treating at least one of the cancers. To the extent the compositions disclosed herein act to prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0230] Also provided herein are methods of foreating cancer, for reducing tumor metastasis, for increasing cytokine production in the tumor microenvironment, for increasing anti-tumor immunity for increasing infiltration of a tumor with immune cells, for reducing T cell tolerance, for enhancing T cell expansion in a subject in thereof, and / or for increasing the number of memory T cells in a subject in need thereof, the method comprising administering to the subject an immune cell expressing a soluble or a membrane-anchored IL2 / IL-12 fusion protein disclosed herein. In some embodiments, the immune cell is a lymphocyte, a dendritic cell, a natural killer cell, or a macrophage. In one embodiment, the immune cell is a T lymphocyte. In one embodiment, the immune cell is a B lymphocyte. In some embodiments, the cell is a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR NK, a T cell receptor (TCR) T lymphocyte, or a tumor infiltrating lymphocyte (TIL). The immune cell may have been isolated from the same subject or a different subject. In some embodiments, provided is a method of isolating immune cells from a patient, optionally expanding the immune cells, genetically modifying the immune cells to express a soluble or a membrane-anchored IL2 / IL- 12 fusion protein disclosed herein, optionally further expanding the immune cells, and reintroducing the genetically modified immune cells into the patient.

[0231] The methods disclosed herein may be conducted in vivo, in vitro or ex vivo, as appropriate.

[0232] Administration

[0233] The IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein 54 168734056.1disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein can be administered according to any known method in the art.

[0234] For example, but not by way of limitation, a method for the delivery of a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, (or a pharmaceutical composition comprising a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein) can be via intratumoral injection. In certain embodiments, alternate methods of administration can also be used, e.g., intravenous, via infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, intravaginal, intranasal, intrathecal, or intraperitoneal. The routes of administration can vary with the location and nature of the tumor. In certain embodiments, the route of administration can be intradental, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, by lavage or orally. In certain embodiments, the modified virus can be administered to the patient from a source implanted in the patient.

[0235] In certain embodiments, administration of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant 55 168734056.1vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can occur by continuous infusion over a selected period of time. In certain embodiments, the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered at a therapeutically effective dose by infusion over a period of about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or longer.

[0236] The IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered as a liquid dosage, wherein the total volume of administration is about 1 ml to about 5 ml, about 5 ml to 10 ml, about 15 ml to about 20 ml, about 25 ml to about 30 ml, about 30 ml to about 50 ml, about 50 ml to about 100 ml, about 100 ml to 150 ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400 ml, about 400 ml to about 450 ml, about 450 ml to 500 ml, about 500 ml to 750 ml or about 750 ml to 1000 ml.

[0237] In some embodiments, a single dose of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition 56 168734056.1thereof can refer to the amount administered to a subject or a tumor over a 1, 2, 5, 10, 15, 20 or 24 hour period. In certain embodiments, the dose can be spread over time or by separate injection. In certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6 or more doses) of the IL- 2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered to the subject, for example, where a second treatment can occur within 1, 2, 3, 4, 5, 6, 7 days or weeks of a first treatment. In certain embodiments, multiple doses of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered to the subject over a period of 1, 2, 3, 4, 5, 6, 7 or more days or weeks. In certain embodiments, the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered over a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or longer. The frequency of administration of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed 57 168734056.1herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding an IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can be, in certain instances, once daily, twice daily, once every week, once every three weeks, once every four weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months).

[0238] An effective amount of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can be determined by methods known in the art. In certain embodiments, the IL-2 / IL- 12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or the pharmaceutical composition thereof can be administered in an amount sufficient to induce oncolysis in at least about 20% of cells in a tumor, in at least about 30% of cells in a tumor, in at least about 40% of cells in a tumor, in at least about 50% of cells in a tumor, in at least about 60% of cells in a tumor, in at least about 70% of cells in a tumor, in at least about 80% of cells in a tumor, or in at least about 90% of cells in a tumor.

[0239] In certain embodiments, the amount of the oncolytic virus (including a recombinant vaccinia virus) comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or a pharmaceutical composition thereof administered can be between about 1×107and 1×1010infectious viral particles or plaque forming units (pfu), or between about 1×107and 1×109pfu / m2surface area of the subject to be treated. In certain embodiments, 58 168734056.1the oncolytic virus (including a recombinant vaccinia virus) comprising a nucleic acid (or set of nucleic acids) encoding the IL-2 / IL-12 fusion protein can be administered at a dose that can comprise about 1×108pfu. In certain embodiments, the amount of virus administered can be between about 1×103and 1×1012viral particles or pfu, or between about 1×105and 1×1010pfu, or between about 1×105and 1×108pfu, or between about 1×108and 1×1010pfu. In certain embodiments, the oncolytic virus (including a recombinant vaccinia virus) comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein can be administered at a dose that can comprise about 1×103pfu / dose to about 1×104pfu / dose, about 1×104pfu / dose to about 1×105pfu / dose, about 1×105pfu / dose to about 1×106about 1×107pfu / dose to about 1×108pfu / dose, about 1×109pfu / dose to about 1×1010pfu / dose to about 1×1011pfu / dose, about 1×101112 12 131×10 pfu / dose, about 1×10 pfu / dose to about 1×10 pfu / dose, about to about 1×1014pfu / dose, or about 1×1014pfu / dose to about 1×1015pfu / dose. In certain embodiments, the oncolytic virus (including a recombinant vaccinia virus) comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein can be administered at a dose that can comprise about 1×103viral particles / dose to about 1×104viral particles / dose, about 1×104viral particles / dose to about 1×105viral particles / dose, about 1×105viral particles / dose to about 1×106viral particles / dose, about 1×107viral particles / dose to about 1×108viral particles / dose, about 1×109viral particles / dose to about 1×1010viral particles / dose, about 1×1010viral particles / dose to about 1×1011viral particles / dose, about 1×1011viral particles / dose to about 1×1012viral particles / dose, about 1×1012viral particles / dose to about 1×1013viral particles / dose, about 1×1013viral particles / dose to about 1×1014viral particles / dose, or about 1×1014viral particles / dose to about 1×1015viral particles / dose.

[0240] Preparation of tumor-infiltrating T lymphocytes induced by oncolytic virus (“OV-induced T cells”)

[0241] Provided herein is a method of generating tumor-infiltrating oncolytic virus-induced T lymphocytes (“OV-induced T cells”), the method comprising (a) administering to a subject having cancer a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia 59 168734056.1virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a pharmaceutical composition thereof, (b) isolating tumor- infiltrating T lymphocytes from the subject, and (c) expanding the tumor-infiltrating T lymphocytes ex vivo.

[0242] The resulting tumor-infiltrating oncolytic virus-induced T lymphocytes may be used for adoptive T cell therapy. In certain embodiments, the OV-induced T cells may be employed to kill cancer cells and associated stromal cells. In certain embodiments, the OV-induced T cells can be transferred to the subject from which the cells were isolated. Alternatively and / or additionally, the OV-induced T cells can be transferred to a different subject. In certain embodiments, the OV-induced T cells are allogeneic.

[0243] In embodiments, the OV-induced T cells are isolated from lymphoid and non- lymphoid tissues or from peripheral blood. In certain embodiments, OV-induced T cells are isolated from cancer tissue. For example, the T cells can be isolated from tissue by digesting the tissue and using density gradient centrifugation, e.g., using a Percoll density gradient.

[0244] In some embodiments, during ex vivo expansion, the isolated OV-induced T cells can be treated with one or more cytokines, lymphokines, and / or one or more agents, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL- 23, IL-24, IL-27, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, T F- alpha, TNF-beta, GM-CSF or combinations thereof. For example, but not by way of limitation, the isolated OV-induced T cells can be treated with IL-7, IL-2 and / or a GSK3b inhibitor. In certain embodiments, the OV-induced T cells can be treated with IL-2. In certain embodiments, the OV-induced T cells can be treated with IL-7. In certain embodiments, the OV- induced T cells can be treated with IL-2 and IL-7. In certain, non-limiting examples the isolated T cells can be treated for about two to about 10 days, e.g., about three days.

[0245] In certain embodiments, the isolated OV-induced T cells are co-cultured with dendritic and cancer cells. In certain embodiments, the OV-induced T cells can be co- cultured with cancer cells that have been infected with an oncolytic virus, e.g., an oncolytic virus disclosed herein. In certain embodiments, the cancer cells can be infected with an oncolytic virus that is different than the oncolytic virus used to generate the OV- induced T cells. In certain embodiments, the isolated OV-induce T cells are co-cultured with dendritic cells and cancer cells for about two to about 10 days, e.g., about two days. In certain embodiments, the co-cultured T cells are then treated with one or more cytokines and / or one or more agents. 60 168734056.1

[0246] In certain embodiments, to quantify the ability of the oncolytic virus to promote T cell infiltration in the tumor microenvironment, isolated OV-induced T cells can be analyzed with antibodies against CD3, CD8, CD4, and 4-1BB. In certain embodiments, the oncolytic virus promotes an infiltration of CD3+, CD8+, CD4+, and 4-1BB+T cells in the tumor microenvironment. In certain embodiments, the isolated OV-induced T cells are CD3+, CD8+, CD4+and / or 4-1BB+. In certain embodiments, the OV-induced T cells are CD3+. In certain embodiments, the OV-induced T cells are CD3+. In certain embodiments, the OV-induced T cells are CD4+. In certain embodiments, the OV- induced T cells are CD8+. In certain embodiments, the OV-induced T cells are CD4+4-1BB+. In certain embodiments, the OV- induced T cells are CD8+4-1BB+.

[0247] A different vector other than an oncolytic vector may be used for the induction of T cells, including, but not limited to, a lentiviral vector.

[0248] Combination therapies

[0249] In one aspect, provided herein is a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or a pharmaceutical composition thereof that is administered with an additional therapeutic agent. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti- inflammatory agents, anti-cancer (antineoplastic) agents, anti-neurodegenerative agents, and anti-infective agents. Agents that are used in such combination therapies may fall into one or more of the preceding categories. In one embodiment, the additional therapeutic agent an antineoplastic agent.

[0250] The administration of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein and the additional therapeutic agent may 61 168734056.1be concurrent or consecutive. The administration of the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein and the additional therapeutic agent may be separately or as a mixture. Further, the methods of treatment contemplated herein can relate to a treatment in combination with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy.

[0251] Non-limiting examples of additional therapeutic agents also include radionuclides with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer). Non-limiting examples of high-energy radionuclides include:90Y,125I,131I,123I,111In,105Rh,153Sm,67Cu,67Ga,166Ho,177Lu,186Re and188Re. These isotopes typically produce high energy α- or β- particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0252] Non-limiting examples of additional therapeutic agents also include cytotoxic agents such as cytostatics (e.g., alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross-linkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti- angiogenesis agents, and the like.

[0253] Non-limiting examples of additional therapeutic agents also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin- A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Non-limiting examples of DNA- intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, 62 168734056.1carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0254] Non-limiting examples of additional therapeutic agents also include transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other non-limiting examples of cytostatic agents that are compatible with the embodiments disclosed herein include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0255] Non-limiting examples of additional therapeutic agents also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5- fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; tubulin binding agents such as taxoids (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g., Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatins (e.g., Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenesis compounds such as Angiostatin K1-3, DL-α- difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0256] Non-limiting examples of additional therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens, (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminogluthetimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8- naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0257] Non-limiting examples of additional therapeutic agents also include enzyme inhibitors such as, S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole 1- β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

[0258] Non-limiting examples of additional therapeutic agents also include gene regulators such as 5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4- 63 168734056.1hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0259] Non-limiting examples of additional therapeutic agents also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.

[0260] Still other additional therapeutic agents that are compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0261] In one embodiment, the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein is administered in combination with an agent that is a checkpoint inhibitor. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrative checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies, or antigen binding fragments thereof, other binding proteins, biologic therapeutics or small molecules, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD-1, BTLA, HVEM, TIM-3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049. Illustrative 64 168734056.1immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti- OX40, and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor), as well as the PD-1 and PD-L1 inhibitors described herein. Checkpoint protein ligands include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

[0262] In some embodiments, the IL-2 / IL-12 fusion protein described herein (or nucleic acid (or set of nucleic acids) encoding the IL-2 / IL-12 fusion) is administered with a TIGIT, LAP, Podoplanin, Protein C receptor, ICOS, GITR, CD226 or a CD160 inhibiting agent.

[0263] In some embodiments, the additional therapeutic agent is a checkpoint inhibitor, a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR natural killer cells, a TCR T lymphocyte, or a tumor infiltration lymphocyte.

[0264] Kits

[0265] Provided herein are kits that comprise a IL-2 / IL-12 fusion protein disclosed herein, a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, an oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, a vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or a recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or a pharmaceutical composition thereof.

[0266] In some embodiments, the kit further includes one or more components such as instructions for use, devices and additional reagents, and components, such as tubes, containers and syringes for performing the methods disclosed herein.

[0267] In some embodiments, the kit includes instructions for use, a device for administering the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL- 2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein or a pharmaceutical composition thereof to a subject, or a device for administering an additional agent or compound to a subject. For example, and not by way of limitation, the instructions can include a description of any of the compositions or methods disclosed herein, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper 65 168734056.1dosage amount and the proper administration method for administering the modified virus. Instructions can also include guidance for monitoring the subject over duration of the treatment time.

[0268] In some embodiments, the kit includes a device for administering the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or a pharmaceutical composition thereof to a subject. Any of a variety of devices known in the art for administering medications and pharmaceutical compositions can be included in the kits provided herein. For example, and not by way of limitation, such devices include, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler and a liquid dispenser, such as an eyedropper.

[0269] In some embodiments, the kit includes one or more additional agents that can be administered in combination with the IL-2 / IL-12 fusion protein disclosed herein, the nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, the oncolytic virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL- 12 fusion protein disclosed herein, the vector comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein, or the recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid (or set of nucleic acids) encoding a IL-2 / IL-12 fusion protein disclosed herein or a pharmaceutical composition thereof. For example, but not by way of limitation, a kit can include a cytokine, e.g., IL-2, and / or an anti-PD-1 and / or an anti-PD-L1 antibody.

[0270] Illustrative embodiments

[0271] A list of illustrative embodiments can be found below. The invention is not limited to these embodiments.

[0272] Embodiment 1. A recombinant viral vector comprising a virus genome comprising a nucleic acid encoding a fusion protein comprising: (a) a first polypeptide comprising an interleukin 2 (IL-2) or an IL-2 variant; (b) a second polypeptide comprising (i) an interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant and (ii) an interleukin 12 subunit p40 (IL- 12p40) or an IL-12p40 variant; and optionally (c) a membrane-anchoring polypeptide. 66 168734056.1

[0273] Embodiment 2. A recombinant viral vector comprising a virus genome comprising a nucleic acid (or set of nucleic acids) encoding: (a) an IL-12p35 or an IL-12p35 variant; and (b) a fusion protein comprising: (i) a first polypeptide comprising an IL-2 or an IL-2 variant; (ii) a second polypeptide comprising an IL-12p40 or an IL-12p40 variant; and optionally (iii) a membrane-anchoring polypeptide.

[0274] Embodiment 3. A recombinant viral vector comprising a virus genome comprising a nucleic acid (or set of nucleic acids) encoding: (a) an IL-12p40 or an IL-12p40 variant; and (b) a fusion protein comprising: (i) a first polypeptide comprising an IL-2 or an IL-2 variant; (ii) a second polypeptide comprising an IL-12p35 or an IL-12p35 variant; and optionally (iii) a membrane-anchoring polypeptide.

[0275] Embodiment 4. The recombinant viral vector of embodiment 2 or 3, wherein the IL- 12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently linked.

[0276] Embodiment 5. The recombinant viral vector of embodiment 4, wherein the IL- 12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently by a disulfide bond.

[0277] Embodiment 6. The recombinant viral vector of any one of embodiments 1, wherein the nucleic acid encoding the fusion protein is operatively linked to a promoter.

[0278] Embodiment 7. The recombinant viral vector of any one of embodiments 2, 4, or 5, wherein the nucleic acid encoding the IL-12p35 or the IL-12p35 variant and / or the nucleic acid encoding is operatively linked to a promoter.

[0279] Embodiment 8. The recombinant viral vector of any one of embodiments 3-5, wherein the nucleic acid encoding the IL-12p40 or the IL-12p40 variant and / or the nucleic acid encoding is operatively linked to a promoter.

[0280] Embodiment 9. The recombinant viral vector of any one of embodiments 6-8, wherein the promoter is a p7.5 or a pSe / l promoter.

[0281] Embodiment 10. The recombinant viral vector of any one of embodiments 1-9, wherein the IL-2 is human or murine IL-2.

[0282] Embodiment 11. The recombinant viral vector of any one of embodiments 1-9, wherein the first polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:40 or SEQ ID NO:39.

[0283] Embodiment 12. The recombinant viral vector of embodiment 11, wherein the first polypeptide comprises SEQ ID NO:40 or SEQ ID NO:39. 67 168734056.1

[0284] Embodiment 13. The recombinant viral vector of embodiment 12, wherein the first polypeptide comprises SEQ ID NO:40.

[0285] Embodiment 14. The recombinant viral vector of any one of embodiments 1-13, wherein the IL-12p35 is human or murine IL-12p35.

[0286] Embodiment 15. The recombinant viral vector of any one of embodiments 1-13, wherein the second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:45 or SEQ ID NO:46.

[0287] Embodiment 16. The recombinant viral vector of embodiment 15, wherein the second polypeptide comprises SEQ ID NO:45 or SEQ ID NO:46.

[0288] Embodiment 17. The recombinant viral vector of embodiment 16, wherein the second polypeptide comprises SEQ ID NO:46.

[0289] Embodiment 18. The recombinant viral vector of any one of embodiments 1-17, wherein the IL-12p40 is human or murine IL-12p40.

[0290] Embodiment 19. The recombinant viral vector of any one of embodiments 1-17, wherein the second polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:47 or SEQ ID NO:48.

[0291] Embodiment 20. The recombinant viral vector of embodiment 19, wherein the second polypeptide comprises SEQ ID NO:47 or SEQ ID NO:48.

[0292] Embodiment 21. The recombinant viral vector of embodiment 20, wherein the second polypeptide comprises SEQ ID NO:48.

[0293] Embodiment 22. The recombinant viral vector of any one of embodiments 1-21, wherein the fusion protein further comprises a first linker connecting (i) the first and (ii) the second polypeptide.

[0294] Embodiment 23. The recombinant viral vector of any one of embodiments 1-22, wherein the fusion protein further comprises a second linker connecting (i) the membrane- anchoring polypeptide and (ii) the first or the second polypeptide.

[0295] Embodiment 24. The recombinant viral vector of any one of embodiments 1 or 6- 23, wherein the IL-12p35 or IL-12p35 variant is located N-terminally of the IL-12p40 or IL- 12p40 variant.

[0296] Embodiment 25. The recombinant viral vector of any one of embodiments 1 or 6- 23, wherein the IL-12p35 or IL-12p35 variant is located C-terminally of the IL-12p40 or IL- 12p40 variant. 68 168734056.1

[0297] Embodiment 26. The recombinant viral vector of embodiment 22, wherein the fusion protein comprises from N-terminus to C-terminus: (a) the first polypeptide comprising IL-2 or the IL-2 variant; (b) the first linker; and (c) the second polypeptide comprising (i) IL-12p35 or the IL-12p35 variant and (ii) IL-12p40 or the IL-12p40 variant.

[0298] Embodiment 27. The recombinant viral vector of embodiment 23; wherein the fusion protein comprises from N-terminus to C-terminus: (a) the first polypeptide comprising IL-2 or the IL-2 variant; (b) the first linker; (c) the second polypeptide comprising (i) IL-12p35 or the IL-12p35 variant and (ii) IL-12p40 or the IL-12p40 variant; (d) the second linker; and (e) the membrane-anchoring polypeptide.

[0299] Embodiment 28. The recombinant viral vector of embodiment 22, wherein the fusion protein comprises from N-terminus to C-terminus: (a) the second polypeptide comprising (i) IL-12p35 or an IL-12p35 variant and (ii) IL-12p40 or an IL-12p40 variant; (b) the first linker; and (c) the first polypeptide comprising IL-2 or an IL-2 variant.

[0300] Embodiment 29. The recombinant viral vector of embodiment 23, wherein the fusion protein comprises from N-terminus to C-terminus: (a) the second polypeptide comprising (i) IL-12p35 or an IL-12p35 variant and (ii) IL-12p40 or an IL-12p40 variant; (b) the first linker; (c) the first polypeptide comprising IL-2 or an IL-2 variant; (d) the second linker; and (e) the membrane-anchoring polypeptide.

[0301] Embodiment 30. The recombinant viral vector of any one of embodiments 1 or 6- 29, wherein the IL-12p35 or IL-12p35 variant and the IL-12p40 or IL-12p40 variant are linked by a third linker.

[0302] Embodiment 31. The recombinant viral vector of any one of embodiments 22-30, wherein the first linker, the second linker, and / or the third linker is a polypeptide linker.

[0303] Embodiment 32. The recombinant viral vector of embodiment 31, wherein the first linker, the second linker, and / or the third linker is a flexible polypeptide linker.

[0304] Embodiment 33. The recombinant viral vector of embodiment 32, wherein the flexible polypeptide linker predominantly comprises glycines and serines.

[0305] Embodiment 34. The recombinant viral vector of embodiment 33, wherein the flexible polypeptide linker comprises SEQ ID NO:41 (GGGGS) or one or more repeats of SEQ ID NO:41 (GGGGS).

[0306] Embodiment 35. The recombinant viral vector of embodiment 33, wherein the flexible polypeptide linker comprises a sequence selected from the group consisting of GGS, 69 168734056.1SEQ ID NO:54 (GGSGGGS), SEQ ID NO:55 ((GGGGS)2), SEQ ID NO:56 ((GGGGS)3), and SEQ ID NO:47((GGGGS)4).

[0307] Embodiment 36. The recombinant viral vector of embodiment 35, wherein the flexible polypeptide linker comprises SEQ ID NO:56 ((GGGGS)3).

[0308] Embodiment 37. The recombinant viral vector of embodiment 31, wherein the first linker and / or the second linker is a rigid polypeptide linker.

[0309] Embodiment 38. The recombinant viral vector of embodiment 37, wherein the rigid polypeptide linker predominantly comprises alanines.

[0310] Embodiment 39. The recombinant viral vector of embodiment 37, wherein the rigid polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO:58 (A(EA3K)1AAA), SEQ ID NO:59 (A(EA3K)2AAA), SEQ ID NO:60 (A(EA3K)3AAA), SEQ ID NO:61 (A(EA3K)4AAA), or SEQ ID NO:62 (A(EA3K)5AAA).

[0311] Embodiment 40. The recombinant viral vector of embodiment 39, wherein the rigid polypeptide linker comprises SEQ ID NO:61 (A(EA3K)4AAA).

[0312] Embodiment 41. The recombinant viral vector of any one of embodiments 1-25, 27, or 29-40, wherein the membrane-anchoring polypeptide is between about 10 and about 50 amino acids in length.

[0313] Embodiment 42. The recombinant viral vector of embodiment 41, wherein the membrane-anchoring polypeptide comprises a glycosylphosphatidylinositol (GPI)-anchor acceptor peptide.

[0314] Embodiment 43. The recombinant viral vector of embodiment 41, wherein the membrane-anchoring polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104.

[0315] Embodiment 44. The recombinant viral vector of embodiment 42, wherein the membrane-anchoring polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104.

[0316] Embodiment 45. The recombinant viral vector of embodiment 43, wherein the membrane-anchoring polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:63.

[0317] Embodiment 46. The recombinant viral vector of embodiment 45, wherein the membrane-anchoring polypeptide comprises SEQ ID NO:63. 70 168734056.1

[0318] Embodiment 47. The recombinant viral vector of embodiment 1, wherein the fusion protein comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:11-34.

[0319] Embodiment 48. The recombinant viral vector of embodiment 47, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs:11-34.

[0320] Embodiment 49. The recombinant viral vector of any one of embodiments 1-48, wherein the viral vector is a vaccinia virus comprising a vaccinia virus genome or a lentiviral vector comprising a lentiviral genome.

[0321] Embodiment 50. The recombinant viral vector of embodiment 49, wherein the vaccinia virus genome has a deletion of the thymidine kinase (TK) gene, the vaccinia growth factor (VGF gene), the A56R gene, or combinations thereof.

[0322] Embodiment 51. The recombinant viral vector of embodiment 49 or 50, wherein the vaccinia virus genome has a deletion of the viral gene for A41L, A44L, A46R, A49, A52R, A53R, B5R, B8R, B13R (SPI-2), B15R, B18R, C3L (VCP), C6, C7L, C12L, E3L, F1L, K1L, K3L, K7R, M1L, N1L, or combinations thereof.

[0323] Embodiment 52. The recombinant viral vector of any one of embodiments 1-48, wherein the viral vector is a lentiviral vector.

[0324] Embodiment 53. A cell comprising the recombinant viral vector of any one of embodiments 1-52.

[0325] Embodiment 54. The cell of embodiment 53, wherein the cell is a mammalian cell.

[0326] Embodiment 55. The cell of embodiment 54, wherein the cell is a human cell.

[0327] Embodiment 56. The cell of any one of embodiments 53-55, wherein the cell is a cancer cell.

[0328] Embodiment 57. The cell of any one of embodiments 53-55, wherein the cell is a stromal cell in a tumor microenvironment.

[0329] Embodiment 58. The cell of any one of embodiments 53-55, wherein the cell is an immune cell.

[0330] Embodiment 59. A pharmaceutical composition comprising the recombinant viral vector of any one of embodiments 1-51 and one or more pharmaceutically acceptable excipients.

[0331] Embodiment 60. A method for treating cancer, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59. 71 168734056.1

[0332] Embodiment 61. The method of embodiment 60, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, gastric cancer, sarcoma, mesothelioma, ovarian cancer, endometrial cancer, or cervical cancer.

[0333] Embodiment 62. A method for reducing tumor growth, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59.

[0334] Embodiment 63. A method for reducing tumor metastasis, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59.

[0335] Embodiment 64. A method for increasing cytokine production in the tumor microenvironment, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-51 or the pharmaceutical composition of embodiment 59.

[0336] Embodiment 65. A method for increasing anti-tumor immunity, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-51 or the pharmaceutical composition of embodiment 59.

[0337] Embodiment 66. A method for increasing infiltration of a tumor with immune cells, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-51 or the pharmaceutical composition of embodiment 59.

[0338] Embodiment 67. A method for reducing T cell tolerance, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59.

[0339] Embodiment 68. A method for enhancing T cell expansion, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59.

[0340] Embodiment 69. A method for increasing the number of memory T cells, the method comprising administering to a subject in need thereof the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59.

[0341] Embodiment 70. The method of any one of embodiments 60-69, wherein the step of administering comprises intraperitoneal administration.

[0342] Embodiment 71. The method of any one of embodiments 60-69, wherein the step of administering comprises systemic administration. 72 168734056.1

[0343] Embodiment 72. The method of any one of embodiments 60-71, the method further comprising administering to the subject an additional antineoplastic agent.

[0344] Embodiment 73. The method of embodiment 72, wherein the antineoplastic agent is an immune checkpoint inhibitor.

[0345] Embodiment 74. The method of embodiment 73, wherein the immune checkpoint inhibitor is selected from the group of a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a CTLA4 inhibitor, or combinations thereof.

[0346] Embodiment 75. The method of embodiment 73 or 74, wherein the immune checkpoint inhibitor is an antibody or fragment thereof.

[0347] Embodiment 76. The method of embodiment 72, wherein the antineoplastic agent is a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR natural killer cell, or a tumor infiltrating lymphocyte.

[0348] Embodiment 77. A method of generating tumor-infiltrating T lymphocytes induced by a viral vector, the method comprising: (a) administering to a subject having cancer the recombinant viral vector of any one of embodiments 1-52 or the pharmaceutical composition of embodiment 59; (b) isolating tumor-infiltrating T lymphocytes from the subject; and (c) expanding the tumor-infiltrating T lymphocytes ex vivo.

[0349] Embodiment 78. The method of embodiment 77, the method further comprising expanding the tumor-infiltrating T lymphocytes ex vivo in the presence of IL-2, IL-7, GSK3b, or a combination thereof.

[0350] Embodiment 79. The method of any one of embodiments 60-78, wherein the subject is a mammal.

[0351] Embodiment 80. The method of embodiment 79, wherein the subject is a human.

[0352] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. It is further to be understood that this disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the disclosure, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments disclosed herein.

[0353] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context 73 168734056.1excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0354] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0355] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure. EXAMPLES

[0356] Example 1: Material and methods for Examples 2-8

[0357] Mice and cell lines

[0358] Female C57BL / 6 (B6 in short) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free conditions in Allegheny Health Network Research Institute Preclinical Facility. All animal studies were approved Allegheny Health Network Research Institute Institutional Animal Care and Use Committee. Mouse colon cancer MC38-luc and renal adenocarcinoma CT26-luc cells were generated by the infection of parental tumor cells with firefly luciferase-carrying lentivirus and antibiotic blasticidin selection. Normal African green monkey kidney fibroblast CV1, Human embryonic kidney 293 (HEK293) cells, human ovarian cancer HeLa cells, and mouse melanoma B16 cells were obtained from American Type Culture Collection. HEK293 cells or Hela cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 15-20% Calf Bovine Serum (CBS), 2 mM L-glutamine, and 1 x penicillin / streptomycin in a 37°C, 5% CO2incubator. Other cell lines were grown in DMEM supplemented with 10% FBS, 2 mM L- glutamine, and 1 x penicillin / streptomycin in a 37°C, 5% CO2incubator.

[0359] Virus generation

[0360] VSC20, a vgf gene-deleted Western Reserve strain vaccinia virus, was used as the parental virus for homologous recombination. Murine IL-2 and / or IL-12 cDNAs were PCR- amplified from pCMS1-mIL-2-RG, pCMS1-mIL-12p30-ires-mIL-12p45-FG, pCMS1-IL-21- F-IL-2-RG, or pCMS1-IL-21-E-IL-2-RG and inserted into pCMS1-ires or pCMS1-P2A to 74 168734056.1generate pCMS1-shuttle plasmids for the expression of the proteins shown in Figs. 1A, 1B, and 1C and Table 1. The glycosylphosphatidylinositol (GPI) anchor sequences were derived from human CD16b Val196-Ile233 and fused to the C-terminus of IL-2 or IL-12 to generate cell membrane-bound proteins (see Tables 2 and 3 for sequence). To make new viruses, CV-1 cells were infected with VSC20 at a MOI of 0.1 and then transfected with the shuttle plasmids, resulting in virus mixture of parental virus and recombinant virus, respectively. Selection of these new recombinant viruses was based on expression of yellow fluorescent protein in CV1 cells 24 hours post-infection of relative virus mixture. vvDD-YFP, or vvDD for short, a double viral gene-inactivated (tk− and vgf−) vaccinia virus carrying yfp cDNA at the tk locus, was the control virus for this work. All genomes contained an inactivated thymidine kinase (TK) gene. TK is involved in the synthesis of deoxyribonucleotides. The viral TK gene is not required for dividing cells, while is essential for infection of resting cells. Disruption of the vaccinia virus TK gene leads to significant attenuation in normal tissues, while dividing cells and tumor tissues are able to complement this gene deletion and support viral replication. Table 1. Viral vectors for the expression of cytokines. *Single chain IL-12 comprises scIL- 12p35 and scIL-12p40 connected by a flexible linker. IL-12p35 and IL-12p40 that are expressed separately pair with each other and form a disulfide bond, like naturally occurring IL-12. See Tables 2-4 for sequences. Viral vector Cytokine(s) to be expressed name Protein 1 Protein 2 ”– ” –75 168734056.1Viral vector Cytokine(s) to be expressed name Protein 1 Protein 2 vvDD-IL-1235- IL-1235 IL-1240 – riid linkr “R” – – ” – – – ” – ” – – id or – id or – –76 168734056.1

[0361] The expression of gene of interest in vitro

[0362] MC38-luc (3×105), CT26-luc (2×105), or B16 (2×105) cells were seeded in 24-well plates overnight and mock-infected or infected with vvDD, vvDD-IL-2-RG, vvDD-scIL-12- FG, or vvDD-scIL-12-R-IL-2-RG, at an MOI of 1 in 0.15 mL 2 % FBS-containing-DMEM for 2 h, respectively.350 μL 10 % FBS-containing-DMEM was added to cells and the cells were cultured until harvest at 24 h post-viral infection. The cell pellets were applied to extract RNA to measure the viral house-keeping gene A34R, transgene IL-2 or IL-12p40 using RT-qPCR, respectively. One microgram of RNA was used for cDNA synthesis, and 25 to 50 ng of subsequent cDNA was used to conduct mRNA expression TaqMan analysis on the Quantagene q225 qPCR System (Kubo Technology, Beijing, China). All primers for the analysis were purchased from Thermo Fisher Scientific (Waltham, Massachusetts). Gene expression was normalized to the housekeeping gene HPRT1 and expressed as fold increase (2−ΔCT), where ΔCT=CT(target gene) – CT (HPRT1).

[0363] Rodent tumor models

[0364] B6 mice were intraperitoneally (i.p.) inoculated with 5×105MC38-luc cancer cells or or BALB / c mice were intraperitoneally (i.p.) inoculated with 5×105Renca-luc cancer cells, respectively, and divided into required groups at the indicated day post-tumor cell inoculation according to tumor size based on live animal IVIS imaging, performed using a Xenogen IVIS 200 optical in vivo imaging system (Caliper Life Sciences, Hopkinton, MA). Grouped mice were i.p. injected with indicated viruses, or PBS.

[0365] MC38-luc-tumor-bearing B6 mice treated with indicated viruses respectively, which had survived for more than 150 days, were subcutaneously (s.c.) challenged with 1×106parental MC38 cancer cells in the left flanks and 6×105B16 cells in the right flanks, respectively. Naïve B6 mice also received the same dose tumor challenge as a control. Subcutaneous tumor size was measured using an electric caliper in two perpendicular diameters.

[0366] Human T cell expansion in vitro

[0367] The following lentiviral transfer plasmids were generated: (a) pLVX-hIL-2-IRES-ZsGreen-P2A-Puro (expressing soluble human IL-2); (b) pLVX-hIL-2-RG-IRES-ZsGreen-P2A-Puro (expressing membrane-bound human IL-2 fused to rigid linker “R” fused to membrane anchor “GPI”); (c) pLVX-hscIL-12-FG-IRES-ZsGreen-P2A-Puro (expressing membrane-bound single chain human IL-12 fused to flexible linker “F” fused to membrane anchor “GPI”); 77 168734056.1(d) pLVX-hscIL-12-R-IL-2-IRES-ZsGreen-P2A-Puro (expressing soluble single chain human IL-12 fused to rigid linker “R” fused to IL-2); and (e) pLVX-hscIL-12-R-IL-2-RG-IRES-ZsGreen-P2A-Puro (expressing membrane-bound single chain human IL-12 fused to rigid linker “R” fused to IL-2 fused to rigid linker “R” fused to membrane anchor “GPI”).

[0368] The parent transfer vector pLVX-IRES-ZsGreen-P2A-Puro was created based on transfer vector pLVX-IRES-ZsGreen1 (Clontech, USA). ZsGreen1 is a human codon- optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen. The vector expresses ZsGreen1 and a protein of interest from a bicistronic mRNA transcript, allowing ZsGreen1 to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry.

[0369] The transfer plasmids were co-transfected with package plasmids pMD2.G and psPAX2 into HEK293T cells using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher) to generated lentiviruses. These lentiviruses were titrated using Lentivirus Titer p24 ELISA Kit (GenScript, USA) and used to transduce T cells to deliver human IL-2, membrane- bound human IL-2, membrane-bound human IL-12, human scIL-12-R-IL-2 and membrane- bound human scIL-12-R-IL-2, respectively. Human PBMC were activated with anti- CD3 / CD28 antibodies for three days in the presence of human IL-2 using TexMACS Medium (Miltenyi Biotech, USA) supplied with 10% FBS and 1 x penicillin / streptomycin in a 37°C, 5% CO2 incubator. Activated T cells (2.5×104) were transduced with indicated lentiviruses with an MOI of 240 in TexMACS Medium supplied with 20% FBS, 1 x penicillin / streptomycin and containing 8 μg / mL polybrene in a 37 °C, 5% CO2 incubator in the absence of human IL- 2 in 96-well plates (total volume 250μL / well). The transduction medium was changed to fresh TexMACS Medium supplied with 10% FBS and 1 x penicillin / streptomycin 24 hours after transduction and culture medium was half-changed every other day since day 3 after transduction. Live and dead cells were counted on day 7 and day 9 after transduction.

[0370] Statistics

[0371] Statistical analyses were performed using unpaired Student’s t test (GraphPad Prism version 9). Data are means ± SD. Animal survival is presented using Kaplan-Meier survival curves and was statistically analyzed using a log-rank test (GraphPad Prism version 9). Tumor growth cures were statistically analyzed using two-way ANOVA (GraphPad Prism version 9). Values of P < 0.05 were considered statistically significant, and all P values were two-sided. 78 168734056.1In the figures, standard symbols are used: * P<0.05; ** P<0.01; *** P<0.001; and **** P <0.0001.

[0372] Example 2: Viral vector replication and cytokine expression in tumor cells

[0373] To demonstrate that oncolytic vaccinia viral vectors are useful for the delivery of genes for the expression of cytokines in tumor cells, the cells were (i) mock-infected or infected with (ii) vvDD or (iii) vvDD expressing membrane-anchored IL-2 (vvDD-IL-2-RG), (iv) vvDD expressing membrane-anchored single chain (sc) IL-12 (vvDD-scIL-12-FG), or (v) vvDD expressing scIL-12 fused to IL-2 with a linker, whereby IL-2 in turn was fused to a membrane anchor, respectively, at a multiplicity of infection (MOI) of 1. The following tumor cell lines were used: MC38-luc (3×105cells), a mouse colon cancer cell line expressing luciferase, B16 (2×105cells), a mouse melanoma cell line, and CT26-luc (2×105cells), a mouse colorectal carcinoma cell line expressing luciferase.

[0374] Expression of control gene A34 (a viral housekeeping gene encoding a vaccinia outer envelope protein), IL-2, and IL-12 was determined by RT-qPCR (Fig.2).

[0375] All vectors expressed their respective transgenes, indicating that the designed viral vectors are useful for the delivery of genes for the expression of membrane-anchored cytokines IL-2 and IL-12, as well as a membrane-anchored fusion protein comprising IL-2 and IL-12, to different tumor cells.

[0376] Example 3: Oncolytic vaccinia viral vectors expressing membrane-anchored cytokines show viral toxicity in vitro

[0377] Tumor cell MC38-luc (1×104cells), CT26-luc (1×104cells) or B16 (8×103cells), were (i) mock-infected or infected with (ii) vvDD or (iii) vvDD expressing membrane- anchored IL-2 (vvDD-IL-2-RG), (iv) vvDD expressing membrane-anchored single chain (sc) IL-12 (vvDD-scIL-12-FG), or (v) vvDD expressing membrane-anchored scIL-12 fused to IL- 2 with a linker, whereby IL-2 in turn was fused to a membrane anchor, respectively, at different MOIs.

[0378] In all cancer cell lines, the different viral constructs induced cancer cell killing in a dose-dependent manner (Fig.3).

[0379] Example 4: Oncolytic vaccinia viral vectors expressing a membrane-anchored IL-2 / IL-12 fusion protein elicit potent therapeutic effects in murine cancer models 79 168734056.1

[0380] To assess the anti-tumor activity of membrane-anchored fusion proteins comprising IL-2 and IL-12, C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells (mouse colon cancer cells) (Fig. 4A) and BALB / c mice were i.p. inoculated with 5×105Renca-luc cells (mouse kidney tumor cells) (Fig.4B). The C57BL / 6 mice were treated with (i) PBS, (ii) vvDD or (iii) vvDD expressing membrane-anchored IL-2 (vvDD-IL-2-RG), (iv) vvDD expressing membrane-anchored single chain (sc) IL-12 (vvDD-scIL-12-FG), or (v) vvDD expressing scIL-12 fused to IL-2 with a linker, whereby IL-2 in turn was fused to a membrane anchor, respectively, at 2×108PFU / mouse nine days after tumor inoculation. The BALB / c mice were treated with the same constructs / control at 1×108PFU / mouse five days after tumor inoculation. Mouse survival was determined.

[0381] In both a colon cancer model (Fig. 4A) and a kidney cancer model (Fig. 4B), membrane-anchored fusion proteins comprising IL-2 and IL-12 elicited more potent antitumor effects, resulting in increased survival, as compared to membrane-anchored versions of IL-2 or IL-12 alone.

[0382] Example 5: Oncolytic vaccinia viral vectors expressing a membrane-anchored IL-2 / IL-12 fusion protein elicit potent therapeutic effects in a murine colon cancer model

[0383] To assess the anti-tumor activity of membrane-anchored fusion proteins comprising IL-2 and IL-12, C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells and treated with PBS, vvDD, or vvDD expressing different membrane-anchored cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. Mouse survival was determined. Vectors comprising an IRES or P2A sequence facilitate production of cytokines or cytokine subunits as separate polypeptides, opposed to as a fusion protein. See Table 1 for vector nomenclature. As in naturally occurring IL-12, the two separately expressed subunits of IL-12 may form the p70 form of IL-12. As such, vectors in Fig.5 containing an IRES site or a P2A sequence result in the production of membrane-anchored heterodimers comprising (1) IL-12p35 and (2) a fusion protein comprising IL12p40, IL-2, and a membrane anchor.

[0384] Membrane-anchored fusion proteins comprising IL-2 and IL-12 elicited potent antitumor effects in the colon cancer model (Fig.5). 80 168734056.1

[0385] Example 6: Oncolytic vaccinia viral vectors expressing a membrane-anchored IL-2 / IL-12 fusion protein elicit potent therapeutic effects at the memory phase

[0386] To demonstrate that oncolytic vaccinia viral vectors expressing a membrane- anchored fusion protein fusion comprising IL-2 and IL-12 elicit potent tumor-specific antitumor effects at the memory phase, MC38-luc-intraperitoneal-bearing C57BL / 6 mice were treated with vvDD expressing the membrane-anchored cytokine(s) indicated in Figs. 6A and 6B. Mice that survived more than 150 days were s.c. injected with 1×106MC38 cells (same type of tumor) in the left flanks and 6×105B16 cells (different type of tumor) in the right flanks, respectively. Tumor growth cures were determined. See Table 1 for vector nomenclature.

[0387] Mice treated with vectors expressing membrane-anchored fusion protein fusion comprising IL-2 and IL-12 rejected a re-challenge with the same type of tumor (Fig.6A) and showed potent antitumor effects in response to a challenge with a different tumor (Fig. 6B). Thus, vaccinia viruses expressing membrane-anchored fusion protein fusion comprising IL-2 and IL-12 elicit long-term, tumor-specific antitumor effects.

[0388] Example 7: Oncolytic vaccinia viral vectors expressing a soluble IL-2 / IL-12 fusion proteins elicit potent therapeutic effects and low toxicity

[0389] To assess the anti-tumor activity and toxicity of soluble fusion proteins comprising IL-2 and IL-12, C57BL / 6 mice were i.p. inoculated with 5×105MC38-luc cells and treated with vvDD expressing the indicated cytokine(s) at 2×108PFU / mouse nine days after tumor inoculation. Mouse survival (a measure of both toxicity and efficacy of an anti-cancer drug) and body weight (a measure of toxicity) was determined. Vectors comprising an IRES or P2A sequence facilitate production of cytokines or cytokine subunits as separate polypeptides, opposed to as a fusion protein. See Table 1 for vector nomenclature. As in naturally occurring IL-12, the two separately expressed subunits of IL-12 may form the p70 form of IL-12.

[0390] Mice treated with vectors expressing (1) IL-12p35 and IL-12p40 fused to IL-2 via a flexible linker (wherein the genes for IL-12p35 and IL-12p40 were separated with an IRES site) or (2) IL-12p35 and IL-12p40 fused to IL-2 via a rigid linker (wherein the genes for IL- 12p35 and IL-12p40 were separated with an IRES site) showed comparable survival and body weight profiles (Fig.7A).

[0391] While vectors expressing IL-12 fused to IL-2 via a flexible linker or IL-12 fused to IL-2 via a rigid linker, respectively, both lead to an initial loss in body weight, mice treated with a vector expressing IL-12 fused to IL-2 via a flexible linker recovered (Fig. 7B). 81 168734056.1Consistent with this, mice treated with a vector expressing IL-12 fused to IL-2 via a flexible linker showed increased survival as compared to mice treated with a vector expressing IL-12 fused to IL-2 via a rigid linker (Fig.7B).

[0392] Mice treated with vectors expressing (1) IL-12p35 and IL-12p40 fused to IL-2 via a flexible linker (wherein the genes for IL-12p35 and IL-12p40 were separated with a P2A site) or (2) IL-12p35 and IL-12p40 fused to IL-2 via a rigid linker (wherein the genes for IL-12p35 and IL-12p40 were separated with P2A site) showed both excellent survival and body weight profiles, with the best body weights and survival observed for the vector expressing IL-12p35 and IL-12p40 fused to IL-2 via a flexible linker (wherein the genes for IL-12p35 and IL-12p40 were separated with a P2A site) (Fig.7C).

[0393] Overall, constructs vvDD-IL-12p35-P2A-IL-12p40-R-IL-2, vvDD-scIL-12-F-IL-2, and vvDD-IL-12p35-P2A-IL-12p40-F-IL-2 showed the lowest toxicity and highest survival (Fig.8).

[0394] Example 8: Transduction of human T cells with lentiviral vectors for the expression of membrane-anchored IL-2 / IL-12 fusion proteins promotes T cell proliferation and improves T cell viability

[0395] The ability of membrane-bound IL-2 / IL-12 fusion proteins on T cell proliferation and viability was assessed. To that end, lentiviral constructs expressing the following soluble proteins were created: (1) human IL-2 and (2) hscIL-12-R-IL-2 (soluble single chain human IL-12 fused to rigid linker “R” fused to IL-2). Further, lentiviral constructs expressing the following membrane-bound proteins were created: (1) hIL-2-RG-IRES-ZsGreen-P2A-Puro (IL-2 fused to rigid linker “R” fused to membrane anchor “GPI”); (2) hscIL-12-FG (single chain human IL-12 fused to flexible linker “F” fused to membrane anchor “GPI”); and (3) hscIL-12-R-IL-2-RG (single chain human IL-12 fused to rigid linker “R” fused to IL-2).

[0396] In the presence of IL-2 / IL-12 fusion proteins, the initial amount of 2.5×104T cells was expanded to about 8.2 ×105on Day 9 after transduction, an about 33-fold expansion. This is comparable to the expansion typically achieved (around 30-fold) when cultivating activated T cells in the presence of IL-2 for 9 days (Figs.9A and 9B). 82 168734056.1Table 2. Nucleic acid sequences. The sequences encoding “GPA” (e.g., GGGCCGGCC) and GAP (e.g., GGCGCGCCT) are cloning artefacts and are not required for (but also do not detract from) function. Any stop codon known in the art can be used in the sequences disclosed herein (this applies to both 3’ and internal stop codons). SEQ Descrip- Sequence ID tion N C C G A C T G G T A A G T G C G A A G T T G C168734056.1SEQ Descrip- Sequence ID tion NO A A G C C A C G A T C G T C A T T C T T A T T C A T A A C T A C84 168734056.1SEQ Descrip- Sequence ID tion NO G T A C T A A G A G C G C T T C T A C C C168734056.1SEQ Descrip- Sequence ID tion NO A C T T A T G G C C C A G T T T C G C A C T C T A A T G C A168734056.1SEQ Descrip- Sequence ID tion NO G C T T A C C C A C A A T A A A A C G T C G168734056.1SEQ Descrip- Sequence ID tion NO C C C G A A G G C G G C C A A C C C A C A A T A168734056.1SEQ Descrip- Sequence ID tion NO A A A C G T C G C C G C A A G A A G T G A T G A A C C C A C168734056.1SEQ Descrip- Sequence ID tion NO A A T A A A A C G T C G C C C C A A A168734056.1SEQ Descrip- Sequence ID tion NO A T A C C C A C A A T A A A A C G T C G C168734056.1SEQ Descrip- Sequence ID tion NO C G C G T T T A A T G T A C C C A C A A T C C A G T168734056.1SEQ Descrip- Sequence ID tion NO C A T C T A C T A A G A G C G C T T T T A C C G T T T A C168734056.1SEQ Descrip- Sequence ID tion NO T C A C C C A C A A T C C A G T C A T C T A C T A A G168734056.1SEQ Descrip- Sequence ID tion NO A G C G C T T A C A A G A A G T G A T G A A C C C A C A A168734056.1SEQ Descrip- Sequence ID tion NO T C C A G T C A T C T A C T A A G A G C G C T T T G168734056.1SEQ Descrip- Sequence ID tion NO C T G A A G C T T G A C C C A C A A T C C A G T C A168734056.1SEQ Descrip- Sequence ID tion NO T C T A C T A A G A G C G C T T C G T C G A G T A T T168734056.1SEQ Descrip- Sequence ID tion NO A C C C A C A A T G T A A G A A C G G C C T G A G G168734056.1SEQ Descrip- Sequence ID tion NO A A G G A G A A A C C C A C A A T G G G C C T168734056.1SEQ Descrip- Sequence ID tion NO C G T C G A T T G T G T G G C A C C C A C A A T168734056.1SEQ Descrip- Sequence ID tion NO G T A A G A A C G G C C T G A G C A G A G G A T C C G168734056.1SEQ Descrip- Sequence ID tion NO A C C C A C A A T G G G C C T C G T C G A T A G C168734056.1SEQ Descrip- Sequence ID tion NO C A A A T A C C C A C A A T A A A A C168734056.1SEQ Descrip- Sequence ID tion NO G T C G C C C G A A G G C G A C C C A C A A T168734056.1SEQ Descrip- Sequence ID tion NO A A A A C G T C G C C G C A A G A A G T A C C C A C168734056.1SEQ Descrip- Sequence ID tion NO A A T A A A A C G T C G C C C C A A A168734056.1SEQ Descrip- Sequence ID tion NO A C C C A C A A T A A A A C G T C G C C G C168734056.1SEQ Descrip- Sequence ID tion NO G T T T A A T A C C C A C A A T C C A G T C A T C T A C168734056.1SEQ Descrip- Sequence ID tion NO T A A G A G C G C T T T T A C C G T T T A A C C C A C A168734056.1SEQ Descrip- Sequence ID tion NO A T C C A G T C A T C T A C T A A G A G C G C T T168734056.1SEQ Descrip- Sequence ID tion NO A C A A G A A G T A C C C A C A A T C C A G T C A T C168734056.1SEQ Descrip- Sequence ID tion NO T A C T A A G A G C G C T T T G C T G A A G C T A C C C A168734056.1SEQ Descrip- Sequence ID tion NO C A A T C C A G T C A T C T A C T A A G A G C G C168734056.1SEQ Descrip- Sequence ID tion NO T T C G T C G A G T A C C C A C A A T G T A A G A A C168734056.1SEQ Descrip- Sequence ID tion NO G G C C T G A G G A A G G A G A C C C A C A A T168734056.1SEQ Descrip- Sequence ID tion NO G G G C C T C G T C G A T T G T G T G G A C C C A168734056.1SEQ Descrip- Sequence ID tion NO C A A T G T A A G A A C G G C C T G A G C A G A G G A168734056.1SEQ Descrip- Sequence ID tion NO T C A C C C A C A A T G G G C C T C G T C G A T168734056.1SEQ Descrip- Sequence ID tion NO A G C C A A A G G C A120 168734056.1SEQ Descrip- Sequence ID tion NOa e . mno ac sequences. e sequences an are conng areacs an are not required for (but also do not detract from) function. SEQ Description Sequence ID R H D F K L V F Y D F K V N F P P L168734056.1SEQ Description Sequence ID NO S E C Q K P N Y S R F L S Y A122 168734056.1SEQ Description Sequence ID NO168734056.1SEQ Description Sequence ID NO C K L S K L S168734056.1SEQ Description Sequence ID NO 69 K L S K E P E L S S 70 K L S K E Y L 71 K L168734056.1SEQ Description Sequence ID NO S E R A K L S Q F F S R H D A R H D E126 168734056.1SEQ Description Sequence ID NO E C Q K W L Y FI K N E C Q K W L Y FI K E C Q K W L Y FI K Q L V A168734056.1SEQ Description Sequence ID NO C E C Q K W L Y FI K E V E C Q K W L Y FI K E F A E C Q K W168734056.1SEQ Description Sequence ID NO L Y FI K L L F A S S R F L A V S R F S R D K V S R168734056.1SEQ Description Sequence ID NO F H K A L S R F L E C Q K W L Y FI K P N Y G S168734056.1SEQ Description Sequence ID NO P S K L S V F Y K Q P R K L S P E168734056.1SEQ Description Sequence ID NO L K L S Y L K L S E R K L168734056.1SEQ Description Sequence ID NO S Q F F E C Q K W L Y FI K Q L E C Q K W L Y FI K E133 168734056.1SEQ Description Sequence ID NO E C Q K W L Y FI K E F E C Q K W L Y FI K L L F S R F H K168734056.1SEQ Description Sequence ID NO S R F168734056.1SEQ Description Sequence ID NO. , nd linker sequences. Description Nucleic Amino acid acid136 168734056.1Table 5. Overview of selected fusion protein amino acid sequences. Membrane-anchored fusion proteins Soluble fusion proteins SEQ Protein SEQ Protein ID ID ) ) ne ne ne ne ”) ne ne ) ne ne ”)168734056.1Membrane-anchored fusion proteins Soluble fusion proteins SEQ Protein SEQ Protein ID ID ne ne ”)138 168734056.1

Claims

CLAIMS We claim:

1. A recombinant vaccinia virus comprising a vaccinia virus genome comprising a nucleic acid encoding: (a) a fusion protein comprising: a first polypeptide comprising an interleukin 2 (IL-2) or an IL-2 variant and a second polypeptide comprising (i) an interleukin 12 subunit p35 (IL-12p35) or an IL-12p35 variant, (ii) an interleukin 12 subunit p40 (IL-12p40) or an IL-12p40 variant, and optionally (iii) a membrane-anchoring polypeptide; (b) a heterodimeric fusion protein comprising a first polypeptide comprising an IL-12p35 or an IL-12p35 variant and a second polypeptide comprising: (i) an IL-2 or an IL-2 variant; (ii) an IL-12p40 or an IL-12p40 variant, and optionally (iii) a membrane- anchoring polypeptide; or (c) a heterodimeric fusion protein comprising first polypeptide comprising an IL-12p40 or an IL-12p40 variant and a second polypeptide comprising: (i) IL-2 or an IL-2 variant; (ii) IL-12p35 or an IL-12p35 variant, and optionally (iii) a membrane-anchoring polypeptide.

2. The recombinant vaccinia virus of claim 1(b) or 1(c), wherein the IL-12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently linked.

3. The recombinant vaccinia virus of claim 2, wherein the IL-12p40 or IL-12p40 variant and the IL-12p35 or IL-12p35 variant are covalently by a disulfide bond.

4. The recombinant vaccinia virus of claim 1(a), wherein the nucleic acid encoding the fusion protein is operatively linked to a promoter.

5. The recombinant vaccinia virus of claim 1(b) or 1(c), wherein the nucleic acid encoding the first polypeptide and / or the nucleic acid encoding the second polypeptide is operatively linked to a promoter.

6. The recombinant vaccinia virus of claim 4 or 5, wherein the promoter is a p7.5 or a pSe / l promoter.

7. The recombinant vaccinia virus of any one of claims 1-6, wherein the IL-2 is human or murine IL-2, optionally wherein IL-2 comprises a sequence that is at least 90% identical to SEQ ID NO:40 or SEQ ID NO:

39.

8. The recombinant vaccinia virus of claim 7, wherein the IL-2 comprises SEQ ID NO:40 or SEQ ID NO:

39. 139 168734056.

19. The recombinant vaccinia virus of claim 8, wherein the IL-2 comprises SEQ ID NO:

40.

10. The recombinant vaccinia virus of any one of claims 1-9, wherein the IL-12p35 is human or murine IL-12p35, optionally wherein the IL-12p35 comprises a sequence that is at least 90% identical to SEQ ID NO:45 or SEQ ID NO:

46.

11. The recombinant vaccinia virus of claim 10, wherein the IL-12p35 comprises SEQ ID NO:45 or SEQ ID NO:

46.

12. The recombinant vaccinia virus of claim 11, wherein the IL-12p35 comprises SEQ ID NO:

46.

13. The recombinant vaccinia virus of any one of claims 1-12, wherein the IL-12p40 is human or murine IL-12p40, optionally wherein the IL-12p40 comprises a sequence that is at least 90% identical to SEQ ID NO:47 or SEQ ID NO:

48.

14. The recombinant vaccinia virus of claim 13, wherein the IL-12p40 comprises SEQ ID NO:47 or SEQ ID NO:

48.

15. The recombinant vaccinia virus of claim 14, wherein the IL-12p40 comprises SEQ ID NO:

48.

16. The recombinant vaccinia virus of any one of claims 1-15, wherein the fusion protein further comprises a first linker connecting (i) the first and (ii) the second polypeptide, optionally wherein the first linker is a polypeptide linker.

17. The recombinant vaccinia virus of any one of claims 1-16, wherein the nucleic acid further comprises a sequence encoding a second linker, wherein the second linker is located N- terminally of the membrane-anchoring polypeptide, optionally wherein the second linker is a polypeptide linker.

18. The recombinant vaccinia virus of any one of claims 1-17, wherein the IL-12p35 or IL- 12p35 variant and the IL-12p40 or IL-12p40 variant are linked by a third linker, optionally wherein the third linker is a polypeptide linker.

19. The recombinant vaccinia virus of any one of claims 16-18, wherein the polypeptide linker is a flexible polypeptide linker.

20. The recombinant vaccinia virus of claim 19, wherein the flexible polypeptide linker predominantly comprises glycines and serines.

21. The recombinant vaccinia virus of claim 20, wherein the flexible polypeptide linker comprises SEQ ID NO:53 (GGGGS) or one or more repeats of SEQ ID NO:53 (GGGGS), optionally wherein the flexible polypeptide linker comprises a sequence selected from the 140 168734056.1group consisting of GGS, SEQ ID NO:54 (GGSGGGS), SEQ ID NO:55 ((GGGGS)2), SEQ ID NO:56 ((GGGGS)3), and SEQ ID NO:47((GGGGS)4).

22. The recombinant vaccinia virus of claim 21, wherein the flexible polypeptide linker comprises SEQ ID NO:56 ((GGGGS)3).

23. The recombinant vaccinia virus of claim 16 or 17, wherein the polypeptide linker is a rigid polypeptide linker.

24. The recombinant vaccinia virus of claim 23, wherein the rigid polypeptide linker predominantly comprises alanines.

25. The recombinant vaccinia virus of claim 23, wherein the rigid polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO:58 (A(EA3K)1AAA), SEQ ID NO:59 (A(EA3K)2AAA), SEQ ID NO:60 (A(EA3K)3AAA), SEQ ID NO:61 (A(EA3K)4AAA), or SEQ ID NO:62 (A(EA3K)5AAA), optionally wherein the rigid polypeptide linker comprises SEQ ID NO:61 (A(EA3K)4AAA).

26. The recombinant vaccinia virus of any one of claims 1-25, wherein the membrane- anchoring polypeptide is between about 10 and about 50 amino acids in length.

27. The recombinant vaccinia virus of claim 26, wherein the membrane-anchoring polypeptide comprises a glycosylphosphatidylinositol (GPI)-anchor acceptor peptide.

28. The recombinant vaccinia virus of claim 26, wherein the membrane-anchoring polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104.

29. The recombinant vaccinia virus of claim 28, wherein the membrane-anchoring polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:63 or 97-104.

30. The recombinant vaccinia virus of claim 29, wherein the membrane-anchoring polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:63, optionally wherein the membrane-anchoring polypeptide comprises SEQ ID NO:

63.

31. The recombinant vaccinia virus of claim 1, wherein the fusion protein comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:11-34.

32. The recombinant vaccinia virus of claim 29, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs:11-34.

33. The recombinant vaccinia virus of any one of claims 1-32, wherein the vaccinia virus genome has a deletion of the thymidine kinase (TK) gene, the vaccinia growth factor (VGF gene), the A56R gene, or combinations thereof. 141 168734056.

134. The recombinant vaccinia virus of any one of claims 1-33, wherein the vaccinia virus genome has a deletion of the viral gene for A41L, A44L, A46R, A49, A52R, A53R, B5R, B8R, B13R (SPI-2), B15R, B18R, C3L (VCP), C6, C7L, C12L, E3L, F1L, K1L, K3L, K7R, M1L, N1L, or combinations thereof.

35. A cell comprising the recombinant vaccinia virus of any one of claims 1-34.

36. The cell of claim 35, wherein the cell is a mammalian cell, optionally, wherein the cell is a human cell.

37. The cell of claim 35 or 36, wherein the cell is a cancer cell, a stromal cell in a tumor microenvironment, or an immune cell.

38. A pharmaceutical composition comprising the recombinant vaccinia virus of any one of claims 1-34 and one or more pharmaceutically acceptable excipients.

39. A method for treating cancer, the method comprising administering to a subject in need thereof the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38.

40. The method of claim 39, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, gastric cancer, sarcoma, mesothelioma, ovarian cancer, endometrial cancer, or cervical cancer.

41. A method for reducing tumor growth, the method comprising administering to a subject in need thereof the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38.

42. A method for reducing tumor metastasis, the method comprising administering to a subject in need thereof the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38.

43. A method for increasing cytokine production in the tumor microenvironment, the method comprising administering to a subject in need thereof the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38.

44. A method for increasing infiltration of a tumor with immune cells, the method comprising administering to a subject in need thereof the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38.

45. The method of any one of claims 39-44, wherein the step of administering comprises intraperitoneal or systemic administration. 142 168734056.

146. The method of any one of claims 39-45, the method further comprising administering to the subject an additional antineoplastic agent.

47. The method of claim 46, wherein the antineoplastic agent is an immune checkpoint inhibitor.

48. The method of claim 47, wherein the immune checkpoint inhibitor is selected from the group of a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a CTLA4 inhibitor, or combinations thereof.

49. The method of claim 47 or 48, wherein the immune checkpoint inhibitor is an antibody or fragment thereof.

50. The method of claim 46, wherein the antineoplastic agent is a chimeric antigen receptor (CAR) T lymphocyte, a CAR macrophage, a CAR natural killer cell, or a tumor infiltrating lymphocyte.

51. A method of generating tumor-infiltrating vaccinia virus-induced T lymphocytes, the method comprising: (a) administering to a subject having cancer the recombinant vaccinia virus of any one of claims 1-34 or the pharmaceutical composition of claim 38; (b) isolating tumor-infiltrating T lymphocytes from the subject; and (c) expanding the tumor-infiltrating T lymphocytes ex vivo.

52. The method of claim 51, the method further comprising expanding the tumor-infiltrating T lymphocytes ex vivo in the presence of IL-2, IL-7, GSK3b, or a combination thereof.

53. The method of any one of claims 39-50, wherein the subject is a mammal.

54. The method of claim 53, wherein the subject is a human. 143 168734056.1

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