Combination of recombinant virus expressing interleukin-12 with PD-1 / PD-L1 inhibitor

Abstract

The present disclosure relates to a therapeutic combination comprising (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor. Also provided are methods of treating cancer and methods of enhancing a tumor-specific immune response, comprising administering the therapeutic combination.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This international application claims priority to U.S. Provisional Application No. 63 / 369,605, filed on July 27, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the electronically submitted XML sequence listing submitted with this application (name: 2943_230PC01_SequenceListing_ST26; size: 48,263 bytes; and creation date: July 19, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to a combination comprising (i) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, and (ii) an inhibitor of PD-1 / PD-L1. Such a combination can be used to treat cancer, including cancers that are resistant to therapy using immune checkpoint inhibitors. Background Art

[0006] Recombinant viruses, including, for example, recombinant poxviruses, represent an emerging therapeutic platform for treating cancer because they have advantages over conventional treatment modalities (e.g., chemotherapy). For example, recombinant viruses can selectively replicate in cancer cells while sparing normal cells and tissues, thereby limiting off-target cell killing and toxicity, thus providing levels of efficacy and specificity that may be far greater than conventional cancer treatments. Recombinant viruses can be engineered to express therapeutic transgenes in cells, such as transgenes that are important in cancer biological pathways. Cancer cells are ideal hosts for many viruses because they can inactivate antiviral interferon pathways or can have mutated tumor suppressor genes that allow viral replication to proceed unimpeded.

[0007] Interleukin-12 (IL-12) is considered a potential candidate for anticancer therapy and has been introduced into viral vectors (e.g., adenoviral vectors) for evaluation. IL-12 is a cytokine with immunomodulatory and anti-angiogenic functions. IL-12 acts as a key regulator of cell-mediated immune responses by inducing T helper 1 cell differentiation and induces cellular immunity by promoting interferon-γ (IFN-γ) production, proliferation, and cytolytic activity of natural killer cells and T cells. The versatility of IL-12 has triggered research on this cytokine as an anticancer agent. However, despite encouraging results in animal models, the antitumor effects of IL-12 are very limited, and unacceptable adverse events have occurred in early clinical trials, which has reduced the hope of successful use of this cytokine.

[0008] Immune checkpoint inhibitors (such as PD-1 and PD-L1 targeting molecules) have demonstrated some success as cancer therapeutics. However, not all cancers respond to checkpoint inhibitors, and some cancers become resistant to checkpoint inhibitors.

[0009] Therefore, there remains a need for new cancer therapies that increase efficacy and / or reduce adverse events. Summary of the invention

[0010] In some aspects, the present disclosure provides a therapeutic combination comprising (a) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, and (b) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some aspects, the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid-promoter. In some aspects, the poxvirus belongs to the genus Orthopoxvirus. In some aspects, the poxvirus belonging to the genus Orthopoxvirus is an oncolytic vaccinia virus. In some aspects, the oncolytic vaccinia virus is selected from the group consisting of Copenhagen (Cop), Western Reserve (WR), Elstree, Wyeth, Lister, TianTan and LIVP virus strains. In some aspects, the genome comprises at least 150 kb, at least about 175 kb, at least about 180 kb, at least about 185 kb, at least about 190 kb, at least about 192 kb, or at least about 194 kb. In some aspects, the poxvirus is attenuated. In some aspects, the poxvirus is not NYVAC.

[0011] In some aspects of the therapeutic combinations disclosed herein, the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG7L and pF17R. In some aspects, the late promoter is selected from pA14L, pA26L and pF17R. In some aspects, the late promoter is pA14L. In some aspects, the late promoter is pF17R. In some aspects, the late promoter comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23. In some aspects, the late promoter comprises a nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23.

[0012] In some aspects, the mid-stage promoter is selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA3L, or pA27L. In some aspects of the therapeutic combinations disclosed herein, the mid-stage promoter comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 25-31.

[0013] In some aspects of the therapeutic combinations disclosed herein, IL-12 is human IL-12. In some aspects, IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit. In some aspects, the IL-12p40 subunit is located at the N-terminus of the IL-12p35 subunit. In some aspects, the IL-12p40 subunit comprises an amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the amino acid sequence of SEQ ID NO: 17. In some aspects, the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 19. In some aspects, the IL-12p40 subunit and the IL-12p35 subunit are fused in a single polypeptide via an amino acid linker. In some aspects, the length of the amino acid linker is about 5 to about 10 amino acids. In some aspects, the length of the amino acid linker is 7 amino acids. In some aspects, the amino acid linker is a glycine-serine linker. In some aspects, the amino acid linker comprises the amino acid sequence of SEQ ID NO: 18. In some aspects, the IL-12 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 20. In some aspects, the IL-12p40 subunit and the IL-12p35 subunit are directly fused in a single polypeptide. In some aspects, the heterologous nucleic acid sequence encoding IL-12 comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 21. In some aspects, the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO: 21.

[0014] In some aspects of the therapeutic combinations disclosed herein, the thymidine kinase (TK) activity of the poxvirus is defective. In some aspects, the poxvirus lacks a functional J2R gene. In some aspects, the ribonucleotide reductase (RR) activity of the poxvirus is defective. In some aspects, the poxvirus lacks a functional I4L gene. In some aspects, the poxvirus lacks a functional F4L gene. In some aspects, a heterologous nucleic acid sequence encoding IL-12 is inserted into the J2R locus of the poxvirus genome. In some aspects, the insertion makes the J2R gene non-functional, optionally wherein the J2R locus is completely deleted by insertion. In some aspects, a heterologous nucleic acid sequence encoding IL-12 is inserted into the I4L locus of the poxvirus genome. In some aspects, the insertion makes the I4L gene non-functional, optionally wherein the I4L locus is not completely deleted by insertion. In some aspects, a heterologous nucleic acid sequence encoding IL-12 is inserted into the F4L locus of the poxvirus genome. In some aspects, the insertion renders the F4L gene non-functional, optionally wherein the F4L locus is not completely deleted by the insertion.

[0015] In some aspects of the therapeutic combinations disclosed herein, the poxvirus further comprises one or more therapeutic genes in its genome. In some aspects, the one or more therapeutic genes are selected from the group consisting of suicide genes, immunomodulatory genes, anti-angiogenesis genes, immune checkpoint blocking genes, antibody encoding genes, extracellular matrix degradation or regulation genes, and combinations thereof.

[0016] In some aspects, the therapeutic combination disclosed herein can lyse one or more cancer cells. In some aspects, the recombinant poxvirus can express at least 50ng / mL, at least 100ng / mL, at least 300ng / mL, at least 500ng / mL, at least 1.0μg / mL, at least 2.0μg / mL, at least 3.0μg / mL, at least 4.0μg / mL, at least 5.0μg / mL, at least 6.0μg / mL, at least 7.0μg / mL, at least 8.0μg / mL or about 8.3μg / mL of IL-12 in cancer cells 72 hours after infection with a multiplicity of infection (MOI) of 10-2. In some aspects, the cancer cell is a renal cancer cell, a prostate cancer cell, a breast cancer cell, a bladder cancer cell, a colorectal cancer cell, a lung cancer cell, a liver cancer cell, a gastric cancer cell, a bile duct cancer cell, an endometrial cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a head and neck cancer cell, a melanoma cell, a glioblastoma cell, a multiple myeloma cell or a malignant glioma cell. In some aspects, the cancer cell is A549, HT29, MIAPaCa-2, A375, RPMI7591, Sk-Mel-5, OVCAR3, OVCAR4, NCI-H292, NCI-H460, SW 780, TCCSUP, T24, Huh7, Hep3B, Panc1, Hup-T3, DAN-G, MDA-MB-435, HCC38, BT20, SW1417, WiDr, HCT-116, SNU5, NCI-N87, Kato III, A CHN, A498, PC-3, or MM.1R cell.

[0017] In some aspects of the therapeutic combinations disclosed herein, the virus is expressed in chicken embryo fibroblasts (CEF), HeLa cells, In some aspects of the therapeutic combinations disclosed herein, the virus is produced in chicken embryo fibroblasts (CEF).

[0018] In some aspects of the therapeutic combinations disclosed herein, the recombinant poxvirus is capable of upregulating interferon (IFN)-γ.

[0019] In some aspects of the therapeutic combinations disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some aspects, the anti-PD-1 antibody or its antigen-binding fragment or the anti-PD-L1 antibody or its antigen-binding fragment is produced in Chinese hamster ovary (CHO) cells. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a small molecule. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a PD-L1 inhibitor. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is selected from the group consisting of Durvalumab, nivolumab, pembrolizumab, lambrolizumab, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, T SR-042, Pidilizumab, GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, atezolizumab, MPDL3280A, RG7446, avelumab, STI-1014, CX-072, KN035, and CK-301. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 33; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 34; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39.

[0020] In some aspects of the therapeutic combinations disclosed herein, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc variant, wherein the Fc variant comprises at least one amino acid substitution selected from the group consisting of 234F, 235F and 331S as numbered by the EU index as set forth in Kabat.

[0021] In some aspects of the therapeutic combinations disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

[0022] In some aspects, the present disclosure provides a therapeutic combination comprising (a) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, and (b) durvalumab. In some aspects, the therapeutic combination is used to (i) treat cancer in a subject, (ii) inhibit the growth of cancer, or (iii) enhance a tumor-specific immune response.

[0023] In some aspects, the present disclosure provides a therapeutic combination, wherein the therapeutic combination is a kit of parts comprising (a) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, and (b) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor.

[0024] In some aspects, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some aspects, the present disclosure provides a method for inhibiting cancer growth in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor. In some aspects, the present disclosure provides a method for enhancing a tumor-specific immune response in a subject suffering from cancer, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor. In some aspects, the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter.

[0025] In some aspects of the methods disclosed herein, the poxvirus belongs to the genus Orthopoxvirus. In some aspects, the poxvirus belonging to the genus Orthopoxvirus is an oncolytic vaccinia virus. In some aspects, the oncolytic vaccinia virus is selected from the group consisting of: Copenhagen (Cop), Western Reserve (WR), Elstree, Wyeth, Lister, Tian Tan, and LIVP strains. In some aspects, the genome comprises at least 150 kb, at least about 175 kb, at least about 180 kb, at least about 185 kb, at least about 190 kb, at least about 192 kb, or at least about 194 kb. In some aspects, the poxvirus is attenuated. In some aspects, the poxvirus is not NYVAC.

[0026] In some aspects of the methods disclosed herein, the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG7L and pF17R. In some aspects, the late promoter is selected from pA14L, pA26L and pF17R. In some aspects, the late promoter is pA14L. In some aspects, the late promoter is pF17R. In some aspects, the late promoter comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23. In some aspects, the late promoter comprises a nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23.

[0027] In some aspects of the methods disclosed herein, the mid-stage promoter is selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA3L, or pA27L. In some aspects, the mid-stage promoter comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 25-31.

[0028] In some aspects of the methods disclosed herein, IL-12 is human IL-12. In some aspects, IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit. In some aspects, IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit. In some aspects, the IL-12p40 subunit comprises an amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 17. In some aspects, the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of SEQ ID NO: 19. In some aspects, the IL-12p40 subunit and the IL-12p35 subunit are fused in a single polypeptide via an amino acid linker. In some aspects, the length of the amino acid linker is about 5 to about 10 amino acids, optionally wherein the length of the amino acid linker is 7 amino acids. In some aspects, the amino acid linker is a glycine-serine linker. In some aspects, the amino acid linker comprises the amino acid sequence of SEQ ID NO: 18. In some aspects, the IL-12 comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 20. In some aspects, the IL-12p40 subunit and the IL-12p35 subunit are directly fused in a single polypeptide.

[0029] In some aspects of the methods disclosed herein, the heterologous nucleic acid sequence encoding IL-12 comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 21, optionally wherein the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO: 21.

[0030] In some aspects of the methods disclosed herein, the recombinant poxvirus is defective in thymidine kinase (TK) activity. In some aspects, the recombinant poxvirus lacks a functional J2R gene. In some aspects, the recombinant poxvirus is defective in ribonucleotide reductase (RR) activity. In some aspects, the recombinant poxvirus lacks a functional I4L gene. In some aspects, the recombinant poxvirus lacks a functional F4L gene. In some aspects, a heterologous nucleic acid sequence encoding IL-12 is inserted into the J2R locus of the poxvirus genome. In some aspects, the insertion renders the J2R gene non-functional, optionally wherein the J2R locus is completely deleted by insertion.

[0031] In some aspects of the methods disclosed herein, the recombinant poxvirus is capable of lysing one or more cancer cells. In some aspects, the recombinant poxvirus is capable of expressing at least 50 ng / mL, at least 100 ng / mL, at least 300 ng / mL, at least 500 ng / mL, at least 1.0 μg / mL, at least 2.0 μg / mL, at least 3.0 μg / mL, at least 4.0 μg / mL, at least 5.0 μg / mL, at least 6.0 μg / mL, at least 7.0 μg / mL, at least 8.0 μg / mL, or about 8.3 μg / mL of IL-12 in cancer cells 72 hours after infection with a multiplicity of infection (MOI) of 10-2.

[0032] In some aspects of the methods disclosed herein, the recombinant poxvirus is capable of upregulating interferon (IFN)-γ.

[0033] In some aspects of the methods disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some aspects, the anti-PD-1 antibody or its antigen-binding fragment or the anti-PD-L1 antibody or its antigen-binding fragment is produced in Chinese hamster ovary (CHO) cells. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a small molecule. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is a PD-L1 inhibitor. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lanolinizumab, MEDI-0680, cemiplizumab, JS001, BGB-A317, INCSHR1210, TSR-042, pidilizumab, GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, atezolizumab, MPDL3280A, RG7446, durvalumab, avelumab, STI-1014, CX-072, KN035, and CK-301. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 33; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 34; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc variant, wherein the Fc variant comprises at least one amino acid substitution selected from the group consisting of 234F, 235F, and 331S, as numbered by the EU index as set forth in Kabat. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

[0034] In some aspects of the methods disclosed herein, the cancer is renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma or malignant glioma. In some aspects, the cancer is resistant to immune checkpoint inhibitor therapy. In some aspects, the cancer is resistant to PD1 inhibitors. In some aspects, the cancer is resistant to PD-L1 inhibitors.

[0035] In some aspects of the methods disclosed herein, an effective amount of an individual dose of the recombinant poxvirus comprises 1 x 10 3 pfu to 1x 10 12 pfu, optionally 1x 10 4 pfu to 1x 10 11 pfu, optionally 1x 10 5 pfu to 1x 10 10 pfu, optionally 5x10 7 pfu to 4x 10 9 pfu.

[0036] In some aspects of the methods disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and the effective amount of an individual dose of durvalumab is 10 mg / kg. In some aspects of the methods disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and the effective amount of an individual dose of durvalumab is 1500 mg. In some aspects, durvalumab is administered at a dose of 10 mg / kg every 2 weeks, 1500 mg every 4 weeks, or 1500 mg every 3 weeks.

[0037] In some aspects of the methods disclosed herein, the administration results in an enhanced therapeutic effect compared to treatment with a recombinant poxvirus alone or treatment with a PD-1 inhibitor or PD-L1 inhibitor alone. In some aspects, the subject is a human. In some aspects, the administration is intratumoral. In some aspects, the administration is intravenous. In some aspects, the intravenous administration is via intravenous infusion. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and the dose of the administration is 10 mg / kg every 2 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and is administered at a dose of 1500 mg every 3 weeks or every 4 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and is administered at a dose of 1500 mg every 4 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and is administered at a dose of 1500 mg every 3 weeks.

[0038] In some aspects of the methods disclosed herein, the recombinant poxvirus and / or the PD-1 inhibitor or PD-L1 inhibitor are administered two or more times. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient before the administration of the recombinant poxvirus. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient simultaneously with the recombinant poxvirus. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor and the recombinant poxvirus are administered in separate pharmaceutical compositions. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor and the recombinant poxvirus are administered in the same pharmaceutical composition. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient after the administration of the recombinant poxvirus.

[0039] In some aspects, provided herein is a composition comprising a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding IL-12, for treating cancer in a subject in need thereof, wherein the composition is for administration in combination with a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the treatment comprises any one of the methods disclosed herein.

[0040] In some aspects, provided herein is a composition comprising a PD-1 inhibitor or a PD-L1 inhibitor for use in treating cancer in a subject in need thereof, wherein the composition is for administration in combination with a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding IL-12 in its genome, optionally wherein the treatment comprises any one of the methods disclosed herein.

[0041] In some aspects, the present invention also provides a pharmaceutical composition comprising a recombinant poxvirus comprising in its genome (i) a heterologous nucleic acid sequence encoding IL-12 and (ii) a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the recombinant poxvirus is a recombinant poxvirus used in any of the methods disclosed herein and / or wherein the PD-1 inhibitor or PD-L1 inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor used in any of the methods disclosed herein. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

[0042] In some aspects, a kit is also provided herein, comprising a unit dose of (i) a pharmaceutical composition comprising a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the recombinant poxvirus is a recombinant poxvirus used in any of the methods disclosed herein and / or wherein the PD-1 inhibitor or the PD-L1 inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor used in any of the methods disclosed herein. In some aspects of the kit disclosed herein, the PD-1 inhibitor or the PD-L1 inhibitor is durvalumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a schematic diagram of plasmid pTG19409.

[0044] Figure 2 Shown is the luciferase expression from six late promoters after infection / transfection of DF-1 cells.

[0045] Figure 3A and 3B Shown is luciferase expression at 6 hours (A) and 24 hours (B) after infection of MIAPaCa-2 cells with ten recombinant vaccinia viruses.

[0046] Figure 4 Shown is the luciferase expression after infection of HeLa cells.

[0047] Figure 5 Shown is the luciferase expression following infection of HCT-116 cells.

[0048] Figure 6 GFP-positive cells at 6 and 24 hours after infection of human PBMCs are shown.

[0049] Figure 7 Shown is luciferase expression at 6 and 24 hours after infection of human PBMCs.

[0050] Figure 8 The sequence of the hIL-12 expression cassette of COPTG19673 is shown (SEQ ID NO: 21).

[0051] Fig. 9 is a schematic diagram of plasmid pTG19673.

[0052] Fig.10 is a schematic diagram of plasmid pTG19674.

[0053] Fig.11Shown is the expression of IL-12 in the supernatant of A549 cells infected with primary study stocks COPTG19673 and COPTG19674 measured by ELISA.

[0054] Fig. 12A -C shows the replication of VACV-IL-12 (COPTG19673 and COPTG19674) and unarmed control VACV (VVTG18058) in human tumor cell lines A549 (A), Mi PaCa-2 (B) and HT-29 (C) at 24 h, 48 h and 72 h after infection.

[0055] Fig.13 Replication of VACV-IL-12 (COPTG19673 and COPTG19674) and unarmed control VACV (VVTG18058) in producer cells (HeLa and CEF) 72 hours after infection is shown. Results are the average of three wells.

[0056] Fig.14A -C shows: Oncolytic activity of COPTG19673, COPTG19674 and empty VACV (VVTG18058) at different MOIs in three different human tumor cell lines A549 (A), MIA PaCa-2 (B) and HT-29 (C). The results shown are the mean + / - SD of 3 measurements and are expressed as a percentage of cell viability (100% corresponds to mock-infected cells).

[0057] Fig.15 Shown are the expression levels of vIL-12 in supernatants of A549, MIA PaCa-2, and HT-29 cells infected with COPTG19673 and COPTG19674 after 3 days of incubation post infection at MOI 0.01. Results are the mean and SD of duplicate measurements of three samples.

[0058] Fig.16A -F shows HEK-Blue compared with rhIL-12 TM IL-12 bioactivity after incubation of IL-12 cells with supernatants of COPTG19673-infected tumor cell lines (AC) and with supernatants of COPTG19674-infected tumor cell lines (DF), as determined using the HEKBlue IL-12 cell reporter gene assay. Results are expressed as mean ± SD of two measurements.

[0059] Fig.17A-F shows IL-12 bioactivity after incubation of NK-92 cells with supernatants of COPTG19673-infected tumor cell lines (AC), with supernatants of COPTG19674-infected tumor cell lines (DF) as compared to rhIL-12, as determined using the NK-92 proliferation assay.

[0060] Fig.18 The replication yields of VACVwt, unarmed control VACV (VVTG18058), COPTG19673 and COPTG19674 on human hepatocytes are shown. The results are expressed as the replication yield corresponding to the ratio between the input / output virus quantities. The results are the average of three wells.

[0061] Fig.19 The replication of VACVwt, VVTG18058 and COPTG19673 and COPTG19674 on human PBMCs is shown. The results are expressed as the replication yield corresponding to the ratio between the input / output virus quantity. The results are the average of three wells.

[0062] Fig. 20 The efficacy of virus-mediated oncolysis in cultured human tumor cells incubated with VACV IL-12 at different MOIs in each of three independent experiments is shown to determine the average EC50 for cell lysis. (See also 29B).

[0063] Fig.21A -B shows virus recovered from tumors by plaque formation assay (viral PFU / gram tumor tissue) (A) and intratumoral IL-12- (ngIL-12 / gram tumor) using human IL-12-specific ELISA (B). Asterisks indicate time points where virus and transgene were not determined.

[0064] Fig.22A -E shows the experimental scheme (A), spider plots of tumor growth in C57BL / 6 mice implanted with subcutaneous MC38 colorectal tumors after multiple doses, intratumoral administration of VACV expressing murine IL-12 (BD), and Kaplan-Meier plots of survival of tumor-bearing mice (E). CR, complete response of the tumor to therapy, with undetectable tumor volume; *, p=0.0024 for comparison of three or more groups by log rank (Mantel-Cox).

[0065] Fig.23A-E shows the expression of murine IL-12 (A) and the expression of cytokines IFNγ (B), CXCL10 (C), IL-6 (D), and TNFα (E) in the peripheral blood of mice 4 and 24 hours after intratumoral administration of VACV muIL-12. *, p<0.05, one-way ANOVA with Tukey post hoc test.

[0066] Fig.24A -H shows the antitumor activity of VACV expressing luciferase (VACVluc) determined using primary tumors from cancer patients transplanted into immunocompromised NOD / SCID mice (PDX: patient-derived xenografts). Each tumor tested was either untreated (open circles, gray line) or administered 1e7 VACVluc virus (filled squares, black line). Each primary patient-derived tumor included only one untreated tumor and one treated tumor, so each open circle and filled square represents a pair, but are drawn together within the tumor type for simplicity.

[0067] Fig.25A -F shows the mRNA expression levels of IL-12RB1 (A), IL-12RB2 (B), NKp46 (NK cells) (C), PD-L1 (D), CXCL9 (E), and CXCL10 (F) genes in mouse stroma isolated from patient-derived xenograft models of bladder, head and neck, liver, colon, lung, and ovarian cancer from VACV-luciferase-treated mice at day 0 and 48 hours after day 14.

[0068] Fig.26A -E shows the efficiency of VACV-IL12 in infecting human tumors in vitro, as measured by detecting IL-12p70 in supernatants from dissociated tumor cell cultures (A), IL-12p70 expression in supernatants from tumor slice cultures (B), IFNγ mRNA levels (C), IFNγ protein levels (D), and B8R mRNA levels (E). ****<0.0001, one-way ANOVA with Tukey's multiple comparison correction. Each point is a single slice, with 1-4 replicates per condition. Fig.26F is a schematic diagram showing the timing of VACV infection, transgene production, and immune activation.

[0069] Fig.27A -C shows that COPTG19673 replicates selectively in tumor cells relative to normal human cells. Relative to cancer cells (SW780), there was no observable expansion in PBMCs, and minimal replication in normal hepatocytes (HUCPG and HUCPI) and normal human dermal fibroblasts (NHDF).

[0070] Fig.28A -C shows the experimental scheme (A), oncolytic VACV efficacy and overall response across multiple tumor indications (B), and replication kinetics observed 48 hours after the 1st and 3rd doses (C). CRC, colorectal cancer; HN, head and neck squamous cell carcinoma.

[0071] Fig.29A -E shows the oncolytic activity of VACV-Luc and VACVIL-12 (COPTG1673) across 30 human cancer cell lines representing 12 tumor indications. Panel A shows the oncolytic activity of VACV-Luc in cultured human cancer cell lines. Panel B shows the oncolytic activity of VACV-IL12 in cultured human cancer cell lines (see also Fig. 20 ). Panel C shows the correlation of the oncolytic effects of VACV Luc and VACV-IL12 on human tumor cell lines. Panels D and E show the production and replication of the VACV-IL12 transgene in tumor cell lines 5 days after infection.

[0072] Fig. 30A -F shows the anti-tumor efficacy of VACVIL-12 (COPTG1673) in a human xenograft tumor model. Figures AC show the average tumor volume changes over time for different doses of VACV-IL12 compared to VACV-Luc in mice carrying SW780 tumors, NCI-H292 tumors, and HCT-116 tumors. Figures D and E show the amount of virus and recovered intratumoral IL-12 over time for each dose in SW780 tumor-transplanted mice. Figure F shows human IL12 in mouse plasma over time for each dose level of VACV-IL12 compared to VACV-Luc in the SW780 tumor model.

[0073] Fig.31A -D shows gene expression analysis of TSC cultures after VACV IL-12 (COPTG1673) infection. Panel A shows the production of IL-12p70 in cells infected with VACV-IL12 compared to mock-infected or VACV GFP-infected groups. Panels BD show the production of IFN proteins (IFNγ in panel B, IFNα2a in panel C, and IFNβ in panel D) in cells infected with VACV-IL12 compared to mock-infected or VACV GFP-infected groups.

[0074] Fig.32A-H shows that VACV-muIL12 exhibits similar oncolytic activity and similar IL-12 bioactivity to VACV IL-12 (COPTG1673) in human tumor cells. Figure A shows the dose-dependent relationship between the dilution of the culture supernatant of cells infected with VACV-IL12 and rat IFNγ neutralization. Figure BC shows the production of viruses (Figure B) and IL12 transgenes (Figure C) across 3 tumor cell lines for VACV-GFP and VACV-muIL12. Figure DF shows the survival percentage with infection multiplicity for VACV-GFP and VACV-muIL12 in each cancer cell line. Figure GH shows the IL12p70 (Figure G) and IFNγ (Figure H) plasma levels over time in rats transplanted with F98 rat gliomas and treated intravenously with vehicle, VACV-luc or three doses of VACV-muIL12.

[0075] Fig.33A -E shows that VACV-muIL12 exhibits similar oncolytic activity and similar IL-12 bioactivity to VACVIL-12 (COPTG1673) in cultured human tumor cells. Figure AC shows the survival percentage with the multiplicity of infection for VACV-LUC, VACV-muIL12 and VACV-huIL12 using cultured SW780 bladder, NCI-H292 lung and HCT-116 colorectal tumor cell lines. Figure D shows the IL-12p70 concentration produced by VACV-Luc, VACV-muIL12 and VACV-huIL12 in human and mouse cells for SW780, NCI-H292 and HCT-116 tumor cell lines. Figure E shows different concentrations of recombinant human IL-12 (rIL-12) and the IL-12 activity of IL-12 measured in the cell culture supernatant of SW780 tumor cells treated with VACV-muIL12, VACV-huIL12, or VACV-Luc.

[0076] Fig.34A -F shows that VACV-muIL12 exhibits oncolytic activity similar to VACV-LUC in mouse tumor cells and produces IL-12 with moderate viral replication across tumor cell lines. Figure AD shows the survival percentage with multiplicity of infection for VACV-Luc and VACV-muIL12 in mouse tumor cell lines. Figure E shows the IL-12 concentration produced in multiple mouse tumor cell lines for VACV-muIL-12 and VACV-Luc over a period of 5 days. Figure F shows the amount of virus recovered in each mouse tumor cell line for VACV-luc and VACV-muIL12 on day 5.

[0077] Fig.35A -G shows that VACV-muIL12 enhances antitumor immune responses in a murine syngeneic CT26 tumor model. Panel A shows the general procedure. Panel B shows vehicle control, VACV-Luc (1 x 10 7 PFU) and VACV-muIL12 (1x 10 7 Figure CE shows the changes in tumor volume for the vehicle control group, VACV-Luc, and VACV-muIL12. Figure F shows the mouse plasma IL12p70 concentrations at 4 and 24 hours after injection for the vehicle control group, VACV-Luc, and VACV-muIL12. Figure G shows the mouse plasma IFN-γ concentrations at 4 and 24 hours after injection for the vehicle control group, VACV-Luc, and VACV-muIL12. ** = p < 0.01; *** = p < 0.001.

[0078] Fig.36A -C shows tumor volume over time in CT26 tumor-transplanted mice after administration of vehicle (A), VACV-LUC (B), or VACV-muIL12 (C). The Y-axis shows tumor volume, and the X-axis shows time in days.

[0079] Fig.37A -B shows the results of an ex vivo splenocyte restimulation assay following VACV administration and stimulation with the VACV-specific peptide A52L (A) or the tumor-associated peptide antigen AH-1 (B). DETAILED DESCRIPTION

[0080] In order to more easily understand the present disclosure, first define certain terms. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0081] definition

[0082] Generally, the terms and techniques used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry as described herein in conjunction with hybridization are well known in the art and commonly used.Amino acids may be referred to herein by their commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.Similarly, nucleotides may be referred to by their commonly accepted single letter codes.

[0083] Throughout this application, the singular forms "a, an," and "the" include plural referents unless the context clearly indicates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably herein. In some aspects, the terms "a" and "an" mean "single." In other aspects, the terms "a" and "an" include "two or more" or "multiple."

[0084] The term "about" includes ±10% of the number recited. Thus, "about 10" means 9 to 11. References herein to an "about" value or parameter include (and describe) aspects relating to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0085] The term "or" is used to mean "and / or", unless it is explicitly indicated that only alternatives are referred to or that the alternatives are mutually exclusive, although the present disclosure supports definitions that only refer to alternatives and "and / or". In addition, "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other when used herein. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this article is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0086] As used in this specification and in one or more claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional unrecited elements or method steps. It is contemplated that any aspect discussed in this specification can be implemented with respect to any recombinant virus (e.g., poxvirus), method, system, host cell, expression vector, and / or composition disclosed herein.

[0087] Unless expressly stated otherwise, use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular terms recited are representative examples of the disclosure and they are not intended to be limited to the particular examples referenced or cited.

[0088] "Nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence", "oligonucleotide" or "polynucleotide" refers to a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA.

[0089] "Gene" refers to an assembly of nucleotides that encodes a gene product (RNA or protein). Genes include cDNA and genomic DNA molecules.

[0090] As used herein, a "functional" gene (including a functional transgenic) refers to a gene that can express an RNA or protein product, wherein the RNA or protein product retains at least one functional activity. In this article, a "non-functional" gene (including a non-functional transgenic) refers to a gene that cannot express an RNA or protein product that retains any functional activity. A non-functional gene can refer to a gene that has been completely removed or replaced. A non-functional gene can also refer to a gene that has been partially removed or replaced, wherein the partial removal or replacement makes the remaining part of the gene unable to express an active RNA or protein product.

[0091] "Coding sequence" is a nucleic acid sequence that can be transcribed and translated into a polypeptide in vitro or in vivo in a cell when placed under the control of an appropriate regulatory sequence."Regulatory sequence" includes a nucleotide sequence that is located upstream (5' non-coding sequence), inside or downstream (3' non-coding sequence) of the coding sequence, and affects transcription, RNA processing or stability or the translation of the relevant coding sequence.Regulatory sequence includes a promoter, a translation leader sequence, an intron, a polyadenylation recognition sequence, an RNA processing site, an effector binding site and a stem-loop structure. The boundary of the coding sequence is determined by the start codon at the 5' (amino) terminal and the translation stop codon at the 3' (carboxyl) terminal. The coding sequence includes but is not limited to prokaryotic sequences, cDNA from mRNA, genomic DNA sequences and synthetic DNA sequences. If the coding sequence is intended to be expressed in a eukaryotic cell, a polyadenylation signal and a transcription termination sequence can be located at the 3' of the coding sequence.

[0092] The abbreviation of “open reading frame” is ORF, which refers to a nucleic acid sequence (DNA, cDNA or RNA) that includes a translation initiation signal or start codon (such as ATG or AUG) and a stop codon and may be translated into a polypeptide sequence.

[0093] "Homologous recombination" refers to the insertion of an exogenous DNA sequence ("inserted DNA sequence") into another DNA molecule ("target DNA sequence"). In some cases, the inserted DNA sequence targets a specific site within the target DNA sequence for homologous recombination. For targeted homologous recombination, the inserted DNA sequence generally contains a sufficiently long homology region with the sequence of the target DNA sequence to allow the inserted DNA sequence to complementally bind and be incorporated into the target DNA sequence. Longer homology regions and greater sequence similarity generally improve the efficiency of homologous recombination.

[0094] "Heterologous" describes the relationship of a nucleic acid or amino acid sequence to one or more different nucleic acid or amino acid sequences, and indicates that these sequences are not found in nature linked together in the same position, structure and orientation. The linking of heterologous sequences produces a non-naturally occurring juxtaposition of sequences. This linking is the product of engineering changes performed in the laboratory. The product of this linking can be called a "recombinant."

[0095] Two heterologous nucleic acids or amino acid sequences can be directly connected (fused) or can be connected by a "joint". In some aspects, the joint is a chemical joint. In some aspects, the joint comprises one or more amino acids. A glycine-serine joint is a joint that contains glycine and serine amino acids in any ratio at the same time, such as GGGS.

[0096] "Operably linked" means that the polynucleotide of interest is linked to a regulatory element in a manner that allows expression of the polynucleotide sequence. In some aspects provided herein, the regulatory element is a promoter.

[0097] "Promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcription of a nucleic acid coding sequence to which it is operably linked.

[0098] An "endogenous promoter" is a promoter naturally associated with a gene or nucleic acid sequence. For example, an endogenous promoter can be obtained by isolating 5' non-coding sequences upstream of a coding segment and / or exon. A "recombinant" or "heterologous" promoter is a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

[0099] "Late promoter" is a promoter naturally associated with the expression of late genes. "Medium promoter" is a promoter that controls gene expression after early gene expression but is not controlled by late promoter. Genes expressed early in the viral life cycle before medium and late gene expression are called "early promoters". The expression of late and medium genes controlled by medium / late promoters depends on viral replication (unlike the expression of early genes, the expression of early genes does not depend on viral replication). The expression time frame of early, medium and late promoters is a distinguishing factor, as discussed in Yang et al. J.Virol. [Journal of Virology], Vol. 85, No. 19, pp. 9899-9908 (2011), citing Baldick et al., J.Virol. [Journal of Virology], 67: 3515-3527, (1993), each of which is incorporated herein by reference. Baldick disclosed that after synchronous infection of HeLa cells with VACV, early, medium and late mRNAs can be detected in 20min, 100min and 140min, respectively. Yang prepared a genome-wide early, intermediate and late transcriptional map, revealing unique features of intermediate and late promoters. As used herein, the terms "intermediate promoter" and "late promoter" may refer to any of the intermediate promoters and late promoters discussed in Yang, particularly those wherein Figure 8 The promoter shown in .

[0100] Exemplary late promoters include, but are not limited to, pA10L, pA11R, pA13L, pA14L, pA26L, pG7L, and pF17R. Exemplary mid-stage promoters include, but are not limited to, pI1L, pA12L, pA19L, pA42R, pD13L, pA3L, or pA27L.

[0101] "Vector" refers to a carrier nucleic acid molecule or vehicle that can be introduced into a cell for replication. "Expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. An expression vector typically contains one or more control sequences necessary for transcription and / or translation of an operably linked coding sequence. The vector can be introduced into a desired host cell by known methods, including but not limited to transfection, transduction, cell fusion, and liposome transfection.

[0102] "Transfection" refers to the introduction of exogenous nucleic acid molecules into cells. A "transfected" cell includes an exogenous nucleic acid molecule inside the cell, while a "transformed" cell is one in which the exogenous nucleic acid molecule inside the cell induces a change in the cell's phenotype.

[0103] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0104] The starting point of a protein or polypeptide is called the "N-terminus" (or amino-terminus, NH2-terminus, N-terminus or amine-terminus), which refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The end of a protein or polypeptide is called the "C-terminus" (or carboxy-terminus, carboxyl-terminus, C-terminus or COOH-terminus), which refers to the free carboxyl (-COOH) group of the last amino acid residue of the protein or peptide.

[0105] As used herein, "amino acid" refers to a compound that includes both a carboxyl group (-COOH) and an amino group (-NH2). "Amino acid" refers to natural amino acids and non-natural amino acids, such as synthetic amino acids. Natural amino acids (using their three-letter and single-letter abbreviations) include: alanine (Ala; A); arginine (Arg, R); asparagine (Asn; N); aspartic acid (Asp; D); cysteine ​​(Cys; C); glutamine (Gln; Q); glutamic acid (Glu; E); glycine (Gly; G); histidine (His; H); isoleucine (Ile; I); leucine (Leu; L); lysine (Lys; K); methionine (Met; M); phenylalanine (Phe; F); proline (Pro; P); serine (Ser; S); threonine (Thr; T); tryptophan (Trp; W); tyrosine (Tyr; Y); and valine (Val; V).

[0106] "Amino acid substitution" in a polypeptide or protein refers to a polypeptide or protein in which one or more wild-type or naturally occurring amino acids are replaced by an amino acid different from the wild-type or naturally occurring amino acid at the amino acid residue. The substituted amino acid can be a synthetic or naturally occurring amino acid. In some aspects, the substituted amino acid is a naturally occurring amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. Substitution mutants can be described using an abbreviation system. For example, a substitution mutation in which the fifth (5th) amino acid residue is substituted can be abbreviated as "X5Y", wherein "X" is a wild-type or naturally occurring amino acid to be replaced, "5" is the position of the amino acid residue in the amino acid sequence of a protein or polypeptide, and "Y" is a substituted or non-wild-type or non-naturally occurring amino acid.

[0107] An "isolated" polypeptide, protein, peptide or nucleic acid has been removed from its natural environment. It is also understood that an "isolated" polypeptide, protein, peptide or nucleic acid can be formulated with an excipient (such as a diluent) or adjuvant and still be considered isolated.

[0108] The term "recombinant" when used in reference to nucleic acid molecules, peptides, polypeptides or proteins means a new combination of or produced from genetic material not known to occur in nature. Recombinant molecules can be produced by any well-known technique currently available in the field of recombinant technology, including but not limited to polymerase chain reaction (PCR), gene splicing (e.g., using restriction endonucleases), and solid phase synthesis of nucleic acid molecules, peptides or proteins.

[0109] "Poxvirus" refers to a virus of the Poxviridae family, including, for example, viruses of the genus Orthopoxvirus. The "genome" of the recombinant poxvirus provided herein includes a poxvirus genome that contains a deletion (removal) of one or more endogenous sequences (genes or nucleotides) and / or an addition of one or more heterologous sequences (genes and / or nucleotides). For example, the genome of the recombinant poxvirus may refer to the genome of an attenuated poxvirus.

[0110] "Oncolytic viruses" refer to DNA or RNA viruses that preferentially infect and kill cancer cells compared to normal cells. Oncolytic viruses can kill cancer cells through a variety of mechanisms, including direct oncolysis or apoptosis of infected cells, apoptotic death of uninfected cells, and by inducing an immune response against cancer cells. In direct oncolysis, the direct result of viral replication or infection leads to host cell lysis or apoptosis.

[0111] "Oncolytic activity" refers to the ability of a virus to preferentially infect and kill cancer cells relative to normal cells. Cancer cell death may be due to preferential infection of cancer cells, replication in cancer cells, and destruction of cancer cells (referred to as "direct cytotoxic activity"), as well as by stimulating and amplifying host anti-cancer immune responses (which can establish lasting immunity in addition to destroying existing cancer cells). Oncolytic activity can be detected by known methods, including but not limited to detecting cell death or apoptosis, inhibition of cell proliferation, and / or by detecting a reduction in tumor size.

[0112] A virus is considered "cytotoxic" if it reduces cell viability in treated target cells relative to untreated target cells. Methods for determining viral cytotoxicity are known and include, for example, cytotoxicity assays that measure cell necrosis and / or apoptosis following viral infection, such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and other related tetrazolium salt-based assays (e.g., XTT, MTS or WST), ATP assays, apoptosis assays (e.g., TUNEL staining of infected cells), DNA fragmentation assays, DNA ladder assays, and cytochrome C release assays. Another method for determining cytotoxicity is to monitor the size and location of the tumor before and after treatment. In some cases, it may be desirable to monitor the size at several time points to obtain information about an increase or decrease in the size of a tumor or metastasis.

[0113] "Attenuated virus" refers to a virus that is not pathogenic and has reduced toxicity to normal or non-cancerous cells. An attenuated virus can be recombinantly modified to be less toxic or non-toxic in normal tissues. In some aspects, the modification does not affect or only minimally affects the oncolytic ability of the virus.

[0114] "Pathogenic virus" refers to a virus that produces a disease. In some aspects, the recombinant poxvirus provided herein is not a pathogenic virus.

[0115] "Replication-competent" refers to the ability of a virus to replicate in a cell or cell line and produce infectious progeny virions. Viruses that are able to produce infectious progeny virions in a cell or cell line are considered "replication-competent," while viruses that are unable to produce infectious progeny virions in a cell or cell line are considered "replication-defective." Viral replication can be represented by the ratio of the amount of virus produced by infected cells to the amount used to infect the cell, called the "amplification ratio." An amplification ratio of 1 or greater means that the amount of virus produced from the infected cell is the same or more than the amount used to infect the cell, indicating that replication has occurred, while an amplification ratio of less than 1 means that the amount of virus produced from the infected cell is less than the amount used to infect the cell, indicating a lack of replication in the cell.

[0116] As used herein, the terms "interleukin-12", "IL-12" and "IL12" refer to a protein comprising a p35 subunit (IL-12A) and a p40 subunit (IL-12B). The p35 subunit and the p40 subunit can be expressed as separate heterodimeric proteins or together as a single fusion protein.

[0117] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. A cell proliferative disorder can include cancer.

[0118] "Cancer" or "cancerous" refers to the physiological condition in mammals characterized by unregulated cell growth, lack of differentiation, local tissue invasion and / or metastasis. "Tumor" refers to the abnormal growth of cells of a tissue. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive.

[0119] "Effective amount" refers to an amount sufficient to reproducibly produce a detectable result, e.g., in vitro or when administered to a patient. "Therapeutically effective amount" refers to an amount sufficient to produce a therapeutically significant change in one or more symptoms of a disorder when administered to a patient suffering from the disorder. In one aspect, the therapeutically effective amount is sufficient to treat cancer.

[0120] "PD-1 inhibitor" refers to an agent that reduces the amount or activity of PD-1. For example, a PD-1 inhibitor can be an agent that binds to the PD-1 protein and inhibits its interaction with PD-L1. The PD-1 inhibitor can be, for example, an antibody or antigen-binding fragment thereof that binds to PD-1, a peptide-based inhibitor, a small molecule, or an antibody-drug conjugate. In some aspects, a PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-1. PD-1 inhibitors include, but are not limited to, pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplizumab (Libtayo).

[0121] "PD-L1 inhibitor" refers to an agent that reduces the amount or activity of PD-L1. For example, a PD-L1 inhibitor can be an agent that binds to the PD-L1 protein and inhibits its interaction with PD-1. The PD-L1 inhibitor can be, for example, an antibody or antigen-binding fragment thereof that binds to PD-L1, a peptide-based inhibitor, a small molecule, or an antibody-drug conjugate. In some aspects, the PD-L1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-L1. PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi).

[0122] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to antibody molecules that recognize and specifically bind to a target (e.g., a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination of the foregoing (e.g., a glycoprotein)) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. The term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and any other immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are referred to as α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant domains. Different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT., 1994, p. 71 and Chapter 6.

[0123] The term "antibody fragment" refers to a part of an antibody. An "antigen-binding fragment" of an antibody refers to a part of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigenic determining region (e.g., a complementary determining region (CDR)) of an antibody. Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies and single-chain antibodies. An antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., divalent). An antigen-binding fragment of an antibody may be derived from any animal species, such as a rodent (e.g., a mouse, a rat or a hamster) and a human, or may be artificially produced.

[0124] An "antigen binding domain" or "antigen binding region" refers to a monovalent portion of an antibody that binds to an antigen. An "antigen binding domain" may include an antigenic determining region (e.g., a complementarity determining region (CDR)) of an antibody. An antibody or antigen binding fragment thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen binding fragments thereof) may include an antigen binding domain.

[0125] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain, referred to as "framework" regions, show less intermolecular variability. The framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structure, and in some cases the loops form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that provides for positioning the CDRs in the correct orientation through non-covalent interactions between chains. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of an antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids that make up the CDR and framework regions, respectively, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), incorporated herein by reference in their entireties).

[0126] The term "Kabat numbering" and similar terms are well-known in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci [Annuals of the New York Academy of Sciences] 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest [Protein sequences with immunological significance], Fifth Edition, US Department of Health and Human Services [U.S. Department of Health and Human Services], NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (optionally may include one or two additional amino acids after position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0127] Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by the Oxford Molecular AbM antibody modeling software.

[0128]

[0129] As used herein, the term "heavy chain" when used to refer to an antibody can refer to any of the different types, e.g., α (a), δ (d), ε (e), γ (g), and μ (m), based on the amino acid sequence of the constant domain, which respectively produce the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In specific aspects provided herein, the heavy chain is a human heavy chain.

[0130] As used herein, the term "light chain" when used with respect to an antibody can refer to any of the different types, e.g., κ or λ, based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In specific aspects provided herein, the light chain is a human light chain.

[0131] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light (VL) chain and the heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distant from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains contain the carboxyl termini of the heavy and light chains, respectively.

[0132] As indicated above, the variable region allows the binding molecule to selectively recognize and specifically bind to an epitope on an antigen. That is, the subclasses of the VL domain and the VH domain or the complementary determining region (CDR) of the binding molecule (e.g., antibody) combine to form a variable region that defines a three-dimensional antigen binding site. The quaternary binding molecule structure forms an antigen binding site present at the end of each arm of the Y. More particularly, the antigen binding site is defined by three CDRs on each of the VH and VL chains.

[0133] "Monoclonal" antibody or its antigen binding fragment refers to a homologous antibody or antigen binding fragment group that is related to a single antigenic determinant or epitope with high specific recognition and binding. This is in contrast to polyclonal antibodies that typically include different antibodies for different antigenic determinants. The term "monoclonal" antibody or its antigen binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibody or its antigen binding fragment refers to antibodies and their antigen binding fragments prepared in any number of ways (including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals).

[0134] As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins, as described below and in, e.g., U.S. Pat. No. 5,939,598 to Kucherlapati et al.

[0135] "Patient," "subject," and "individual" are used interchangeably and refer to animals, including humans and non-human animals, including, for example, primates, cattle, pigs, sheep, goats, dogs, cats, rabbits, and rodents, as well as non-mammals such as chickens, amphibians, and reptiles, that are treated. In one aspect, the subject is a human. In one aspect, the subject is a human with cancer. In another aspect, the subject is an experimental animal or an animal disease model.

[0136] The term "treat" or "treatment" refers to therapeutic treatment in which the purpose is to alleviate or eliminate one or more symptoms. Beneficial or desired results include, but are not limited to, elimination of symptoms, alleviation of symptoms, reduction in the extent of the condition, stabilization of the condition state (e.g., not worsening), delay or slowing of the progression of the condition. Treating cancer may include inducing cell death of cancer cells or cells within a tumor.

[0137] "Tumor progression" refers to the stage of a tumor, including tumorigenesis, tumor growth and proliferation, invasion and metastasis. "Inhibiting tumor progression" refers to inhibiting the development, growth, proliferation or spread of a tumor, including, for example: inhibiting or reducing tumor growth; reducing the number of cancer cells; reducing tumor size; inhibiting or reducing cancer cell infiltration into adjacent peripheral organs and / or tissues; inhibiting or reducing metastasis; increasing the survival time of a patient or patient group after treatment; and / or reducing the mortality rate of a patient or patient group at a given time point after treatment.

[0138] As used herein, the terms "combination", "therapeutic combination", "combination composition", "combination therapy" or "pharmaceutical combination" may include a fixed combination in the form of one dosage unit, a separate dosage unit, or a kit or instructions for combined administration, wherein the recombinant poxvirus and the PD-1 / PD-L1 inhibitor may be administered independently at the same time or separately at a certain time interval. The combined pharmaceutical composition may be suitable for simultaneous, separate or sequential administration. Thus, administration "in combination" with one or more additional therapeutic agents includes simultaneous (concurrent) or sequential administration in any order.

[0139] Recombinant poxvirus, method for preparing recombinant poxvirus and composition containing the same

[0140] Provided herein is a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. Such recombinant poxviruses are particularly advantageous because they exhibit cell specificity, preferentially killing cancer cells while minimizing any deleterious effects on healthy non-cancerous cells.

[0141] In some aspects, the poxvirus belongs to the genus Orthopoxvirus. In some aspects, the poxvirus belonging to the genus Orthopoxvirus is a vaccinia virus. In some aspects, the poxvirus belonging to the genus Orthopoxvirus is an oncolytic vaccinia virus. In some aspects, the oncolytic vaccinia virus is selected from the group consisting of: Western Reserve (WR), Elstree, Wyeth, Lister, TianTan, LIVP and Copenhagen (Cop) strains. In some aspects, the oncolytic vaccinia virus is selected from the Copenhagen (Cop) strain.

[0142] In some aspects of the recombinant poxviruses provided herein, the genome of the recombinant poxvirus comprises at least 150 kilobases (kb), at least 175 kb, at least 180 kb, at least 185 kb, at least 190 kb, at least 192 kb, or at least 194 kb. In some aspects of the recombinant poxviruses provided herein, the genome of the recombinant poxvirus comprises about 150 kb to 200 kb.

[0143] In some aspects, the recombinant poxvirus is attenuated.

[0144] As provided herein, a variety of late promoters can be used in recombinant poxviruses. In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxvirus comprises a late promoter selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG7L, and pF17R. In some aspects, the late promoter is selected from pA14L, pA26L, and pF17R. In some aspects, the late promoter is pA14L or pF17R. In some aspects, the late promoter is pA14L. In some aspects, the late promoter is pF17R.

[0145] The sequences of the late promoters pA10L, pA11R, pA13L, pA14L, pA26L, pG7L, and pF17R are provided in Table 1 below.

[0146] Table 1.

[0147]

[0148]

[0149] As provided herein, a variety of mid-stage promoters can be used in recombinant poxviruses. In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxvirus comprises a mid-stage promoter selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA3L, or pA27L.

[0150] The sequence of the mid-stage promoter plL, pA12L, pA19L, pA42R, pD13L, pA3L or pA27L is provided in Table 2 below.

[0151] Table 2.

[0152]

[0153] In some aspects, the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 11. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO: 11.

[0154] In some aspects, the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 22. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO: 22.

[0155] In some aspects, the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 13. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO: 13.

[0156] In some aspects, the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 23. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO: 23.

[0157] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 25. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 25.

[0158] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 26. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 26.

[0159] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 27. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 27.

[0160] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 28. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 28.

[0161] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 29. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 29.

[0162] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 30. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 30.

[0163] In some aspects, the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 31. In some aspects, the mid-stage promoter comprises the nucleotide sequence of SEQ ID NO: 31.

[0164] As described above, the recombinant poxvirus provided herein comprises in its genome a heterologous nucleic acid sequence encoding IL-12. IL-12 can be human IL-12. IL-12 can be mouse IL-12.

[0165] In some aspects of the recombinant poxvirus disclosed herein, IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit. The IL-12p40 subunit may be located at the N-terminus of the IL-12p35 subunit. Alternatively, the IL-12p40 subunit may be located at the C-terminus of the IL-12p35 unit. The IL-12p40 subunit and the IL-12p35 subunit may be fused directly (i.e., without a joint) or may be fused by a joint. The joint may be, for example, a chemical joint or an amino acid joint. The amino acid joint may be a glycine-serine joint. In some aspects, the length of the joint is about 5 to about 10 amino acids. In some aspects, the length of the joint is 7 amino acids. In some aspects, the joint comprises the amino acid sequence of SEQ ID NO: 18.

[0166] Widely used vaccinia virus vectors include highly attenuated strains, such as New York vaccinia virus (NYVAC). In some aspects of the recombinant poxvirus disclosed herein, the recombinant poxvirus is not NYVAC.

[0167] In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 17. In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit has the amino acid sequence of SEQ ID NO: 17.

[0168] In some aspects of the recombinant poxviruses disclosed herein, the IL-12p35 subunit comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 19. In some aspects of the recombinant poxviruses disclosed herein, the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19.

[0169] In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 17, and the IL-12p35 subunit comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 19. In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit comprises the amino acid sequence of SEQ ID NO: 17, and the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19.

[0170] In some aspects of the recombinant poxviruses disclosed herein, the heterologous nucleic acid sequence encoding IL-12 comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 21. In some aspects, the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO: 21.

[0171] In some aspects of the recombinant poxviruses disclosed herein, the heterologous nucleic acid sequence encodes the IL-12 amino acid sequence of SEQ ID NO:20, or an amino acid sequence that is at least 75%, 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 the amino acid sequence of SEQ ID NO:20.

[0172] In some aspects of the recombinant poxvirus disclosed herein, the poxvirus is deficient in thymidine kinase (TK). In some aspects of the recombinant poxvirus disclosed herein, the poxvirus lacks a functional J2R gene.

[0173] In some aspects of the recombinant poxvirus disclosed herein, the poxvirus is deficient in ribonucleotide reductase (RR) activity. In some aspects of the recombinant poxvirus disclosed herein, the poxvirus lacks a functional I4L gene. In some aspects of the recombinant poxvirus disclosed herein, the poxvirus lacks a functional F4L gene. In some aspects of the recombinant poxvirus disclosed herein, the poxvirus lacks a functional I4L gene and lacks a functional F4L gene.

[0174] In some aspects of the recombinant poxvirus disclosed herein, the poxvirus is deficient in thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity. In some aspects of the recombinant poxvirus disclosed herein, the poxvirus lacks a functional J2R gene and lacks a functional I4L gene. In some aspects, the poxvirus lacks a functional J2R gene and lacks a functional F4L gene. In some aspects, the poxvirus lacks a functional J2R gene, lacks a functional I4L gene, and lacks a functional F4L gene.

[0175] In some aspects of the recombinant poxvirus disclosed herein, a heterologous nucleic acid sequence encoding IL-12 is inserted into the J2R locus of the poxvirus genome. In some aspects, the insertion renders the J2R gene non-functional. In some aspects, the JR2 locus is completely deleted by the insertion. In some aspects, the JR2 locus is not completely deleted by the insertion.

[0176] In some aspects of the recombinant poxvirus disclosed herein, a heterologous nucleic acid sequence encoding IL-12 is inserted into the I4L locus of the poxvirus genome. In some aspects, the insertion renders the I4L gene non-functional. In some aspects, the I4L locus is completely deleted by the insertion. In some aspects, the I4L locus is not completely deleted by the insertion.

[0177] In some aspects of the recombinant poxvirus disclosed herein, a heterologous nucleic acid sequence encoding IL-12 is inserted into the F4L locus of the poxvirus genome. In some aspects, the insertion renders the F4L gene non-functional. In some aspects, the F4L locus is completely deleted by the insertion. In some aspects, the F4L locus is not completely deleted by the insertion.

[0178] In some aspects of the recombinant poxvirus disclosed herein, in addition to encoding IL-12, the poxvirus further encodes one or more therapeutic genes. In some aspects, the one or more therapeutic genes are selected from the group consisting of suicide genes, immunomodulatory genes, anti-angiogenesis genes, immune checkpoint blocking genes, antibody encoding genes, extracellular matrix degradation or regulation genes, or a combination thereof.

[0179] In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxvirus is capable of lysing one or more cancer cells.

[0180] In some respects, -2The recombinant poxvirus is capable of expressing at least 50 ng / mL, at least 100 ng / mL, at least 300 ng / mL, at least 500 ng / mL, at least 1.0 μg / mL, at least 2.0 μg / mL, at least 3.0 μg / mL, at least 4.0 μg / mL, at least 5.0 μg / mL, at least 6.0 μg / mL, at least 7.0 μg / mL, at least 8.0 μg / mL, or about 8.3 μg / mL of IL-12 in cancer cells (e.g., A549 cells) 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 50 ng / mL to about 50 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 1 μg / mL to about 50 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 2 μg / mL to about 50 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 3 μg / mL to about 50 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 1 μg / mL to about 40 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 2 μg / mL to about 40 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 3 μg / mL to about 40 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 1 μg / mL to about 30 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 2 μg / mL to about 30 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 3 μg / mL to about 30 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2The recombinant poxvirus is capable of expressing about 1 μg / mL to about 25 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus is capable of expressing about 2 μg / mL to about 25 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 -2 The recombinant poxvirus was able to express about 3 μg / mL to about 25 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI).

[0181] In some aspects, cancer cells that can be lysed by the recombinant poxvirus provided herein and / or can express IL-12 from the recombinant poxvirus provided herein include, but are not limited to, renal cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, bile duct cancer cells, endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, head and neck cancer cells, melanoma cells, glioblastoma cells, multiple myeloma cells, or malignant glioma cells. In some aspects, such cancer cells are A549, HT29 or MIA PaCa-2 cells.

[0182] In some aspects of the recombinant poxvirus disclosed herein, the virus is expressed in chicken embryo fibroblasts (CEF), HeLa cells, In some aspects of the recombinant poxvirus disclosed herein, the virus is produced in chicken embryo fibroblasts (CEF).

[0183] In some aspects, the recombinant poxvirus is capable of upregulating interferon (IFN)-γ.

[0184] In some aspects, the recombinant poxviruses disclosed herein are produced in a suitable host cell line or a suitable producer cell using conventional techniques, including culturing the transfected or infected host cells under suitable conditions to allow for the production and recovery of infectious poxvirus particles.

[0185] Also provided herein is a method for producing a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. In some aspects, the method for producing a recombinant poxvirus comprises the following steps: a) obtaining or preparing a production cell; b) infecting the obtained or prepared production cell with the recombinant poxvirus; c) culturing the infected production cell under suitable conditions to allow the production of the recombinant poxvirus. In some aspects, such a method further comprises d) recovering the produced recombinant poxvirus from the culture of the production cell. In some aspects, such a method further comprises e) purifying the recovered recombinant poxvirus. In some aspects, the production cell is a chicken embryo fibroblast (CEF), HeLa, Vero, HEK293, PerC6, BHK21 or MRC5 cells. In some aspects, the production cell is a chicken embryo fibroblast (CEF). Also provided herein is a recombinant poxvirus produced by such a method.

[0186] In some aspects, the production cells can be cultured in a suitable culture medium in step a), which may or may not be supplemented with serum and / or appropriate one or more growth factors, if necessary (e.g., a chemically defined culture medium containing no animal-derived or human-derived products can be used). Those skilled in the art can select a suitable culture medium based on the production cells. Such culture medium is commercially available. Before infection, the production cells are cultured at a temperature between +30°C and +38°C (e.g., about 37°C) for 1 to 8 days. If necessary, multiple passages can be performed within 1 to 8 days to increase the total number of cells.

[0187] Also provided herein are pharmaceutical compositions comprising a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. In some aspects, the disclosure provides pharmaceutical compositions comprising the recombinant poxvirus described herein and a pharmaceutically acceptable carrier. In some aspects, the disclosure provides compositions comprising a therapeutically effective amount of the recombinant poxvirus and a pharmaceutically acceptable carrier. In some aspects, the therapeutically effective amount of an individual dose of the recombinant poxvirus described herein comprises 1 x 10 3 pfu to 1x 10 12 In some aspects, the therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 1 x 10 4 pfu to 1x 10 11 In some aspects, the therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 1 x 10 5 pfu to 1x 10 10In some aspects, the therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 5 x 10 7 pfu to 4x 10 9 pfu.

[0188] In some aspects, the disclosure provides a pharmaceutical composition comprising a recombinant poxvirus for treating or preventing a proliferative disease such as cancer, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. In some aspects, the cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma and malignant glioma.

[0189] PD-1 and PD-L1 inhibitors

[0190] PD-1 is a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified, which are expressed on antigen presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion after binding PD-1. Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743), and since May 2006, the FDA has approved six immune checkpoint inhibitors for the PD-1 / PD-L1 pathway, including three for PD-1 (pembrolizumab, nivolumab, and cemiplimab), and three for PD-L1 (atezolizumab, avelumab, and avelumab) (Ai et al., Drug Des Devel Ther., 14:3625-3649, 2020).

[0191] Any of the PD-L1 inhibitors and / or PD-1 inhibitors known in the art for use in treating one or more cancers are suitable for inclusion in the therapeutic combinations and methods described herein.

[0192] In some aspects, the inhibitor is a PD-1 inhibitor. In some aspects, a PD-1 inhibitor is an agent that binds to a PD-1 protein and inhibits its interaction with PD-L1. In some aspects, a PD-1 inhibitor is an antibody or an antigen-binding fragment thereof that binds to PD-1. In some aspects, a PD-1 inhibitor is a peptide-based inhibitor. In some aspects, a PD-1 inhibitor is a small molecule. In some aspects, a PD-1 inhibitor is an antibody drug conjugate comprising an antibody or an antigen-binding fragment thereof that binds to PD-1.

[0193] In some aspects, the inhibitor is a PD-L1 inhibitor. In some aspects, a PD-L1 inhibitor is an agent that binds to a PD-L1 protein and inhibits its interaction with PD-1 and / or CD80. In some aspects, a PD-L1 inhibitor is an antibody or an antigen-binding fragment thereof that binds to PD-L1. In some aspects, a PD-L1 inhibitor is a peptide-based inhibitor. In some aspects, a PD-L1 inhibitor is a small molecule. In some aspects, a PD-L1 inhibitor is an antibody drug conjugate comprising an antibody or an antigen-binding fragment thereof that binds to PD-L1.

[0194] In some aspects, the anti-PD-1 therapy is an antibody selected from the group consisting of: nivolumab (also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as lanlorizumab and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplizumab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-YangLiu et al., J. Hematol. Oncol. 70:136 (2017)), BGB-A317 (BeiGene; see WO 2015 / 112900), and sirolimus (Regeneron; also known as REGN-2810; see WO 2015 / 112800). 2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847; Si-Yang Liu et al., J Hematol. Oncol. 70:136 (2017)), TSR-042 (Tesaro Biopharmaceuticals; also known as ANB011; see WO 2014 / 179664), pidilizumab (Medivation / CureTech; see U.S. Pat. No. 8,686,119 B2 or WO 2013 / 014668 Al); GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J Hematol. Oncol. 70:136 (2017)). Liu et al., J. Hematol. Oncol. 70: 136 (2017), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics, see WO 2017 / 19846), and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540).In some aspects, the anti-PD-1 therapy is the PD-1 antagonist AMP-224, which is a recombinant fusion protein consisting of the extracellular domain of the PD-1 ligand programmed cell death ligand 2 (PD-L2) and the Fc region of human IgG. AMP-224 is discussed in U.S. Publication No. 2013 / 0017199. The contents of each of these references are incorporated herein by reference in their entirety.

[0195] In some aspects, the anti-PD-L1 therapy is an antibody selected from the group consisting of: durvalumab (AstraZeneca; also known as IMFINZI TM , MEDI-4736; see WO 2011 / 066389), BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(Suppl):3000), Avelumab (Pfizer; also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3DMed / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (April 2016), the contents of each of which are incorporated herein by reference in their entirety.

[0196] In some aspects of the therapeutic combinations, methods and uses described herein, the PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some aspects, the anti-PD-L1 antibody or an antigen-binding fragment thereof comprises the CDR sequences provided in Table 3 (ie, SEQ ID NOs: 32-37).

[0197] Table 3: Durvalumab antibody sequences

[0198]

[0199] In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 38. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain (e.g., a human IgG1 heavy chain). In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a kappa light chain (e.g., a human kappa light chain). In some aspects, the anti-PD-L1 antibody is a human IgG1 kappa monoclonal antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof is produced in Chinese hamster ovary (CHO) cells. In some aspects, the anti-PD-L1 antibody is a human IgG1κ monoclonal antibody produced in CHO cells, comprising a VH comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody comprises a variant Fc comprising at least one amino acid substitution selected from the group consisting of 234F, 235F, and 331S, as numbered by the EU index as set forth in Kabat. In some aspects, the anti-PD-L1 antibody comprises a variant Fc comprising amino acid substitutions 234F, 235F, and 331S, as numbered by the EU index as set forth in Kabat. In some aspects, the anti-PD-L1 antibody is a human IgG1κ monoclonal antibody comprising a variant Fc comprising amino acid substitutions 234F, 235F, and 331S (as numbered by the EU index as set forth in Kabat), a VH comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising the amino acid sequence of SEQ ID NO: 39. In some aspects, the anti-PD-L1 antibody is a human IgG1κ monoclonal antibody produced in CHO cells, comprising a variant Fc comprising amino acid substitutions 234F, 235F, and 331S (as numbered by the EU index as set forth in Kabat), a VH comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising the amino acid sequence of SEQ ID NO: 39.

[0200] In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof is provided in 10 mL of solution, wherein each mL of solution comprises the antibody or antigen-binding fragment thereof (e.g., durvalumab) (50 mg), L-histidine (2 mg), L-histidine hydrochloride monohydrate (2.7 mg), α,α-trehalose dihydrate (104 mg), polysorbate 80 (0.2 mg), and water for injection, USP. The solution may comprise, for example, 120 mg of the antibody or antigen-binding fragment thereof (e.g., durvalumab) in 2.4 mL or 500 mg of the antibody or antigen-binding fragment thereof (e.g., durvalumab) in 10 mL.

[0201] In some aspects of the therapeutic combinations, methods, and uses described herein, the PD-L1 inhibitor is durvalumab ( AstraZeneca). Durvalumab is a human monoclonal antibody that selectively binds to PD-L1 and blocks the binding of PD-L1 to PD-1 and CD80 receptors, as disclosed in U.S. Patent No. 9,493,565, which is incorporated herein by reference in its entirety. The fragment crystallizable (Fc) domain of Durvalumab contains a triple mutation in the constant domain of the IgG1 heavy chain that reduces binding to complement component C1q and Fcγ receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0202] Reagent test kit

[0203] Provided herein are kits comprising a recombinant poxvirus or a pharmaceutical composition comprising a recombinant poxvirus as described herein, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter as described herein. In certain aspects, the kit comprises a unit dose of such a recombinant virus or pharmaceutical composition. In certain aspects, provided herein is a kit comprising one or more containers filled with one or more ingredients of a composition as described herein (such as a recombinant poxvirus as described herein), optionally together with instructions for use.

[0204] In some aspects, a kit is provided herein, comprising one or more containers filled with one or more ingredients of the composition described herein (such as the recombinant poxvirus described herein), and one or more PD-1 or PD-L1 inhibitors described herein, optionally together with instructions for use. In some aspects, the kit described herein comprises the recombinant poxvirus described herein and an anti-PD1 antibody (e.g., pembrolizumab or nivolumab). In some aspects, the kit described herein comprises the recombinant poxvirus described herein and an anti-PD-L1 antibody (e.g., durvalumab).

[0205] Therapeutic uses and methods

[0206] Provided herein is a method for inducing apoptosis of a cancer cell, the method comprising contacting the cancer cell with a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome or a pharmaceutical composition comprising the recombinant poxvirus described herein under conditions that induce apoptosis, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. In some aspects, the cancer cell may include, but is not limited to, a renal cancer cell, a prostate cancer cell, a breast cancer cell, a bladder cancer cell, a colorectal cancer cell, a lung cancer cell, a liver cancer cell, a gastric cancer cell, a bile duct cancer cell, an endometrial cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a head and neck cancer cell, a melanoma cell, a glioblastoma cell, a multiple myeloma cell, or a malignant glioma cell.

[0207] Also provided herein is a method of inhibiting cancer cell growth or promoting cancer cell death, the method comprising contacting cancer cells with a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome or a pharmaceutical composition comprising the recombinant poxvirus described herein under conditions that inhibit growth or promote cancer cell death, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter. In some aspects, cancer cells may include, but are not limited to, renal cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, bile duct cancer cells, endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, head and neck cancer cells, melanoma cells, glioblastoma cells, multiple myeloma cells, or malignant glioma cells.

[0208] In some aspects of the present disclosure, the method of inducing apoptosis of cancer cells is performed in vitro. In some aspects of the present disclosure, the method of inhibiting the growth of cancer cells or promoting the death of cancer cells is performed in vitro. In some aspects of the present disclosure, the method of inducing apoptosis of cancer cells is performed in vivo. In some aspects of the present disclosure, the method of inhibiting the growth of cancer cells or promoting the death of cancer cells is performed in vivo.

[0209] Also provided herein is a method of treating cancer in a subject, the method comprising administering to the subject a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome or a pharmaceutical composition comprising the recombinant poxvirus described herein in an amount effective to treat the cancer, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter.

[0210] Also provided herein is a method of reducing the amount of cancer cells in a subject, the method comprising administering to the subject a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome or a pharmaceutical composition comprising the recombinant poxvirus described herein to reduce the amount of cancer cells in the subject, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter.

[0211] Also provided herein is a method for inducing an anti-cancer immune response in a subject, the method comprising contacting a cancer cell with a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome or a pharmaceutical composition comprising a recombinant poxvirus as described herein in an amount effective to induce an anti-cancer immune response, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid-stage promoter. In some aspects, the anti-cancer immune response comprises activation of an innate immune response or an adaptive immune response to cancer. In some aspects, the anti-cancer immune response comprises activation of an innate immune response to cancer. In some aspects, the anti-cancer immune response comprises activation of an adaptive immune response to cancer. In some aspects, the anti-cancer immune response comprises activation of an innate immune response and an adaptive immune response to cancer.

[0212] In some aspects of the methods described herein that include administration, administration includes systemic administration. In some aspects systemic administration is selected from subcutaneous administration, intramuscular administration, oral administration, intravenous administration, intranasal administration, transdermal administration, subcutaneous administration, and intramuscular administration. In some aspects, the recombinant poxvirus is administered two or more times.

[0213] In some aspects of the methods described herein that include administration, administration includes local administration. In some aspects, local administration includes intratumoral administration. In some aspects, the recombinant poxvirus is administered two or more times.

[0214] In some aspects, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some aspects, the present disclosure provides a method for inhibiting cancer growth in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor. In some aspects, the present disclosure provides a method for enhancing a tumor-specific immune response in a subject suffering from cancer, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus, the recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor.

[0215] In some aspects, compared with that observed after treatment with recombinant poxvirus alone or with immune checkpoint inhibitors alone, immune checkpoint inhibitors are administered in combination with recombinant poxviruses comprising heterologous nucleic acid sequences encoding interleukin-12 (IL-12) in their genome as described herein, resulting in enhanced therapeutic effects (e.g., more significant reductions in tumor growth, increases in tumor infiltration by lymphocytes, increases in the length of progression-free survival, etc.). The enhanced therapeutic effect can be, for example, an enhancement of tumor-specific immune responses. This can be characterized, for example, by an increase in the production of IFN-γ and / or an increase in the number of IFN-γ secreting cells.

[0216] In addition, some cancers are resistant to treatment with immune checkpoint inhibitors (i.e., insensitive or unresponsive). In addition, some cancers that were initially responsive (i.e., sensitive) to treatment with immune checkpoint inhibitors develop inhibitor resistance during treatment. Therefore, in some aspects, a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome as described herein is administered in combination with a PD-1 inhibitor or a PD-L1 inhibitor to treat a cancer that is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with one or more immune checkpoint inhibitors. In some aspects, a recombinant poxvirus or a composition thereof comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome as described herein is administered to a subject with a cancer that is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with one or more immune checkpoint inhibitors, resulting in cancer being treated (e.g., tumor growth is reduced, the length of progression-free survival is increased, etc.). In some aspects, the cancer is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with a PD1 inhibitor.

[0217] In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient prior to administration of the recombinant poxvirus. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient simultaneously with the recombinant poxvirus. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient after administration of the recombinant poxvirus.

[0218] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of inducing apoptosis of cancer cells described herein.

[0219] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of inhibiting the growth of cancer cells described herein or promoting the death of cancer cells described herein.

[0220] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of treating cancer in a subject described herein.

[0221] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of reducing the amount of cancer cells in a subject described herein.

[0222] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of reducing the amount of cancer cells in a subject described herein.

[0223] In some aspects, the present disclosure provides (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, or a pharmaceutical composition comprising the recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or a PD-L1 inhibitor or a pharmaceutical composition comprising a PD-1 inhibitor or a PD-L1 inhibitor, for use in a method of eliciting an anti-cancer immune response in a subject described herein.

[0224] Examples

[0225] The examples in this Examples section are offered by way of illustration and not by way of limitation.

[0226] The generation of COPTG19673 and COPTG19674 vectors, which are recombinant vaccinia viruses (VACV) expressing human IL-12 (hIL-12) under the control of two different promoters, pF17R and pA14L, respectively, is described below. Experiments and analyses related to the selection of the pF17R and pA14L promoters are also described.

[0227] The COPTG19673 and COPTG19674 vectors encode human IL-12 (hIL-12) as a fusion of the p40 and p35 subunits connected by a glycine-serine (GS)-linker. The same hIL-12 coding sequence is inserted into both viruses, but is controlled by two different late promoters: COPTG19673 contains the pF17R promoter, while COPTG19674 contains the pA14L promoter. The IL-12 transgene is vectored in the Copenhagen strain of vaccinia virus, which, as described in WO 2009 / 065546 (which is incorporated herein by reference in its entirety), lacks both the thymidine kinase gene (J2R) and the ribonucleotide reductase gene (I4L). These two deletions limit viral replication to highly proliferative cells (containing high concentrations of nucleotides), such as tumor cells. Thus, transgene expression, which is directly dependent on viral genome replication, is restricted to tumor cells (see, e.g., Foloppe et al., 2019, Mol Ther Oncolytics 14:1-14; and Kleinpeter et al., 2016, Oncoimmunology 5:e1220467). An expression cassette containing the promoter and IL-12 transgene was inserted into the J2R locus of the Copenhagen strain of double-deleted vaccinia virus.

[0228] In vitro characterization of interleukin-12-expressing oncolytic vaccinia viruses COPTG19673 and COPTG19674 is also described below.

[0229] Material

[0230] Virus

[0231] VVTG18058 (empty VACV, VACV control or unarmed control VACV) is a vaccinia virus (Copenhagen strain) with the J2R and I4L genes deleted. VVTG18058 was used as an unarmed control virus. VVTG18058 was produced in chicken embryo fibroblasts (CEFs). Titers were performed on Vero cells by plaque assay.

[0232] COPTG19104 is a vaccinia virus (Copenhagen strain) expressing the fluorescent protein mCherry under the control of the pH5R promoter at the J2R locus. It has the I4L gene deleted. It was used as the starting parental virus for the generation of recombinant viruses.

[0233] VACVwt (also called COPwt) is a wild-type vaccinia virus (Copenhagen strain) without deletions. VACVwt is produced in CEFs.

[0234] Cells and cell lines

[0235] Chicken embryo fibroblasts (CEF): CEF cells were isolated from 11-day-old specific pathogen-free (SPF) embryonated eggs (Charles River).

[0236] Vero cells: African green monkey (Cercopithecus aethiops) (African green) kidney cell line Vero ( CCL-81 TM ) were grown in DMEM (Gibco) 4.5 g / l glucose supplemented with 10% FBS, 2 mM L-glutamine and containing gentamicin at a final concentration of 40 mg / L. Growth conditions were 37°C-5% CO2.

[0237] Human tumor cell lines

[0238] Human lung cancer cell line A549 ( CCL-185 TM ) were grown in DMEM (Gibco) supplemented with 10% FBS, 2 mM L-glutamine and 4.5 g / l glucose containing gentamicin at a final concentration of 40 mg / L. The growth conditions were 37°C-5% CO2.

[0239] Human cervical tumor cell line HeLa ( CCL-2 TM ) were grown at 37°C, 5% CO2 in DMEM (Gibco) supplemented with 10% FBS and 40 mg / L gentamicin.

[0240] Human pancreatic tumor cell line MIAPaCa-2 ( CCL-1420 TM ) were cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS and containing gentamicin at a final concentration of 40 mg / L. Growing in.

[0241] Human colorectal tumor cell line HCT116 ( CCL-247TM ) were cultured at 37°C, 5% CO2 in Mc Coys' 5A medium supplemented with 10% FBS and containing gentamicin at a final concentration of 40 mg / L. Growing in.

[0242] Human colorectal cancer cell line HT-29 ( HTB-38) were grown in McCoy's 5A (Gibco) supplemented with 10% FBS and containing gentamicin at a final concentration of 40 mg / L.

[0243] HEK-Blue TM IL-12 cells (InvivoGen, reference number hkb-il12) were cultured in a PBS solution supplemented with 10% inactivated FBS, 100 μg / ml Normocin TM (Invitrogen), HEK Blue TM Selection (Invitrogen) and grown in DMEM (Gibco) containing penicillin and streptomycin at final concentrations of 100 U / mL and 100 μg / mL, respectively.

[0244] Natural killer cell line NK-92 ( CRL-2407 TM ) were grown in alpha minimal essential medium (Giboco) containing 1X Glutamax (Giboco), 1.5 g / L sodium bicarbonate (Giboco), 0.2 mM inositol (Sigma), 0.1 mM β-mercaptoethanol (Sigma), 0.02 mM folic acid (Sigma), 150 U / ml recombinant IL-2 and 25% fetal bovine serum.

[0245] Chicken fibroblasts and human cell lines

[0246] Chicken embryo fibroblast DF-1 ( CRL-12203 TM ) continuous cell lines were grown at 39°C, 10% CO2 in DMEM (Gibco) supplemented with 10% FBS, 2 mM L-glutamine and 40 mg / L gentamicin.

[0247] From healthy donors (National Blood Service (EFS: Etablissement Peripheral blood mononuclear cells (PBMCs) from human dermatologist Du Sang were prepared on a polysucrose gradient and cultured in RPMI supplemented with 10% FBS and containing gentamicin and glutamine at final concentrations of 40 mg / L and 2 mM, respectively. Growing in.

[0248] bacteria

[0249] Escherichia coli DH5α strain (genotype: F-Φ80lacZΔM15Δ(lacZYA-argF)U169 recA1endA1 hsdR17(rk-,mk+)phoA supE44 thi-1gyrA96 relA1λ-; Invitrogen, 1826312) was used for cloning and plasmid amplification in LB medium supplemented with 100 μg / mL ampicillin.

[0250] DNA cloning

[0251] Cloning, plasmid amplification, and other molecular biology procedures were performed according to standard procedures.

[0252] DNA Sequencing

[0253] DNA sequencing was performed using the Sanger method.

[0254] Example 1: Comparative evaluation of vaccinia virus promoters

[0255] Construction of reporter transfer plasmid

[0256] The reporter transfer plasmid is designed to evaluate the strength of different poxvirus promoters to be evaluated. The reporter gene encodes a fusion between Renilla luciferase (RLuc) and Aequorea green fluorescent protein (GFP) separated by the linker (Ala) 5-Thr (GenBank: ABZ79968.1). Different poxvirus promoters are inserted upstream of the RLuc-GFP fusion (SEQ ID NO: 1). The expression cassette is inserted into a poxvirus transfer plasmid designed to allow the insertion of nucleotide sequences into the J2R locus of the vaccinia virus genome by homologous recombination. The plasmid is derived from the plasmid pUC18, in which the flanking sequences left arm (L arm) and right arm (R arm) surrounding the J2R locus are cloned.

[0257] SEQ ID NO: 1: Nucleic acid sequence of RLuc / GFP

[0258]

[0259] A synthetic fragment named "RLuc-GFP" containing a fusion gene encoding the RLuc-GFP protein was synthetically generated and inserted into a set of transfer plasmids containing poxvirus early / late promoters by homologous recombination and restricted with PvuII to generate the plasmids described in Table 4. Figure 1 A schematic diagram of plasmid pTG19409 containing the RLuc-GFP encoding gene under the expression control of the pH5R promoter is shown.

[0260] Table 4. Nucleic acid sequences of early and early / late promoters and corresponding plasmid names containing the early and early / late promoters

[0261]

[0262] To evaluate the late promoter, the coding sequence of the RLuc-GFP fusion was cloned downstream of the promoter using its native start codon. A synthetic fragment containing the late promoter and the start point of the RLuc-GFP fusion was generated by synthesis and inserted into pTG19409 restricted by SmaI-MscI by homologous recombination, resulting in the plasmids described in Table 5.

[0263] Table 5: Nucleic acid sequences of late promoters and corresponding plasmid names containing the late promoters

[0264]

[0265] The reporter transfer plasmids obtained were first tested in transient infection / transfection expression studies to identify the best candidates. In a second stage, some of them were used to generate recombinant poxviruses as described below.

[0266] Cloning, plasmid amplification, and other molecular biology procedures were performed according to standard procedures.

[0267] Transient infection / transfection with luciferase reporter plasmid

[0268] The reporter transfer plasmid using the RLuc-GFP reporter allows the assessment of cell infection and simultaneous precise measurement of reporter expression levels.

[0269] To better control expression levels and minimize off-target expression of different transgenes, several VACV promoters can be used depending on their strength or timing of expression during poxvirus infection (early, middle, or late). Some poxvirus promoters (such as p7.5K or pH5R) have both early and late elements, allowing transgene expression early after viral infection and late after viral genome replication, respectively. Therefore, to further minimize off-target expression of potentially toxic transgenes (such as IL-12), transgene expression can be driven by a late promoter that is active only after viral genome replication.

[0270] Six late promoters were selected for preliminary evaluation in transient infection transfection experiments: pA10L (SEQ ID NO: 9), pA11R (SEQ ID NO: 10), pA14L (SEQ ID NO: 11), pA26L (SEQ ID NO: 12), pF17R (SEQ ID NO: 13) and pG7L (SEQ ID NO: 14). They were cloned upstream of the gene encoding the fusion RLuc-GFP in the transfer plasmid. These plasmids were evaluated by transient infection transfection experiments in DF-1 cells. A plasmid encoding Firefly luciferase under the control of the p11K7.5 promoter was co-transfected to normalize for transfection variability. Both luciferases were measured after 24 hours (h).

[0271] In brief, DF-1 cells were cultured in 24-well culture plates and then infected with empty vaccinia virus (VVTG18058) without any transgene at MOI 1. After 2 hours, 0.5ng of different Renilla reporter plasmids (see Table 2) compounded with 0.625μL of lipofectamine 2000 (Invitrogen) in opti-MEM medium, and 0.5ng of control reporter plasmids encoding firefly luciferase under the control of p11K7.5 and 250ng of control plasmid pTG15839 encoding GFP under the control of CMV promoter were added to each well. Transfection was performed in triplicate. The plate was then incubated at 37°C and 5% CO2 for 24 hours. For luciferase measurement, the supernatant was removed, the cells were lysed and processed according to the "Dual Luciferase Reporter Analysis System" (Promega).

[0272] Figure 2 The results obtained using six late promoters are illustrated. The Renilla luciferase / firefly luciferase (R / F) ratios for each promoter are reported, and the results of two independent experiments are shown. The highest expression level was obtained in the case of the pF17R promoter, which was 3 to 4 times stronger than the pA14L and pA10L promoters. The pA11R, pA26L and pG7L promoters were 8 to 10 times weaker than the pF17R promoter.

[0273] Three promoters of different strengths were selected for further evaluation in the context of recombinant poxviruses: pF17R (SEQ ID NO: 13), pA14L (SEQ ID NO: 11), and pA26L (SEQ ID NO: 12).

[0274] Generation of recombinant poxviruses for comparative evaluation of vaccinia virus promoters

[0275] Transfer plasmids containing the fusion reporter gene RLuc-GFP under the control of various promoters were constructed. Four early / late promoters were tested: p7.5K (SEQ ID NO: 4), pH5R (SEQ ID NO: 2), p11K7.5 (SEQ ID NO: 3) and pSE / L (SEQ ID NO: 8); and three early promoters were also evaluated: pB2R (SEQ ID NO: 5), pA35R (SEQ ID NO: 7) and pC11R (SEQ ID NO: 6). Three late promoters (pF17R (SEQ ID NO: 13), pA14L (SEQ ID NO: 11) and pA26L (SEQ ID NO: 12) previously tested in transient infection-transfection were also tested in recombinant VACV. See Tables 4 and 5.

[0276] Ten different VACV-RLuc-GFP vectors were generated by homologous recombination in CEFs by inserting the RLuc-GFP expression cassette into the J2R locus of the double-deleted vaccinia virus Copenhagen strain under the transcriptional control of different poxvirus promoters described herein. All of these viruses are deficient in thymidine kinase (TK, J2R locus) and ribonucleotide reductase (RR, I4L locus) activities.

[0277] Using COPTG19104 as the starting parental virus, and a shuttle plasmid containing an expression cassette to be integrated with the flanking sequences (L arms and R arms) around the J2R locus, recombinant vaccinia virus was generated by homologous recombination in CEF. Homologous recombination between the shuttle plasmid and the parental vaccinia virus (Copenhagen strain) can generate recombinant vaccinia virus, which has lost the mCherry expression cassette and obtained an expression cassette that produces white (non-fluorescent) plaques. More specifically, CEFs in F175 flasks were infected with COPTG19104 at room temperature with an MOI of 0.05 for 1 hour. The virus suspension was then discarded, and the infected cells were incubated in MBE+5% FBS at 37°C+5% CO2 for 2 hours, followed by trypsinization and counting. Ten million infected cells were then transfected with 2 μg I-SceI restriction enzyme transfer plasmid by nuclear transfection. The transfected cells were then transferred to wells of a 6-well plate, incubated at 37°C for 48 hours, and then frozen. After sonication, serial dilutions of the transfer mixture were used to infect CEFs to select for recombinant virus. Non-fluorescent white plaques were picked and used for a second round of plaque purification. Selected non-fluorescent white plaques were picked and amplified in a 6-well plate at 37°C, 5% CO2 for 72 hours. The amplified products were used for PCR analysis and then recombinant vaccinia virus was selected.

[0278] Primary stock solution is produced by infection of CEF grown within 72 hours before infection with 100 μL selected clones. Virus amplification was performed at 37°C 5% CO2 for 72 hours in MBE supplemented with 5% FBS. Infected cells and culture medium are subjected to freeze / thaw cycles and then homogenized by ultrasonic treatment. This so-called primary stock solution is then characterized and stored in equal portions until use. After virus amplification in F500 flasks inoculated with CEF, purified material is produced. Infected cells and culture medium are harvested to generate a crude harvest and stored at -80°C. Virus is purified according to the procedure described in WO 2007 / 147528 (which is incorporated herein in its entirety by reference).

[0279] The identification was performed with reference to the following recombinant vaccinia viruses: COPTG19409 (VACV comprising the promoter pH5R), COPTG19410 (VACV comprising the promoter p11K7.5), COPTG19411 (VACV comprising the promoter p7.5K), COPTG19412 (VACV comprising the promoter pB2R), COPTG19415 (VACV comprising the promoter pA14L), COPTG19416 (VACV comprising the promoter pA26L), COPTG19417 (VACV comprising the promoter pF17R), COPTG19431 (VACV comprising the promoter pC11R), COPTG19436 (VACV comprising the promoter pA35R), and COPTG19437 (VACV comprising the promoter pSE / L).

[0280] Comparative evaluation of vaccinia virus promoters in human tumor cell lines and human PBMCs

[0281] Human tumor cell lines HeLa, MIAPaCa-2, and HCT116 were infected with ten previously obtained recombinant vaccinia viruses in 96-well plates at an MOI of 0.1 or 1 as described in the following protocol. After 6 and 24 hours, cells were harvested for quantification of luciferase expression and detection of cells expressing GFP.

[0282] For these human tumor cell lines, cells were seeded into 96-well plates at 1E+05 cells / well / 200 μL the day before. Prior to infection, the medium was removed and replaced with 200 μL of medium with FBS containing virus, and infection was performed at an MOI of 0.1 or an MOI of 1.

[0283] For human PBMC, cells were inoculated into 96-well plates with 2E+05 cells / well / 125 μL the day before. For infection, 50 μl of virus dilutions in the culture medium containing FBS were added to each well, and infection was carried out with MOI of 1. Infection was carried out in triplicate and in two independent plates (one for luciferase measurement and the other for GFP analysis). For luciferase measurement, supernatant was removed, and lysed cells were processed according to " Renilla reporter analysis system" (Promega).

[0284] For GFP quantification, the supernatant was removed, and the cells were then trypsinized, centrifuged, washed with 100 μL of PBS, stained with 100 μL of 100-fold diluted live / dead near-IR, and incubated in the dark for 15 minutes at room temperature. The cells were then centrifuged, washed, and resuspended in 100 μL PBS. GFP detection was then performed by flow cytometry using a MACS Quant 16 instrument (Miltenyi Biotec) and analyzed using Kaluza software (Beckman Coulter). The results are expressed as the percentage of live GFP-positive cells (infected cells). FACS (fluorescence activated cell sorting) analysis showed that approximately 70% to 90% of cells were GFP-positive for all MOIs and cell lines after 24 hours of infection (data not shown). All viruses produced the same results, indicating that their levels of infectivity were similar.

[0285] Renilla luciferase was measured after 6 and 24 h, and the results obtained in MIAPaCa-2 cells were as follows, respectively. Figure 3A and Figure 3B As shown. Expression was normalized relative to the weakest promoter pA26L. Six hours after infection, late promoters pF17R, pA14L and pA26L produced very low levels of expression. The early / late promoter pH5R obtained the highest expression level, followed by early promoters pB2R and pC11R and early / late promoters pSE / L and p11K7.5. Similar results were obtained using the other two cell lines (Hela and HCT116) regardless of the MOI of infection (data not shown). After 24 hours of infection, the results of the early promoters pB2R, pC11R and pA35R, which resulted in low expression levels, were different. Higher expression levels were detected when using promoters p11K7.5, pSE / L, pF17R, and pH5R (11 to 21 times higher than pA26L), while p7.5K and pA14L produced moderate levels of expression (7 to 11 times higher than pA26L).

[0286] The results obtained after 24 h of infection of HeLa and HCT-116 cells are shown in Figure 4 and Figure 5 The results were similar to those obtained in MIAPaca-2 cells. The difference in strength between the promoters was more pronounced at MOI 0.1 than at MOI 1 (up to 14-fold and up to 7-fold relative to pA26L, respectively).

[0287] The results obtained in the three cell lines allow the promoters to be divided into three strength groups. The weak promoters include the three early promoters (pB2R, pA35R and pC11R) and the late promoter A26L. The medium promoters are 2 to 5 times more efficient than the weak promoters. They correspond to the early / late promoters p7.5K and pH5R and the late pA14L promoter. The strongest promoters are 2 to 3 times more potent than the medium promoters. They correspond to the early / late promoters p11K7.5 and pSE / L and the late pF17R promoter.

[0288] Human PBMCs were then infected with ten recombinant vaccinia viruses at an MOI of 1 in 96-well plates. After 6 and 24 hours, cells were harvested for quantification of luciferase expression and detection of GFP-expressing cells.

[0289] The results of flow cytometry analysis are shown in Figure 6 After 6 hours, approximately 12% of cells were detected as GFP-positive after infection with viruses containing early or early / late promoters. In contrast, after infection with viruses containing late promoters, very few cells were detected as GFP-positive (less than 4% for pA26L and less than 1% for pF17R and pA26L). The percentage of GFP-positive cells decreased 24 hours after infection due to infected cell death and lack of recombinant vaccinia virus replication. For viruses containing early and early / late promoters, the percentage of infected cells was approximately 5%, while for viruses containing late promoters, the percentage of infected cells was negligible.

[0290] Renilla luciferase was measured after 6 and 24 h, and the results are shown in Figure 7 The expression was normalized to the weakest promoter, pA26L. The levels of luciferase were lower than those detected after infection of human tumor cells (about 100-fold lower). In addition, expression decreased between 6 and 24 hours due to death of infected cells. Cells infected with viruses containing late promoters (pF17R, pA14L, and pA26L) expressed very low levels of luciferase, about 100-fold to 300-fold lower than the expression detected with early or early / late promoters.

[0291] The vaccinia virus used in this study is derived from the Copenhagen strain and contains deletions in the thymidine kinase gene (J2R) and the ribonucleotide reductase gene (I4L). These two deletions restrict viral replication to highly proliferative cells (containing high concentrations of nucleotides), such as tumor cells. These viruses are unable to replicate efficiently in primary human PBMCs, and therefore the late promoter is not active in these cells.

[0292] in conclusion

[0293] This study allowed the identification of two late promoters, pF17R and pA14L, which drive strong or moderate expression in human tumor cells, while little expression was detected in primary human cells. These promoters were therefore chosen to minimize off-target expression of potentially toxic transgenes such as IL-12.

[0294] Example 2: Generation and production of recombinant vaccinia virus encoding IL-12 by homologous recombination

[0295] Construction of transfer plasmids pTG19673 and pTG19674

[0296] Plasmids pTG19673 and pTG19674 contain the human IL-12 gene under the control of the pF17R and pA14L promoters, respectively.

[0297] Endogenous human IL-12 is unique among cytokines in that it is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40). The single-chain IL-12 protein is expressed from a vaccinia construct in which the full-length p40 subunit is fused to a p35 subunit with its leader sequence truncated via a G6S linker (i.e., IL-12.p40.δp35) (see Lieschke et al., 1997, Nat Biotechnol. [Nature Biotechnology] 1997 January; 15(1):35-40).

[0298] The primary protein structure of the hIL-12 fusion protein contains IL-12p40 linked to IL-12p35 via a polypeptide linker having 7 amino acids, as shown in the following sequence (SEQ ID NO: 15).

[0299] Fusion IL-12.p40.δp35 (SEQ ID NO: 15):

[0300]

[0301]

[0302] Underlined sequence: signal peptide (SEQ ID NO: 16)

[0303] Sequence in bold and not underlined: IL-12-p40 subunit (SEQ ID NO: 17)

[0304] Underlined and bold sequence: linker (SEQ ID NO: 18)

[0305] Italic sequence: IL-12p35 subunit (SEQ ID NO: 19)

[0306] The mature IL-12 fusion protein comprises amino acids 23-532 of SEQ ID NO: 15, as set forth below in SEQ ID NO: 20.

[0307] Mature fusion IL-12.p40.δp35 (SEQ ID NO: 20):

[0308]

[0309] The nucleotide sequence of the fusion IL-12.p40.δp35 was optimized for human codon usage and optimal gene expression using Geneart's GeneOptimizer algorithm. The sequence of the expression cassette present in pTG19673 is shown in Figure 8 (SEQ ID NO: 21). SEQ ID NO: 21 is provided below.

[0310]

[0311] Vaccinia virus transfer plasmids pTG19535 and pTG19537 are designed to allow insertion of nucleotide sequences into the J2R locus of the vaccinia virus genome by homologous recombination. They are derived from plasmid pUC18, in which flanking sequences (L arm and R arm) are cloned on both sides of the J2R locus. Plasmid pTG19535 contains the pF17R promoter, while pTG19537 contains the pA14L promoter.

[0312] The fragment containing the IL-12 fusion was synthetically generated by Geneart and inserted into a plasmid. The corresponding plasmid was restricted by SnaB1 and the resulting fragment "hIL-12" was inserted by homologous recombination into pTG19535 (pF17R promoter) or pTG19537 (pA14L promoter) restricted by PvuII, generating pTG19673 ( Fig. 9 ) or pTG19674( Fig.10 ). In these plasmids, the expression cassette is inserted between the recombination arms, allowing homologous recombination in the J2R locus of the vaccinia virus genome. Maxipreparations of both plasmids were generated and analyzed by sequencing the recombination arms and the expression cassette inserted between them. Alignment of the analyzed sequences with the theoretical sequence showed that the two plasmids have 100% homology.

[0313] Two different late poxvirus promoters were used. The native start codon of the pA14L and pF17R genes is not used for the expression of IL-12. Therefore, the sequence ATA was added downstream of the promoter instead of the native ATG.

[0314] The sequences of the two promoters pA14L and pF17R and the downstream ATA are as follows: pA14L (SEQ ID NO: 22)

[0315] TTTGTTCATTCGGCGATTTAAAATTTTTATTAGTTAAATApF17R(SEQ ID NO:23)

[0316] AAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATA Generation of recombinant vaccinia virus by homologous recombination

[0317] Using COPTG19104 as the starting parental virus and the shuttle plasmids pTG19673 and pTG19674 encoding hIL-12 under the control of pF17R and pA14L, respectively, recombinant COPTG19673 and COPTG19674 were generated by homologous recombination in CEF. COPTG19104 contains an expression cassette for mCherry in its J2R locus. Homologous recombination between the shuttle plasmid and the parental vaccinia virus can generate recombinant vaccinia viruses (COPTG19673 and COPTG19674) that lose the mCherry expression cassette and gain the hIL-12 expression cassette (see Example 1 for the generation method of recombinant vaccinia virus).

[0318] The primary research stock of the recombinant virus COPTG19673 was obtained from homologous recombination between the transfer plasmid pTG19673 and the parent COPTG19104, hereinafter referred to as the COPTG19673 primary stock.

[0319] The primary research stock of the recombinant virus COPTG19674 was obtained from homologous recombination between the transfer plasmid pTG19674 and the parental COPTG19104, hereinafter referred to as the COPTG19674 primary stock. The expression of IL-12 in the supernatant of A549 cells infected with COPTG19673 and COPTG19674 as measured by ELISA was as follows Fig.11 shown.

[0320] These primary research stocks were used for in vitro characterization in Example 3.

[0321] Example 3: Recombinant oncolytic vaccinia virus COPTG19673 expressing interleukin-12 and

[0322] In vitro characterization of COPTG19674

[0323] Vaccinia viruses COPTG19673, COPTG19674 and VVTG18058 described in Example 2 herein were used in the following studies.

[0324] VACVwt (also called COPwt) is a wild-type vaccinia virus (Copenhagen strain) without deletions. VACVwt was produced in CEFs. Infectious titer determinations in the following assays were performed using plaque assays on Vero cells.

[0325] Virus replication assay

[0326] Replication in human tumor cell lines

[0327] The replication of COPTG19673 and COPTG19674 was evaluated in three human tumor cell lines (A549, HT-29, and MIA PaCa-2) and compared to the replication of the unarmed control VACVVVTG18058 as a benchmark. The A549, HT-29, and MIA PaCa-2 tumor cell lines were infected with each virus at an MOI of 10 -3 Infection. The infected cells were then plated into 6-well plates and incubated at 37°C in a 5% CO2 atmosphere for 24, 48 and 72 hours. The amount of virus produced in each cell line was determined at each time point by plaque assay on Vero cells.

[0328] Fig. 12A -C shows that the replication of COPTG19673, COPTG19674 and VVTG18058 in three human tumor cell lines is similar.

[0329] Replication in producer cells

[0330] The replication of COPTG19673 and COPTG19674 was evaluated in CEF and in a human cell line (HeLa) as cells for virus production and compared with the replication of an unarmed control VACV as a benchmark. In 6-well plates, HeLa or CEF were infected with each virus at an MOI of 0.05. The plates were then incubated at 37°C in a CO2 atmosphere for 72 hours. Virus production in each cell type was determined by titration on Vero cells using a plaque assay. Fig.13 It was shown that the replication of COPTG19673, COPTG19674, and VVTG18058 in each of the two cells (CEF and HeLa) was similar (ie, the log difference at 72 h was less than 0.7).

[0331] Oncolytic activity assay

[0332] The oncolytic activity of COPTG19673 and COPTG19674 was evaluated on three human tumor cell lines (A549, HT-29, and MIA PaCa-2) and compared with the activity of an unarmed control VACV (VVTG18058). Oncolytic activity was assessed by quantifying cell viability after 5 days of incubation.

[0333] Comparison of two viruses: Tumor cell lines were infected with each virus at 10 different MOIs depending on the cell line used (i.e. A549 and HT-29: 3.10 -5 to 1, MIA PaCa-2:10 -5 To 3.10 -1The infected tumor cells were then incubated in 96-well plates in a CO2 environment at 37°C for 5 days. Cell viability was determined using the CellTiter Blue cell viability assay according to the manufacturer's protocol.

[0334] Oncolytic activity expressed as cell viability represents the lytic activity of the tested virus samples against tumor cells. The oncolytic activity of each sample is expressed as a percentage of the viability of mock-infected cells. Fig.14A -C shows the oncolytic activity of COPTG19673, COPTG19674 and VVTG18058 at different MOIs. Based on these results, the EC50 value (MOI when 50% of cells were killed) of each virus in each cell line was calculated. Comparison of EC50 showed that in MIAPaCa-2, the oncolytic activity of COPTG19673, COPTG19674 and the unarmed control VACV was very similar. In A549 and HT-29, the EC50 of COPTG19673 was higher than that of VVTG18058, but at high MOI, both VACVs showed comparable strong oncolytic activity, with less than 20% of viable cells remaining. The EC50 of the two viruses was calculated using GraphPadPrism. As shown,

[0335] For A549: EC50 of COPTG19673 is 6.3.10 -3 ;EC50 of COPTG19674 is 1.9.10 -3 ; EC50 of VVTG18058 is 3.8.10 -3 ;

[0336] For Mia PaCa-2: EC50 of COPTG19673 is 4.8.10 -3 ; EC50 of COPTG19674 is 2.7.10 -3 ; EC50 of VVTG18058 is 4.4.10 -3 ;and

[0337] For HT29: EC50 of COPTG19673 is 1.2.10 -2 ; EC50 of COPTG19674 is 7.6.10 -3 ; EC50 of VVTG18058 is 4.9.10 -3

[0338] Expression level of vIL-12 determined by ELISA

[0339] Expression levels of the cytokine vhIL-12 were measured in the supernatants of the three infected tumor cell lines 3 days after infection at MOI 0.01. The terms vIL-12 and vhIL-12 refer to IL-12 expressed with VACV according to the present disclosure.

[0340] Supernatant preparation

[0341] The starting material for measuring the expression level of vIL-12 was the supernatant recovered from MIAPaCa-2, A549 and HT-29 cells. Tumor cell lines were seeded in 6-well plates at an MOI of 10 -2 Infection was performed and incubated for 72 hours in 3 mL of adequate medium without fetal bovine serum. The supernatant was collected and then filtered to remove the virus.

[0342] Determination of IL-12 expression in supernatants by ELISA

[0343] use The ELISA Development System Human IL-12 (R&D Systems Reference DY1270-05) determined the IL-12 concentration in the supernatant of infected tumor cell lines. Three tumor cell lines A549, HT-29 and MIA PaCa-27 were infected with VVTG18058, COPTG19673 or COPTG19674 at an MOI of 0.01 for 2 hours. The IL-12 concentration in the supernatant of the infected cells was then measured by ELISA. The supernatant of VVTG18058 infected cells was used as a negative control. The results are shown in Fig.15 In the Figures, the mean and standard deviation (SD) are the mean values ​​and standard deviations (SD) of duplicate measurements of three samples. Both the cell line and the promoter controlling transgene transcription affect the expression level of the transgene. The highest expression was obtained in A549 cells infected with COPTG19673. However, for both viruses and the three tumor cell lines tested, high levels of expression of vIL-12 were obtained in the concentration range of 0.3 to 8.3 μg / mL.

[0344] Functional assay of vIL-12 protein in the supernatant of infected tumor cells

[0345] Using HEK-Blue TM The functionality of virally produced IL-12 during the transgene expression assay in the "Supernatant Preparation" section of this example was assessed using IL-12 reporter cells and a cell proliferation assay using NK-92 cells.

[0346] vIL-12 bioactivity in HEKBlue IL-12 reporter cells

[0347] Using HEK-BlueTM IL-12 reporter cell assay for the bioactivity of vhIL-12 produced in the supernatant of human tumor cell lines The bioactivity of vIL-12 produced in three human tumor cell lines infected with COPTG19673 and COPTG19674 was measured and compared with the bioactivity of human recombinant hIL-12 (hereinafter referred to as rhIL-12).

[0348] HEK-Blue TM The IL-12 cell was designed to detect biologically active human and mouse IL-12 by monitoring activation of the STAT-4 pathway. TM Binding of IL-12 receptors on the cell surface triggers a signaling cascade that activates STAT-4 and subsequently produces the secretory alkaline phosphatase (SEAP) marker protein. TM HEK-Blue can be easily evaluated TM Detection of SEAP in IL-12 cell supernatants.

[0349] In short, HEK-Blue TM IL-12 cells were plated at 5E+04 cells / well in a 96-well flat-bottom microtiter plate. Different dilutions of culture supernatant and rhIL-12 standards (ranging from 10 pg / mL to 100 ng / mL) were added to a 96-well cell culture microtiter plate. The plate was incubated at 37°C. After 24 hours of incubation, QUANTI-Blue TM (Invitrogen, reference rep-qbs) to identify HEK-Blue TM Detection of SEAP in supernatants of IL-12 cells. Supernatants of mock-infected cells and unarmed control VACV-infected cells were used as negative controls.

[0350] Fig.16A-F presents the result that all 3 different samples tested contain biologically active vhIL-12.Add culture medium without rIL-12, supernatant (SN) from simulated infected cells and SN from unarmed control VACV infected cell line as control, which does not show the expression of SEAP.After all samples are administered, the same ELISA is used to compare the biological activity of vhIL-12 and rhIL-12.At equal concentrations, the vhIL-12 and rhIL-12 generated by the three infected tumor cell lines induce relatively close absorbance levels.The results show that the vIL-12 produced by two VACV-IL12 activates the HEK-Blue IL-12 cell line, and EC50 is better than rhIL-12.These results show that the IL-12 produced by the tumor cells infected by VACV retains its cytokine activity.

[0351] Bioactivity of vIL-12 on NK-92 cells

[0352] The bioactivity of vIL-12 produced in the supernatant of infected human tumor cell lines was determined using a cytokine-dependent cell proliferation assay of the NK-92 cell line. IL-12 can induce NK-92 cell proliferation. The bioactivity of vhIL-12 produced by three human tumor cell lines infected with COPTG19673 and COPTG19674 was measured and compared with the bioactivity of human recombinant hIL-12.

[0353] NK-92 is an interleukin-2-dependent natural killer cell line derived from peripheral blood mononuclear cells. NK-92 cells are also dependent on IL-12 for proliferation and can then be used to control IL-12 function.

[0354] Briefly, NK-92 cells were plated at 1E+04 cells / well in 96-well flat-bottom microtiter plates. Different dilutions of culture supernatant and rhIL-12 standards (ranging from 1 pg / mL to 100 ng / mL) were added to the 96-well cell culture microtiter plates. The plates were incubated at 37°C. After 48 hours of incubation, Supernatants from mock-infected cells and unarmed control VACV-infected cells were used as negative controls.

[0355] Fig.17A-The results presented in E show that all 3 different samples tested for the two viruses COPTG19673 and COPTG19674 contain biologically active vhIL-12. SN from mock-infected cells and SN from empty VACV-infected cell lines were added as negative controls, and they did not stimulate the proliferation of NK-92. After all samples were administered, the same ELISA was used to compare the biological activity of vhIL-12 with rhIL-12. At the same concentration, vhIL-12 from three tumor cell lines infected with viruses and rhIL-12 induced similar levels of NK-92 proliferation. As with the HEK-Blue IL12 cell line, the EC50 value of vIL-12 was lower than that of rhIL-12. These results also demonstrate the functionality and powerful efficiency of IL-12 produced by VACV.

[0356] In vitro safety assay

[0357] Normal human hepatocyte replication rate

[0358] Normal healthy human hepatocytes were chosen to monitor the safety profile of COPTG19673 and COPTG19674 because these primary cells can be routinely obtained directly from donors.

[0359] Hepatocytes were provided by Biopredic in 6-well plates. Hepatocytes were infected with each virus at an MOI of 10 -3 Infection, and incubated for 72 hours at 37 ° C, CO2 atmosphere. The amount of virus produced within 72 hours of cultivation was determined by virus titration in each well by plaque assay in Vero cells. The results are expressed as the replication yield corresponding to the ratio between the input / output virus quantity. The results are the average of three wells.

[0360] VACVwt propagated well with a replication yield of 837 (the ratio between output and input viruses). In the case of the unarmed control virus VVTG18058, the replication rate dropped sharply to 1 ( Fig.18 ). Similarly, the results showed that COPTG19673 and COPTG19674 were absent from replication in human hepatocytes, with replication ratios below 1. These results indicate that the attenuated replication against normal cells provided by the two deletions (TK and RR) is conserved between the unarmed control VACV (VVTG18058) and IL-12-expressing VACV (COPTG19673 and COPTG19674).

[0361] Viral replication in hPBMCs

[0362] Human PBMCs were selected as the second normal primary cells to evaluate the safety profile of the newly generated VACV. Wild-type VACV or unarmed VACV does not usually replicate in hPBMCs. The presence of cytokines or immunostimulatory molecules expressed by VACV can activate or stimulate immune cells, thereby changing the replication of VACV in these cells. Therefore, the replication of COPTG19673 and COPTG19674 in these cells (i.e., hPBMCs) was evaluated and compared with the benchmarks VACVwt and VVTG18058. After infection with MOI 1 and incubation for 3 days, the production of the virus was measured by plaque assay in Vero cells.

[0363] Virus replication yield was determined as the ratio between total infectious particles detected 72 h after infection (output) and viral particles used for PBMC infection (input).

[0364] Fig.19 It was shown that two IL-12 expressing VACV, COPTG19673 and COPTG19674, as well as VACVwt and unarmed control VACV did not replicate in hPBMCs. In other words, vectorization of human IL-12 did not alter the replication behavior of VACV on hPBMCs. Fig.27A -C shows that no COPTG19673 amplification was observed in human PBMCs, and minimal replication was observed in normal human hepatocytes and dermal fibroblasts, indicating that replication is relatively specific to tumor cells.

[0365] Example 4: Efficacy of COPTG19673 ("VACV IL-12") across human tumor cell lines

[0366] VACV expressing IL-12COPTG19673 (hereinafter referred to as "VACV IL-12") was further evaluated.

[0367] Cultured human tumor cells were grown on tissue culture plastic and incubated with VACVIL-12 at MOIs ranging from 6.4E-06 to 10 to determine the efficacy of virus-mediated oncolysis. After seven days of incubation with virus, cells were lysed using CellTiter Cell viability assay measures cell viability in culture. Data were analyzed using GraphPad Prism software version 9.0.0 to determine the viral MOI (PFU) required for half-maximal cell killing (EC50 value) inferred from the sigmoidal dose-response curve. Fig. 20In the graphs, the y-axis represents the mean and standard deviation of the EC50 values ​​for each cell line determined in independent experiments. The tumor type from which each cell line was derived is indicated below the x-axis. The dashed line is arbitrarily set to an MOI of 0.1 PFU, which represents the amount of virus that infects and lyses one-tenth of the cynomolgus monkey Vero cells in the plaque formation assay used to measure PFU. These results, as well as Fig.29B and 29C The results shown in demonstrate that VACV IL-12 is potent across a variety of human tumor cell lines.

[0368] To further characterize the oncolytic activity of VACV IL-12, in vitro cell killing was evaluated in 30 human cancer cell lines representing 12 tumor indications. Tumor cells were infected with VACV-LUC or VACV IL-12 at multiple MOIs and cell killing was evaluated on day 5 ( Fig.29A -B; see also Fig. 20 ). The IL-12 receptor is primarily expressed on immune cells and not on tumor cells; therefore, no difference in cell killing between VACV-LUC and VACV IL-12 was expected using this in vitro model. VACV-LUC and VACV IL-12 effectively killed tumor cells at low MOI and showed similar EC50 values ​​in tumor lines (Pearson r2 = 0.89; p-value < 0.0001), further suggesting that encoded IL-12 does not interfere with VACV replication ( Fig.29C VACVIL-12-mediated oncolysis was widely observed across tumor cell lines of different cancer types, with 27 / 30 tumor cell lines showing average EC50 values ​​of 0.1 MOI or lower. Transgene production and viral replication were also evaluated in tumor cell lines 5 days after infection (MOI 0.004). Fig.29D -E).

[0369] Example 5: VACV recovered from human bladder tumors following IV administration to tumor-bearing mice

[0370] Amount of IL-12 virus and human IL-12

[0371] Tumors derived from SW780 human bladder cancer cells were implanted in the flanks of immunodeficient NOD / SCID mice. 5 , 10 6 or 10 7 After a single dose of 10 PFU of VACV IL-12, tumors were removed and analyzed for viral infiltration by plaque formation assay (viral PFU / gram tumor tissue) and for IL-12 production by virally infected cells using a human IL12-specific ELISA (ngIL-12 / gram tumor) ( Fig.21A ).exist Fig. 21B In the figure, the X-axis represents the time points after virus administration. Asterisks indicate time points at which virus and transgene were not determined. Zeros above the X-axis indicate that no measurable virus or transgene was recovered. Fig.21A The results in showed that the amount of virus isolated from the tumor correlated with the amount of IL-12 detected in the tumor (Pearson correlation coefficient 0.20, p = 0.03).

[0372] Additional experiments were performed to evaluate transgene production and replication in mice bearing SW780 tumors. A dose- and time-dependent increase in viral replication was observed in treated tumors. 5 2.5×10 4 ±4.3×10 4 PFU / g; from 1×10 6 9.5×10 7 ±1.0×10 8 PFU / g; and from 1×10 7 1.3×10 8 ±1.2×10 8 PFU / g( Fig. 30A ). Similarly, IL-12 production increases over time in tumors ( Fig.30E ), while IL-12 can be detected in the periphery, but at a lower concentration ( Fig.30F ).

[0373] To further determine the antitumor efficacy of VACV IL-12 in NOD / SCID mice bearing subcutaneous tumors from NCI-H292, SW780, or HCT-116 cell lines, a single intravenous dose (10 5 , 10 6 and 10 7 PFU) of VACV-LUC or VACV IL-12 treated mice ( Fig. 30A -C). In the NCI-H292 and HCT-116 models, administration of 10 5 Significant tumor control was observed at a single dose of 10 PFU (p value < 0.001). 6 Significant tumor control was observed after a single dose of 10 PFU of VACV IL-12 (p value < 0.001). Due to the lack of an intact immune system, VACV-LUC and VACV IL-12 showed similar tumor control, indicating that the oncolytic activity of VACV is a key factor in tumor control.

[0374] Example 6: Activity of VACV muIL-12 against murine syngeneic tumors

[0375] The activity of VACV encoding murine IL-12 was evaluated after multiple doses of intratumoral administration to C57BL / 6 mice implanted with subcutaneous MC38 colorectal tumors ( Fig.22A ). Murine IL-12 was chosen as the cytokine encoded by VACVIL-12 in these experiments because human IL-12 does not bind to the mouse IL-12 receptor. Tumors grew to a median size of approximately 80 mm 3 After randomization to treatment group. Over a 14-day period, 1e was administered twice weekly for a total of 5 doses. 7 PFU of virus. Blood samples were collected 4 hours and 24 hours after the first dose and tumor volume was subsequently recorded. Tumor growth was evaluated in vehicle-treated controls, mice administered VACV without the transgene (empty VACV), and mice administered VACV encoding muIL-12 (VACV muIL-12). Tumor growth is shown in the form of individual tumor spider plots in Fig. 22B -D, and the survival of tumor-bearing mice is shown in the form of a Kaplan-Meier plot. Fig.22E =Moderate (CR, complete response of tumor to therapy with undetectable tumor volume); *, comparison of three or more groups by log-rank (Mantel-Cox), p=0.0024, performed in GraphPad Prism 9. These results indicate that VACV muIL-12 is effective against murine syngeneic tumors.

[0376] Example 7: Expression of murine IL-12 by VACV muIL-12 and induction of pro-inflammatory cytokines

[0377] Expression of murine IL-12 was detected in the peripheral blood of mice 4 and 24 hours after intratumoral administration of VACV muIL-12, but not after administration of empty VACV or vehicle. Fig.23A Similarly, peripheral blood IFNγ levels were significantly higher in mice treated with VACV muIL-12 compared with mice administered empty VACV or vehicle ( Fig. 23B Other proinflammatory cytokines detected in peripheral blood tended to be higher in VACVmuIL-12-treated mice compared with controls and included the IFNγ-inducible cytokine CXCL10 ( Fig.23C ) and the pro-inflammatory cytokine IL6 ( Fig.23D ) and TNFα( Fig.23E ).

[0378] Example 8: Activity of VACV luc in primary human patient-derived tumor xenografts

[0379] The antitumor activity of luciferase-expressing VACV (VACVluc) was determined using primary tumors from 47 cancer patients transplanted into immunocompromised NOD / SCID mice (Table 6) (PDX: patient-derived xenografts). Fig.24A -H). Each tumor tested was either untreated (open circles, gray line) or treated when the tumor reached approximately 200 mm 3 The average volume of 1x 10 was administered by intravenous (IV) route starting at the beginning of a total of three doses per week. 7 pfuVACVluc virus (solid squares, black lines) (scheme as Fig.28A ). Each primary patient-derived tumor included only one untreated tumor and one treated tumor, so each open circle and filled square represents a pair but are plotted together within tumor type for simplicity. Responses were determined based on tumor volume relative to untreated controls (complete responders (CR): no measurable tumor; partial responders (PR): >30% reduction in tumor volume; stable disease (DS): less than 100% tumor increase; and progressive disease (PD): progressive growth). Overall, VACV Luc virus-treated animals exhibited antitumor activity across tumor types compared to untreated controls. ( Fig.28B and Table 6).

[0380] To evaluate VACV replication kinetics, tumors were evaluated for viral replication 48 hours after the 1st and 3rd doses. Infectious virus was recovered from all tumor models and a significant increase in viral replication was demonstrated between the 1st and 3rd doses (p value = 0.0079), indicating that VACV was accumulating and replicating in the treated tumors ( Fig.28C ).

[0381] Table 6: Tumor types represented by implanted PDX

[0382] Tumor type Number of PDX tumors evaluated Bladder cancer 5 Colorectal cancer (CRC) 7 Head and Neck Cancer (HNSCC) 7 Hepatocellular carcinoma 1 Malignant melanoma 7 Merkel cell carcinoma 2 Non-small cell lung cancer (SCLC) 8 Ovarian cancer 4 Small cell lung cancer (SCLC) 6 Total number of PDX tumors evaluated 47

[0383] Example 9: IL-12R, NK cells, PDL1, CXCL9 and 10 are upregulated after VACV infection

[0384] The mRNA expression levels of IL12RB1, IL12RB2, NKp46, PD-L1, CXCL9, and CXCL10 genes were measured in mouse stroma isolated from patient-derived xenograft (PDX) models of bladder, head and neck, liver, colon, lung, and ovarian cancer from VACV-luciferase-treated mice at day 0 and 48 h after day 14. Fig.25ATotal RNA was isolated from fresh-frozen PDX tissue and depleted of ribosomal RNA and globin transcripts. Stranded RNAseq libraries were created and paired-end sequencing was performed.

[0385] Paired-end reads were aligned to the mouse genome reference build mm10 using the STAR aligner, and gene-level read counts were generated using Salmon. Read counts were further normalized for sequencing depth and gene length using tximport to generate transcripts per kilobase million (TPM) values ​​for plotting. All plots were generated using R version 4.1. These results indicate that IL-12R, NK cells, PDL1, and CXCL9 / 10 are upregulated after VACV infection, thereby priming the tumor microenvironment (TME) and triggering effective antitumor immunity.

[0386] Example 10: VACV-IL-12 efficiently infects human tumors in vitro, leading to IL-12 and early B8R-dependent IFN-γ blockade.

[0387] The efficiency of VACV-IL12 infection in human tumors in vitro was studied. In particular, the effectiveness of VACV IL-12 was assessed in tumor tissue slice cultures (TSC) derived from melanoma, bladder cancer, colorectal cancer, lung cancer and ovarian cancer, and in dissociated human tumor cells (DTC) derived from melanoma, head and neck squamous cell carcinoma and ovarian cancer patients. DTCs from different tumor types were thawed in a culture medium supplemented with 1X CTL anti-aggregation washing solution. 50,000 cells were plated in 96-well u-bottom flasks and cultured with PBS (control), VACV-luciferase (MOI 1) or VACV-IL-12 (MOI 1). Supernatant was harvested from cultured DTCs 72 hours after treatment. IL-12 (i.e., IL12p70) concentrations were measured using a custom U-Plex human IL-12p70 assay or U-PLEX mouse IL-12p70 assay (Cat. Nos. K151UAK; K152UAK; Meso Scale Diagnostics, Inc.) according to the manufacturer's protocol, respectively. Human IL-12 expression was detected in supernatants from tumor tissue slice cultures (TSCs) 3 days after infection with VACVIL-12, but not after infection with VACV-GFP or in mock-infected tissues ( Fig.26A ), and the same is true for DTC ( Fig.26B Increased levels of IFNγ mRNA were detected in tissue slice cultures treated with VACVIL-12 compared with slices treated with VACV-GFP or mock-infected, although no differences in IFNγ protein expression were detected in supernatants from the same tissues ( Fig.26C -D).

[0388] Vaccinia virus encodes several immunomodulatory genes that inhibit immune cell activation, such as B8R, which is able to sequester and neutralize IFNγ. VACV-IL-12 infection of TSCs results in the expression of B8R, which has been shown to sequester IFNγ in the supernatant. Significantly higher levels of B8R were detected in TSCs infected with VACV-GFP or VACV-IL-12 compared to mock-infected tissues. Fig.26E ).exist Fig.26E Each point is a single slice, and there were 1-4 replicates per condition. Data were analyzed using GraphPad Prism software version 9.0.0. Schematic diagram of VACV infection, transgene production, and tumor cell lysis is shown in Fig.26F shown.

[0389] VACVIL-12 (COPTG1673) infection resulted in IL-12p70 production in 19 / 19 DTC samples tested (17984 pg / mL ± SD 49523). In contrast, IL-12p70 was not significantly increased in DTC samples from mock infected (6.15 pg / mL ± SD 9.13) or VACVGFP treated (4.99 pg / mL ± SD 6.36) ( Fig.31A -D) IL-12 concentrations in the supernatants of DTCs were negligible. Although IFNγ RNA transcripts were significantly increased after VACV IL-12 infection compared to mock infection (1.5-fold; p value < 0.001) and VACV GFP (1.4-fold; p < 0.001) in TSCs, evaluation of the supernatants of both DTCs and TSCs showed that IFNγ protein was not produced after VACV IL-12 treatment ( Fig.26B , Fig.26D and Fig.31A -D).

[0390] Example 11: VACV encoding murine IL-12 overcomes the inhibitory effects of B8R and leads to IFN-γ induction in an in vivo rat model

[0391] Since IFNγ signaling is critical for the efficacy of IL-12, the effect of B8R on downstream IL-12 and IFNγ activation was evaluated. The VACV-encoded B8R does not bind murine IFNγ; however, B8R binds and neutralizes IFNγ from humans and rats. To confirm the binding of B8R to rat IFNγ, a competitive ELISA was performed. Recombinant B8R protein or filtered supernatant from VACV-infected HeLa cells (MOI 1) was incubated with recombinant IFNγ for 1 hour at 37°C. Next, an ELISA was performed to measure available rat IFNγ. Incubation with rB8R resulted in greater than 50% neutralization, while supernatants from infected cells were able to inhibit detection of IFNγ in a dose-dependent manner, confirming that B8R binds to rat IFNγ ( Fig.32A ).

[0392] Based on the ability of B8R to bind rat IFNγ, VACV IL-12 was evaluated in an immunocompetent model using a syngeneic rat tumor model (COPTG1673). An alternative VACV-muIL12 virus was used because muIL12 has been shown to cross-react in rats. In addition, VACV-muIL12 replicated efficiently and produced the muIL-12 transgene in rat tumor cells compared to murine tumor cells ( Fig.32B -C). Similarly, VACV-muIL12 mediated potent oncolysis in rat tumor cell lines, whereas cell-killing activity was low in mouse tumor cell lines ( Fig.32D -F). Rats were implanted with F98 tumor cells and treated with VACV-LUC (1x 10 7 PFU) or VACV-muIL12 (1x 10 5 , 1x 10 6 or 1x 10 7 ) were treated by intravenous administration on days 0, 4, and 7. Cytokine analysis in plasma confirmed the production of IL12p70. 5 and 1x 10 6 There was a modest increase in the number of treated rats ( Figure 32G In addition, IFN-γ was detected in the plasma of rats treated with VACV-muIL12, but not in the VACV-LUC control group ( Fig.32H ). These results indicate that VACV-encoded IL12 can induce peripheral IFNγ production, whereas B8R does not inhibit this production.

[0393] Interferon-γ competitive ELISA

[0394] Recombinant rat IFN-γ (1 ng / ml) (R&D) was incubated with PBS, recombinant B8R (50 ng / ml) (supplier) or 0.2 μm filtered supernatant of HeLa cells infected with VACV IL-12 (MOI 1). The mixture was incubated at 37°C for 1 hour. Since B8R binds to IFN-γ and prevents the antibody from binding and being detected by ELISA, an ELISA was performed to determine the percent recovery of IFN-γ as described below. The percent recovery was calculated relative to IFN-γ alone.

[0395] ELISA

[0396] Rat interferon-γ was measured in the plasma of rats treated with VACV at the indicated time points. Plasma was diluted and measured using the rat IFN-γ DuoSet Elisa according to the manufacturer's protocol (R&D Systems). This demonstrates that the recombinant poxvirus is able to upregulate interferon (IFN)-γ. Example 12: VACV expressing IL12 improves therapeutic efficacy in a murine syngeneic tumor model

[0397] To evaluate the immunomodulatory effects of IL-12, the murine surrogate VACV-muIL12 virus was tested in murine tumor cell lines and tumor models. VACV-muIL12 exhibited replication, oncolytic, and transgenic bioactivity similar to VACV IL-12 in human and mouse tumor cell lines ( Fig.33A -E and 34A-F). Five intratumoral (it) doses (10 7 PFU) to treat mice bearing subcutaneous CT26 tumors ( Fig.35A -D). Treatment with VACV-LUC did not result in control of CT26 tumors (0 / 10 complete responders) and was similar to the vehicle group ( Fig.35B -D). However, treatment with VACV-muIL12 resulted in regression of CT26 tumors, with 6 / 10 complete responders ( Fig.35E ).

[0398] VACV-LUC and VACV-muIL12 showed similar oncolytic activity and replication in murine tumor cells ( Fig.34A -F). Therefore, it was hypothesized that the observed differences in tumor control could be attributed to the immunomodulatory effects of muIL12. Fig.35A Animals were treated with the indicated regimens. Evidence of IL12 production was detected in serum as early as 4 hours after injection ( Fig.35F ). Induction of IFNγ is an important feature of IL-12 signaling and was detected in VACV-muIL12-treated mice at 4 and 24 h after injection ( Figure 35G Similar results were observed in the MC38 syngeneic tumor model ( Fig.22A Overall, these results suggest that VACV encoding IL-12 participates in the adaptive immune response to control tumor growth, whereas the oncolytic activity of VACV-LUC alone is insufficient to control tumor burden.

[0399] Example 13: In vivo and ex vivo study methods

[0400] In vivo studies

[0401] By resuspending 5 × 10 6 Cell line-derived xenografts were established by subcutaneous (sc) injection of 10 cells / 200 μL into the right flank of 8- to 12-week-old animals. Tumors reached 150-250 mm before randomization. 3 Animal body weights were measured twice weekly for 4 weeks and then weekly thereafter, and any weight loss was monitored for health purposes.

[0402] To establish the MC38 murine syngeneic tumor model, cells were removed from tissue culture plastic using accutase solution. Harvested cells were kept on ice from harvest to implantation (no more than 3 hours). The implantation site of each animal was shaved at least 24 hours before cell injection. 5 × 10 5 Syngeneic tumors were established by subcutaneously injecting 10 cells into the right flank of 7- to 9-week-old C57BL / 6J female animals. When tumors reached 200 mm 3 The mice were randomized at 4 hrs. The body weight of mice was monitored throughout the study.

[0403] To establish CT26 tumors, cells were removed from T-150 flasks using trypsin solution prior to implantation and trypsin was neutralized by adding RPMI + 10% FBS. Harvested cells were kept on ice from harvest to implantation (no more than 3 hours). The implantation site of each animal was shaved at least 24 hours prior to cell injection. 5 × 10 5 Syngeneic tumors were established by subcutaneously injecting 10 cells into the right flank of 9- to 12-week-old Balb / c females. When tumors reached a volume of 150-250 mm 3 The mice were randomized at 4 hrs. The body weight of mice was monitored throughout the study.

[0404] To establish 47 PDX models, cryogenic vials containing tumor cells were thawed and prepared for injection into mice. Thawed cells were washed in RPMI medium, counted, and resuspended in cold RPMI at a concentration of 50,000-100,000 viable cells / 50 μL. The cell suspension was mixed with an equal volume of CULTUREX TM Extracellular matrix (ECM) was mixed and kept on ice during transport to the animal facility. Cells were prepared for injection by drawing the ECM-cell mixture into a frozen 1 ml sliding tip syringe. The filled syringe was kept on ice to prevent ECM solidification. Animals were shaved before injection. One mouse was fixed at a time and the injection site was disinfected with an alcohol cotton swab. 100 mL of cell suspension (50,000-100,000 cells) in ECM was subcutaneously injected into the posterior flank of 9 to 29 week old NOD-SCID mice. Each syringe was injected with 100 ml of cell suspension up to 5 animals.

[0405] For tumor mass inoculation, frozen tumor fragments were thawed and cut into tumor masses of approximately 2-3 mm in diameter. Each mouse received a single buprenorphine injection 30 minutes prior to tumor mass implantation. One tumor mass was loaded into a trocar and injected into the right anterior flank of the mouse to promote tumor growth. Mice were ear-tagged and animals were left undisturbed for up to seven days before observing tumor growth. Tumors were allowed to reach approximately 130-230 mm before assignment to treatment groups. 3 The anticancer effect of VACV Luc was determined by comparing the growth curve of each treated tumor with that of a size-matched untreated tumor. In this manner, the best overall response of the treated tumor compared with the untreated tumor was designated as complete response (CR), partial response (PR), stable disease (DS), or progressive disease (PD). Tumors that did not meet the classification were considered not evaluable (NE).

[0406] To establish F98 tumors in rats, cells were removed from 10 stacked flasks using trypsin solution and trypsin was neutralized by adding DMEM + 10% FBS. Harvested cells were kept on ice from harvest to implantation (no more than 3 hours). 5.0 × 10 6 F98 cell line was implanted by subcutaneous (SC) injection of 10 cells into the right flank of 7- to 9-week-old animals. Tumors were allowed to reach approximately 300-350 mm before randomization. 3 .

[0407] In vitro analysis

[0408] To assess viral replication in tumors, mice were euthanized and blood was collected, and tumors and normal tissues were excised and snap-frozen. Frozen tumors and normal tissues were weighed and suspended in ice-cold homogenization buffer (PBS supplemented with 1x antibiotic-antimycotic and 1x HALT TM Protease-phosphatase inhibitors). The tissue containing homogenization buffer was transferred to a MatrixA tube and homogenized using the Fast-prep-24 lysis system at 4 m / s for 20 s. The tissue homogenate was then subjected to two freeze-thaw cycles, aliquoted and stored at -80°C. Two additional aliquots were prepared without freeze-thaw for DNA isolation or cytokine analysis by MSD.

[0409] Cytokine analysis

[0410] Aliquots from tumor and spleen homogenates were centrifuged at 1,500RPM for 5 minutes at 4°C. Supernatants were collected and stored at -80°C to measure cytokines. Blood was collected from mice at designated time points and centrifuged at 13,000RPM for 10 minutes at 4°C. Plasma was collected and stored at -80°C. Plasma, tumor, and spleen lysates were diluted and measured using mouse and human IL12p70 U-Plex assays (Meso Scale Diagnostics) or custom U-Plex mouse cytokine panels according to the manufacturer's protocol.

[0411] Statistical analysis

[0412] GraphPad Prism software (San Diego, California, USA) was used to evaluate the differences in tumor volume size at specified times during the experiment using one-way analysis of variance (ANOVA) and multiple comparison Tukey correction. The statistical differences in the calculated tumor growth rates were evaluated using the two-sided Mann-WhitneyU test (also known as the Wilcoxon rank sum test). Growth rate means and growth rate inhibition statistics were reported together with the Mann-Whitney U test p value. Using ANOVA and multiple comparison Tukey correction, differences in virus recovery (PFU) of tumors, human IL-12 transgenes detected in tumors (pg / mL), and IL-12 transgenes detected in mouse plasma (pg / mL) were determined. Significant p values ​​were obtained from the ANOVA analysis.

[0413] Example 14: Combination therapy using VACV-muIL12 and immune checkpoint inhibitors

[0414] As demonstrated in the examples above, VACV-muIL12 resulted in significant tumor control. However, it was hypothesized that combining the murine VACV-muIL12 surrogate with a PD-L1 inhibitor could enhance anti-tumor T cell immunity. To test this, mice were implanted with CT26 tumor cells and treated with vehicle, VACV-LUC, or VACV-muIL12. Each treatment group also received vehicle, isotype control, or a commercially available anti-muPD-L1 blocking antibody, which prevents mouse PD-L1 from binding to PD-1. Because tumors were well controlled following VACV-muIL12 therapy, no significant enhancement of tumor control was observed when delivered in combination with an anti-PD-L1 antibody ( Fig.36A -C). However, a significant anti-tumor immune response was observed.

[0415] The antitumor immune response was further characterized by ex vivo splenocyte restimulation assay. Splenocytes were collected on day 7 after VACV administration and stimulated with tumor-associated peptide antigen (AH-1) or VACV-specific peptide (A52L). Combining VACV-muIL12 with anti-PD-L1 resulted in a significant increase in the number of cells secreting IFN-γ after AH-1 peptide stimulation (p value 0.041)( Fig.37A ). In contrast, VACV-LUC alone or in combination did not result in a significant increase in IFN-γ-secreting cells relative to the other treatment groups. Additionally, VACV-specific immune responses were not affected by IL-12 or anti-PD-L1 ( Fig.37B ). This example demonstrates that VACV-muIL12 enhances tumor-specific immune responses when combined with a PD-L1 inhibitor in a tumor model resistant to anti-PD-L1 therapy. These results also suggest that the combination of VACV-muIL12 and an anti-PD-L1 blocking antibody engages the immune system to elicit a T cell-specific immune response against tumor cells.

Claims

1. A therapeutic combination comprising (a) a recombinant poxvirus comprising a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) in its genome, and (b) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor.

2. The therapeutic combination of claim 1, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter.

3. The therapeutic combination of claim 1 or 2, wherein the poxvirus belongs to the genus Orthopoxvirus.

4. The therapeutic combination of claim 3, wherein the poxvirus belonging to the genus Orthopoxvirus is an oncolytic vaccinia virus.

5. The therapeutic combination of claim 4, wherein the oncolytic vaccinia virus is selected from the group consisting of Copenhagen (Cop), Western Reserve (WR), Elstree, Wyeth, Lister, Tian Tan and LIVP virus strains.

6. The therapeutic combination of any one of claims 1-5, wherein the genome comprises at least 150 kb, at least about 175 kb, at least about 180 kb, at least about 185 kb, at least about 190 kb, at least about 192 kb, or at least about 194 kb.

7. The therapeutic combination of any one of claims 1-6, wherein the poxvirus is attenuated.

8. The therapeutic combination of any one of claims 1-7, wherein the poxvirus is not NYVAC.

9. The therapeutic combination of any one of claims 1-8, wherein the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG7L and pF17R.

10. The therapeutic combination of claim 9, wherein the late promoter is selected from the group consisting of pA14L, pA26L and pF17R.

11. The therapeutic combination of claim 10, wherein the late promoter is pA14L.

12. The therapeutic combination of claim 10, wherein the late promoter is pF17R.

13. The therapeutic combination of any one of claims 1-12, wherein the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23.

14. The therapeutic combination of any one of claims 1-13, wherein the late promoter comprises the nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23.

15. The therapeutic combination of any one of claims 1-8, wherein the mid-stage promoter is selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA3L or pA27L.

16. The therapeutic combination of claim 15, wherein the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 25-31.

17. The therapeutic combination of any one of claims 1-16, wherein the IL-12 is human IL-12.

18. The therapeutic combination of any one of claims 1-17, wherein the IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit.

19. The therapeutic combination of claim 18, wherein the IL-12p40 subunit is located at the N-terminus of the IL-12p35 subunit.

20. The therapeutic combination of claim 18 or 19, wherein the IL-12p40 subunit comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

17.

21. The therapeutic combination of any one of claims 18-20, wherein the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

19.

22. The therapeutic combination of any one of claims 18-21, wherein the IL-12p40 subunit and the IL-12p35 subunit are fused in a single polypeptide via an amino acid linker.

23. The therapeutic combination of claim 22, wherein the amino acid linker is about 5 to about 10 amino acids in length.

24. The therapeutic combination of claim 22 or 23, wherein the amino acid linker is 7 amino acids in length.

25. The therapeutic combination of any one of claims 22-24, wherein the amino acid linker is a glycine-serine linker.

26. The therapeutic combination of any one of claims 22-25, wherein the amino acid linker comprises the amino acid sequence of SEQ ID NO:

18.

27. The therapeutic combination of any one of claims 1-26, wherein the IL-12 comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

20.

28. The therapeutic combination of any one of claims 18-21, wherein the IL-12p40 subunit and the IL-12p35 subunit are directly fused in a single polypeptide.

29. The therapeutic combination of any one of claims 1-28, wherein the heterologous nucleic acid sequence encoding IL-12 comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO:

21.

30. The therapeutic combination of claim 29, wherein the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO:

21.

31. The therapeutic combination of any one of claims 1-30, wherein the poxvirus is deficient in thymidine kinase (TK) activity.

32. The therapeutic combination of any one of claims 1-31, wherein the poxvirus lacks a functional J2R gene.

33. The therapeutic combination of any one of claims 1-32, wherein the poxvirus is deficient in ribonucleotide reductase (RR) activity.

34. The therapeutic combination of any one of claims 1-33, wherein the poxvirus lacks a functional I4L gene.

35. The therapeutic combination of any one of claims 1-34, wherein the poxvirus lacks a functional F4L gene.

36. The therapeutic combination of any one of claims 1-35, wherein the heterologous nucleic acid sequence encoding IL-12 is inserted into the J2R locus of the poxvirus genome.

37. The therapeutic combination of claim 36, wherein the insertion renders the J2R gene non-functional, optionally wherein the J2R locus is completely deleted by the insertion.

38. The therapeutic combination of any one of claims 1-35, wherein the heterologous nucleic acid sequence encoding IL-12 is inserted into the I4L locus of the poxvirus genome.

39. The therapeutic combination of claim 38, wherein the insertion renders the I4L gene non-functional, optionally wherein the I4L locus is not completely deleted by the insertion.

40. The therapeutic combination of any one of claims 1-35, wherein the heterologous nucleic acid sequence encoding IL-12 is inserted into the F4L locus of the poxvirus genome.

41. The therapeutic combination of claim 40, wherein the insertion renders the F4L gene non-functional, optionally wherein the F4L locus is not completely deleted by the insertion.

42. The therapeutic combination of any one of claims 1-41, wherein the poxvirus further comprises one or more therapeutic genes in its genome.

43. The therapeutic combination of claim 42, wherein the one or more therapeutic genes are selected from the group consisting of suicide genes, immunomodulatory genes, anti-angiogenic genes, immune checkpoint blockade genes, antibody encoding genes, extracellular matrix degradation or regulation genes, and combinations thereof.

44. The therapeutic combination of any one of claims 1-43, which is capable of lysing one or more cancer cells.

45. The therapeutic combination of claim 44, wherein the recombinant poxvirus is capable of expressing at least 50 ng / mL, at least 100 ng / mL, at least 300 ng / mL, at least 500 ng / mL, at least 1.0 μg / mL, at least 2.0 μg / mL, at least 3.0 μg / mL, at least 4.0 μg / mL, at least 5.0 μg / mL, at least 6.0 μg / mL, at least 7.0 μg / mL, at least 8.0 μg / mL, or about 8.3 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10-2.

46. ​​The therapeutic combination of claim 44 or 45, wherein the cancer cells are renal cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, bile duct cancer cells, endometrial cancer cells, pancreatic cancer cells, ovarian cancer cells, head and neck cancer cells, melanoma cells, glioblastoma cells, multiple myeloma cells or malignant glioma cells.

47. The therapeutic combination of any one of claims 44-46, wherein the cancer cells are A549, HT29, MIAPaCa-2, A375, RPMI7591, Sk-Mel-5, OVCAR3, OVCAR4, NCI-H292, NCI-H460, SW 780, TCCSUP, T24, Huh7, Hep3B, Pancl, Hup-T3, DAN-G, MDA-MB-435, HCC38, BT20, SW1417, WiDr, HCT-116, SNU5, NCI-N87, Kato III, A CHN, A498, PC-3, or MM.1R cells.

48. The therapeutic combination of any one of claims 1-47, wherein the virus is expressed in chicken embryo fibroblasts (CEF), HeLa cells, cells, Vero cells, HEK 293 cells, PerC6 cells, BHK21 cells, or MRC5 cells.

49. The therapeutic combination of any one of claims 1-48, wherein the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

50. The therapeutic combination of claim 49, wherein the anti-PD-1 antibody or antigen-binding fragment thereof or anti-PD-L1 antibody or antigen-binding fragment thereof is produced in Chinese Hamster Ovary (CHO) cells.

51. The therapeutic combination of any one of claims 1-48, wherein the PD-1 inhibitor or the PD-L1 inhibitor is a small molecule.

52. The therapeutic combination of any one of claims 1-51, wherein (b) is a PD-1 inhibitor.

53. The therapeutic combination of any one of claims 1-51, wherein (b) is a PD-L1 inhibitor.

54. The therapeutic combination of any one of claims 1-53, wherein the PD-1 inhibitor or PD-L1 inhibitor is selected from the group consisting of durvalumab, nivolumab, pembrolizumab, lanlorizumab, MEDI-0680, cemiplizumab, JS001, BGB-A317, INCSHR1210, TSR-042, pidilizumab, GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, atezolizumab, MPDL3280A, RG7446, avelumab, STI-1014, CX-072, KN035, and CK-301.

55. The therapeutic combination of any one of claims 1-51 and 53-54, wherein the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 33; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 34; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

37.

56. The therapeutic combination of claim 55, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

39.

57. The therapeutic combination of claim 55 or 56, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc variant, wherein the Fc variant comprises at least one amino acid substitution selected from the group consisting of: 234F, 235F and 331 S as numbered by the EU index as set forth in Kabat.

58. The therapeutic combination of any one of claims 1-51 and 53-57, wherein the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

59. A therapeutic combination comprising (a) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter, and (b) durvalumab.

60. The therapeutic combination of any one of claims 1-59, wherein the therapeutic combination is used to (i) treat cancer in a subject, (ii) inhibit the growth of cancer, or (iii) enhance a tumor-specific immune response.

61. The therapeutic combination of any one of claims 1-60, wherein the therapeutic combination is a kit of parts comprising (a) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (b) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor.

62. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a programmed death protein 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor.

63. A method of inhibiting cancer growth in a subject, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor.

64. A method for enhancing a tumor-specific immune response in a subject having cancer, the method comprising administering to the subject an effective amount of (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor.

65. The method of any one of claims 62-64, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid promoter.

66. The method of any one of claims 62-65, wherein the poxvirus belongs to the genus Orthopoxvirus.

67. The method of claim 66, wherein the poxvirus belonging to the genus Orthopoxvirus is an oncolytic vaccinia virus.

68. The method of claim 67, wherein the oncolytic vaccinia virus is selected from the group consisting of Copenhagen (Cop), Western Reserve (WR), Elstree, Wyeth, Lister, Tian Tan and LIVP virus strains.

69. The method of any one of claims 62-68, wherein the genome comprises at least 150 kb, at least about 175 kb, at least about 180 kb, at least about 185 kb, at least about 190 kb, at least about 192 kb, or at least about 194 kb.

70. The method of any one of claims 62-69, wherein the poxvirus is attenuated.

71. The method of any one of claims 62-70, wherein the poxvirus is not NYVAC.

72. The method of any one of claims 62-71, wherein the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG7L and pF17R.

73. The method of claim 72, wherein the late promoter is selected from the group consisting of pA14L, pA26L, and pF17R.

74. The method of claim 73, wherein the late promoter is pA14L.

75. The method of claim 73, wherein the late promoter is pF17R.

76. The method of any one of claims 62-75, wherein the late promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 11, 13, 22, or 23.

77. The method of any one of claims 62-76, wherein the late promoter comprises the nucleotide sequence of SEQ ID NO: 11, 13, 22 or 23.

78. The method of any one of claims 62-71, wherein the mid-stage promoter is selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA3L or pA27L.

79. The method of claim 78, wherein the mid-stage promoter comprises a nucleotide sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 25-31.

80. The method of any one of claims 62-79, wherein the IL-12 is human IL-12.

81. The method of any one of claims 62-80, wherein the IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit.

82. The method of claim 81, wherein the IL-12 is a fusion protein comprising an IL-12p40 subunit and an IL-12p35 subunit.

83. The method of claim 81 or 82, wherein the IL-12p40 subunit comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

17.

84. The method of any one of claims 81-83, wherein the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

19.

85. The method of any one of claims 81-84, wherein the IL-12p40 subunit and the IL-12p35 subunit are fused in a single polypeptide via an amino acid linker.

86. The method of claim 85, wherein the amino acid linker is about 5 to about 10 amino acids in length, optionally wherein the amino acid linker is 7 amino acids in length.

87. The method of claim 85 or 86, wherein the amino acid linker is a glycine-serine linker.

88. The method of any one of claims 85-87, wherein the amino acid linker comprises the amino acid sequence of SEQ ID NO:

18.

89. The method of any one of claims 62-88, wherein the IL-12 comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO:

20.

90. The method of any one of claims 65-89, wherein the IL-12p40 subunit and the IL-12p35 subunit are directly fused in a single polypeptide.

91. The method of any one of claims 62-90, wherein the heterologous nucleic acid sequence encoding IL-12 comprises a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 21, optionally wherein the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO:

21.

92. The method of any one of claims 62-91, wherein the recombinant poxvirus is deficient in thymidine kinase (TK) activity.

93. The method of any one of claims 62-92, wherein the recombinant poxvirus lacks a functional J2R gene.

94. The method of any one of claims 62-93, wherein the recombinant poxvirus is deficient in ribonucleotide reductase (RR) activity.

95. The method of any one of claims 62-94, wherein the recombinant poxvirus lacks a functional I4L gene.

96. The method of any one of claims 62-95, wherein the recombinant poxvirus lacks a functional F4L gene.

97. The method of any one of claims 62-96, wherein the heterologous nucleic acid sequence encoding IL-12 is inserted into the J2R locus of the poxvirus genome.

98. The method of claim 97, wherein the insertion renders the J2R gene non-functional, optionally wherein the J2R locus is completely deleted by the insertion.

99. The method of any one of claims 62-98, wherein the recombinant poxvirus is capable of lysing one or more cancer cells.

100. The method of any one of claims 62-99, wherein the recombinant poxvirus is capable of expressing at least 50 ng / mL, at least 100 ng / mL, at least 300 ng / mL, at least 500 ng / mL, at least 1.0 μg / mL, at least 2.0 μg / mL, at least 3.0 μg / mL, at least 4.0 μg / mL, at least 5.0 μg / mL, at least 6.0 μg / mL, at least 7.0 μg / mL, at least 8.0 μg / mL, or about 8.3 μg / mL of IL-12 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10-2.

101. The method of any one of claims 62-100, wherein the recombinant poxvirus is capable of upregulating interferon (IFN)-γ.

102. The method of any one of claims 62-101, wherein the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

103. The method of claim 102, wherein the anti-PD-1 antibody or antigen-binding fragment thereof or anti-PD-L1 antibody or antigen-binding fragment thereof is produced in Chinese hamster ovary (CHO) cells.

104. The method of any one of claims 62-103, wherein the PD-1 inhibitor or the PD-L1 inhibitor is a small molecule.

105. The method of any one of claims 62-104, wherein (ii) is a PD-1 inhibitor.

106. The method of any one of claims 62-105, wherein (ii) is a PD-L1 inhibitor.

107. The method of any one of claims 62-106, wherein the PD-1 inhibitor or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lanlorizumab, MEDI-0680, cemiplizumab, JS001, BGB-A317, INCSHR1210, TSR-042, pidilizumab, GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, atezolizumab, MPDL3280A, RG7446, durvalumab, avelumab, STI-1014, CX-072, KN035, and CK-301.

108. The method of any one of claims 1-103 and 107, wherein the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 33; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 34; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

37.

109. The method of claim 108, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 38, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

39.

110. The method of claim 108 or 109, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc variant, wherein the Fc variant comprises at least one amino acid substitution selected from the group consisting of: 234F, 235F and 331 S as numbered by the EU index as set forth in Kabat.

111. The method of claim 110, wherein the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

112. The method of any one of claims 62-111, wherein the cancer is renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, or malignant glioma.

113. The therapeutic combination of claim 60 or the method of any one of claims 62-112, wherein the cancer is resistant to immune checkpoint inhibitor therapy.

114. The therapeutic combination of claim 60 or 113 or the method of any one of claims 62-113, wherein the cancer is resistant to a PD1 inhibitor.

115. The therapeutic combination of claim 60, 113, or 1114, or the method of any one of claims 62-114, wherein the cancer is resistant to a PD-L1 inhibitor.

116. The method of any one of claims 62-115, wherein the effective amount of an individual dose of the recombinant poxvirus comprises 1 x 10 3 pfu to 1x10 12 pfu, optionally 1x10 4 pfu to 1x10 11 pfu, optionally 1x10 5 pfu to 1x10 10 pfu, optionally 5x10 7 pfu to 4x10 9 pfu.

117. The method of any one of claims 62-116, wherein the PD-1 inhibitor or PD-L1 inhibitor is durvalumab, and the durvalumab is administered at a dose of 10 mg / kg every 2 weeks, 1500 mg every 4 weeks, or 1500 mg every 3 weeks.

118. The method of any one of claims 62-117, wherein the administration results in an enhanced therapeutic effect compared to treatment with the recombinant poxvirus alone or treatment with the PD-1 inhibitor or PD-L1 inhibitor alone.

119. The method of any one of claims 62-118, wherein the subject is a human.

120. The method of any one of claims 62-119, wherein the administration is intratumoral.

121. The method of any one of claims 62-120, wherein the administration is intravenous.

122. The method of claim 121, wherein the intravenous administration is via intravenous infusion.

123. The method of claim 122, wherein the administered dose is 10 mg / kg every 2 weeks.

124. The method of any one of claims 62-123, wherein the recombinant poxvirus and / or the PD-1 inhibitor or PD-L1 inhibitor is administered two or more times.

125. The method of any one of claims 62-124, wherein the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient prior to administration of the recombinant poxvirus.

126. The method of any one of claims 62-124, wherein the PD-1 inhibitor or PD-L1 inhibitor and the recombinant poxvirus are administered to the patient simultaneously.

127. The method of any one of claims 62-126, wherein the PD-1 inhibitor or PD-L1 inhibitor and the recombinant poxvirus are administered in separate pharmaceutical compositions.

128. The method of any one of claims 62-124 or 126, wherein the PD-1 inhibitor or PD-L1 inhibitor and the recombinant poxvirus are administered in the same pharmaceutical composition.

129. The method of any one of claims 62-124, wherein the PD-1 inhibitor or PD-L1 inhibitor is administered to the patient after administration of the recombinant poxvirus.

130. A composition comprising a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding IL-12, for use in treating cancer in a subject in need thereof, wherein the composition is for administration in combination with a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the treatment comprises the method of any one of claims 62-129.

131. A composition comprising a PD-1 inhibitor or a PD-L1 inhibitor for use in treating cancer in a subject in need thereof, wherein the composition is for administration in combination with a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding IL-12, optionally wherein the treatment comprises the method of any one of claims 62-129.

132. A pharmaceutical composition comprising a recombinant poxvirus comprising in its genome (i) a heterologous nucleic acid sequence encoding IL-12 and (ii) a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the recombinant poxvirus is a recombinant poxvirus used in the method of any one of claims 62-129, and / or wherein the PD-1 inhibitor or the PD-L1 inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor used in the method of any one of claims 62-129.

133. The compound for use according to any one of claims 130-132, wherein the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

134. A kit comprising unit doses of (i) a pharmaceutical composition comprising a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the recombinant poxvirus is a recombinant poxvirus used in the method of any one of claims 62-127, and / or wherein the PD-1 inhibitor or the PD-L1 inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor used in the method of any one of claims 62-127.

135. The kit of claim 132, wherein the PD-1 inhibitor or PD-L1 inhibitor is durvalumab.

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