CN119923267A

CN119923267A - Combination of recombinant virus expressing interleukin-12 and PD-1/PD-L1 inhibitor

Info

Publication number: CN119923267A
Application number: CN202380068582.4A
Authority: CN
Inventors: M·D·奥伯斯特; S·伯克; C·黑川
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2022-07-27
Filing date: 2023-07-26
Publication date: 2025-05-02
Also published as: AU2023313118A1; MX2025001073A; EP4561599A1; JP2025526369A; KR20250044313A; CA3262167A1; WO2024023740A1

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 tumor-specific immune responses comprising administering the therapeutic combination.

Description

Combination of recombinant viruses expressing interleukin-12 with PD-1/PD-L1 inhibitors

Cross Reference to Related Applications

The international application claims priority from U.S. provisional application No. 63/369,605 filed on 7.27 of 2022, which is incorporated herein by reference in its entirety.

Reference to an electronically submitted sequence Listing

The contents of the electronically submitted XML sequence listing (name: 2943_230pc01_sequencelisting_st26; size: 48,263 bytes; and creation date: 2023, 7, 19) submitted with the present application are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to a combination comprising (i) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (ii) an inhibitor of PD-1/PD-L1. Such combinations may be used to treat cancer, including cancer that is resistant to therapy with immune checkpoint inhibitors.

Background

Recombinant viruses, including, for example, recombinant poxviruses, represent an emerging therapeutic platform for the treatment of cancer because of their advantages over conventional modes of treatment (e.g., chemotherapy). For example, recombinant viruses can replicate selectively in cancer cells without damaging normal cells and tissues, thereby limiting off-target cell killing and toxicity, thus providing efficacy and specificity levels that may be far higher than conventional cancer therapies. Recombinant viruses can be engineered to express therapeutic transgenes in cells, such as those important in cancer biological pathways. Cancer cells are ideal hosts for many viruses because they can inactivate the antiviral interferon pathway, or can have mutated tumor suppressor genes, enabling unimpeded viral replication.

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 induces cellular immunity by inducing T helper 1 differentiation, acting as a key regulator of cell-mediated immune responses, and by promoting natural killer and T cell interferon-gamma (IFN-gamma) production, proliferation, and cytolytic activity. The versatility of IL-12 has led to the study of this cytokine as an anticancer agent. However, despite encouraging results in animal models, IL-12 has very limited anti-tumor effects and unacceptable adverse events in early clinical trials have occurred, which reduces the hope of successful use of this cytokine.

Immune checkpoint inhibitors (such as PD-1 and PD-L1 targeting molecules) have proven to have some success as cancer therapeutics. However, not all cancers respond to checkpoint inhibitors, and some cancers develop resistance to checkpoint inhibitors.

Thus, there remains a need for new cancer therapies that increase efficacy and/or reduce adverse events.

Disclosure of Invention

In some aspects, the disclosure provides a therapeutic combination comprising (a) a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12), and (b) an inhibitor of apoptosis protein 1 (PD-1) or an inhibitor of apoptosis ligand 1 (PD-L1). In some aspects, the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid-term 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), WESTERNRESERVE (WR), elstree, wyeth, lister, tianTan, and LIVP virus strain. In some aspects, the genome comprises at least 150kb, at least about 175kb, at least about 180kb, at least about 185kb, at least about 190kb, at least about 192kb, or at least about 194kb. In some aspects, the poxvirus is attenuated. In some aspects, the poxvirus is not NYVAC.

In some aspects of the therapeutic combinations disclosed herein, the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA26L, pG L and pF17R. In some aspects, the late promoter is selected from pA14L, pA L 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 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. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO. 11, 13, 22 or 23.

In some aspects, the metaphase promoter is selected from pI1L, pA12L, pA19L, pA42R, pD 3513L, pA L or pA27L. In some aspects of the therapeutic combinations disclosed herein, the metaphase 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 of SEQ ID NOs 25-31.

In the present disclosure of the treatment combination of some aspects, 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-12p40 subunit is located at the N-terminus of IL-12p35 subunit. In some aspects, IL-12p40 subunit comprises the 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 IL-12p35 subunit are fused via an amino acid linker in a single polypeptide. In some aspects, the amino acid linker is about 5 to about 10 amino acids in length. In some aspects, the amino acid linker is 7 amino acids in length. 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, 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 IL-12p35 subunit are fused directly into a single polypeptide. In some aspects, 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. In some aspects, the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO. 21.

In some aspects of the therapeutic combinations disclosed herein, the Thymidine Kinase (TK) activity of the poxvirus is deficient. In some aspects, the poxvirus lacks a functional J2R gene. In some aspects, the poxvirus has defective Ribonucleotide Reductase (RR) activity. 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 renders the J2R gene nonfunctional, optionally wherein the J2R locus is completely deleted by the 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 renders the I4L gene nonfunctional, optionally wherein the I4L locus is not completely deleted by the 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 nonfunctional, optionally wherein the F4L locus is not completely deleted by the insertion. In some aspects of the present invention,

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-angiogenic genes, immune checkpoint blocking genes, antibody encoding genes, extracellular matrix degradation or regulation genes, and combinations thereof.

In some aspects, the therapeutic combinations disclosed herein are capable of lysing one or more cancer cells. In some aspects, the recombinant poxvirus is capable of expressing at least 50ng/mL, at least 100ng/mL, at least 300ng/mL, at least 500ng/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 IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 "2. In some aspects, the cancer cell is a kidney 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 stomach 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.

In some aspects of the therapeutic combinations disclosed herein, the virus is in Chicken Embryo Fibroblasts (CEF), heLa cells,Cells, vero cells, HEK293 cells, perC6 cells, BHK21 cells, or MRC5 cells. In some aspects of the therapeutic combinations disclosed herein, the virus is produced in Chicken Embryo Fibroblasts (CEF).

In some aspects of the therapeutic combinations disclosed herein, the recombinant poxvirus is capable of up-regulating Interferon (IFN) - γ.

In some aspects of the therapeutic combinations disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof or an anti-PD-L1 antibody or antigen-binding fragment thereof. In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof or the anti-PD-L1 antibody or antigen-binding fragment thereof 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 Dewaruzumab (Durvalumab), na Wu Liyou mab (nivolumab), pabrizumab (pembrolizumab), lanrolizumab (lambrolizumab), MEDI-0680, semiphene Li Shan antibody (cemiplimab), JS001, BGB-A317, INCSHR1210, TSR-042, pilizumab (Pidilizumab), GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, abilizumab (atezolizumab), MPDL3280A, RG7446, avelumab (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 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, an 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 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.

In some aspects of the therapeutic combinations disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is dewaruzumab.

In some aspects, the 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 or mid-term promoter, and (b) dewaruzumab. 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.

In some aspects, the disclosure provides a therapeutic combination, 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) an inhibitor of apoptosis protein 1 (PD-1) or an inhibitor of apoptosis ligand 1 (PD-L1).

In some aspects, the disclosure provides a method of 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) an inhibitor of apoptosis protein 1 (PD-1) or an inhibitor of apoptosis ligand 1 (PD-L1). In some aspects, the disclosure provides 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. In some aspects, the disclosure provides a method of 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. In some aspects, the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late promoter or a mid-term promoter.

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), WESTERNRESERVE (WR), elstree, wyeth, lister, tian Tan, and LIVP virus strains. In some aspects, the genome comprises at least 150kb, at least about 175kb, at least about 180kb, at least about 185kb, at least about 190kb, at least about 192kb, or at least about 194kb. In some aspects, the poxvirus is attenuated. In some aspects, the poxvirus is not NYVAC.

In some aspects of the methods disclosed herein, the late promoter is selected from pA10L, pA11R, pA13L, pA14L, pA 3526L, pG L and pF17R. In some aspects, the late promoter is selected from pA14L, pA L 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 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. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO. 11, 13, 22 or 23.

In some aspects of the methods disclosed herein, the metaphase promoter is selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA L or pA27L. In some aspects, the metaphase 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 of SEQ ID NOs 25-31.

In the methods of the present disclosure of some aspects, 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, IL-12p40 subunit comprises the 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 IL-12p35 subunit are fused via an amino acid linker in a single polypeptide. In some aspects, 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. 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, 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 IL-12p35 subunit are fused directly into a single polypeptide.

In some aspects of the methods disclosed herein, a 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, optionally wherein the heterologous nucleic acid sequence encoding IL-12 comprises the nucleotide sequence of SEQ ID NO. 21.

In some aspects of the methods disclosed herein, the Thymidine Kinase (TK) activity of the recombinant poxvirus is deficient. In some aspects, the recombinant poxvirus lacks a functional J2R gene. In some aspects, the Ribonucleotide Reductase (RR) activity of the recombinant poxvirus is defective. 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 nonfunctional, optionally wherein the J2R locus is completely deleted by the insertion.

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 50ng/mL, at least 100ng/mL, at least 300ng/mL, at least 500ng/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 IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 "2.

In some aspects of the methods disclosed herein, the recombinant poxvirus is capable of up-regulating Interferon (IFN) - γ.

In some aspects of the methods disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof or an anti-PD-L1 antibody or antigen-binding fragment thereof. In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof or the anti-PD-L1 antibody or antigen-binding fragment thereof 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 Na Wu Liyou mab, palivizumab, lanloplizumab, MEDI-0680, cimetidine Li Shan mab, JS001, BGB-A317, INCSHR1210, TSR-042, pierizumab, GLS-010, STI-1110, AGEN2034, MGA012, IBI308, AMP-224, BMS-936559, abilizumab, MPDL3280A, RG7446, devaluzumab, 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 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, an 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 dewarfarin.

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, cholangiocarcinoma, 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.

In some aspects of the methods disclosed herein, an effective amount of an individual dose of the recombinant poxvirus comprises 1x 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 5x 10 ⁷ pfu to 4x 10 ⁹ pfu.

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

In some aspects of the methods disclosed herein, the administration results in an enhanced therapeutic effect compared to treatment with the recombinant poxvirus alone or with the 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 Dewaruzumab, and the dose of this administration is 10mg/kg every 2 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is dewarfarin and is administered at a dose of 1500mg every 3 weeks or every 4 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is dewarfarin and is administered at a dose of 1500mg every 4 weeks. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is dewarfarin and is administered at a dose of 1500mg every 3 weeks.

In some aspects of the methods disclosed herein, the recombinant poxvirus and/or PD-1 inhibitor or PD-L1 inhibitor is administered two or more times. 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 concurrently with the recombinant poxvirus. In some aspects, the PD-1 inhibitor or the PD-L1 inhibitor and the recombinant poxvirus are administered in separate pharmaceutical compositions. In some aspects, the PD-1 inhibitor or the 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 administration of the recombinant poxvirus.

In some aspects, provided herein is also 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 of the methods disclosed herein.

In some aspects, provided herein is also 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 any one of the methods disclosed herein.

In some aspects, provided herein is also 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 one 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 one of the methods disclosed herein. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is dewarfarin.

In some aspects, provided herein is also a kit comprising a unit dose 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 for use in any one 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 for use in any one of the methods disclosed herein. In some aspects of the kits disclosed herein, the PD-1 inhibitor or PD-L1 inhibitor is dewaruzumab.

Drawings

FIG. 1 is a schematic diagram of plasmid pTG 19409.

FIG. 2 shows luciferase expression from six late promoters after infection/transfection of DF-1 cells.

FIGS. 3A and 3B show luciferase expression at 6 hours (A) and 24 hours (B) after infection of MIAPaCa-2 cells with ten recombinant vaccinia viruses.

FIG. 4 shows luciferase expression after infection of HeLa cells.

FIG. 5 shows luciferase expression after infection of HCT-116 cells.

Figure 6 shows GFP positive cells at 6 hours and 24 hours after infection with human PBMCs.

Fig. 7 shows luciferase expression at 6 hours and 24 hours after infection of human PBMC.

FIG. 8 shows the sequence of COPTG19673 hIL-12 expression cassette (SEQ ID NO: 21).

FIG. 9 is a schematic diagram of plasmid pTG 19673.

FIG. 10 is a schematic diagram of plasmid pTG 19674.

FIG. 11 shows the expression of IL-12 in supernatants of A549 cells infected with COPTG19673 and COPTG19674 of primary study stocks, as measured by ELISA.

FIGS. 12A-C show replication of VACV-IL-12 (COPTG 19673 and COPTG 19674) and unarmed control VACV (VVTG 18058) in human tumor cell lines A549 (A), mi PaCa-2 (B) and HT-29 (C) 24 hours, 48 hours and 72 hours after infection.

FIG. 13 shows replication of VACV-IL-12 (COPTG 19673 and COPTG 19674) and unarmed control VACV (VVTG 18058) in producer cells (HeLa and CEF) 72 hours after infection. The results are the average of three wells.

FIGS. 14A-C show the oncolytic activity of COPTG19673, COPTG19674 and empty VACV (VVTG 18058) at different MOI 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).

FIG. 15 shows the expression levels of vIL-12 in supernatants of A549, MIA PaCa-2, and HT-29 cells infected with COPTG19673 and COPTG19674 after incubation for 3 days at MOI 0.01. The results are the average and SD of duplicate measurements of three samples.

FIGS. 16A-F show IL-12 bioactivity after incubation of HEK-Blue ^TM IL-12 cells with the supernatant of COPTG 19673-infected tumor cell line (A-C) and with the supernatant of COPTG 19674-infected tumor cell line (D-F), as determined using the HEKBlueIL-12 cell reporter assay, as compared to rhIL-12. Results are expressed as the mean ± SD of the two measurements.

FIGS. 17A-F show IL-12 bioactivity after incubation of NK-92 cells with supernatant of COPTG 19673-infected tumor cell line (A-C) and supernatant of COPTG 19674-infected tumor cell line (D-F) as compared to rhIL-12, as determined using NK-92 proliferation assay.

FIG. 18 shows the replication yields of VACVwt, unarmed control VACV (VVTG 18058), COPTG19673 and COPTG19674 on human hepatocytes. The results are expressed as replication yields corresponding to the ratio between the input/output virus numbers. The results are the average of three wells.

Figure 19 shows replication of vacvswt, VVTG18058, and COPTG19673 and COPTG19674 on human PBMC. The results are expressed as replication yields corresponding to the ratio between the input/output virus numbers. The results are the average of three wells.

Figure 20 shows the efficacy of virus-mediated oncolysis in cultured human tumor cells incubated with VACV IL-12 at different MOI to determine the average EC50 of cell lysis in each of three independent experiments. (see also 29B).

FIGS. 21A-B show virus recovered from tumor by plaque formation assay (virus PFU/gram tumor tissue) (A) and intratumoral IL-12- (ngIL-12/gram tumor) using human IL-12 specific ELISA (B). Asterisks indicate time points at which no virus and transgene were determined.

FIGS. 22A-E show spider plots (B-D) of tumor growth in C57BL/6 mice transplanted with subcutaneous MC38 colorectal tumors following multidose intratumoral administration of VACV expressing murine IL-12, and Kaplan-Meier plots (E) of survival of tumor-bearing mice. CR, complete response of tumor to therapy, where tumor volume was undetectable, by log rank (Mantel-Cox) comparison of three or more groups, p=0.0024.

FIGS. 23A-E show expression of murine IL-12 (A) in peripheral blood of mice 4 and 24 hours after intratumoral administration VACV muIL-12, as well as expression of cytokines IFNγ (B), CXCL10 (C), IL-6 (D) and TNF α (E). * P <0.05, one-way analysis of variance and Tukey post hoc test.

FIGS. 24A-H show the anti-tumor activity of luciferase-expressing VACV (VACVluc) determined using primary tumors (PDX: patient-derived xenografts) from cancer patients transplanted into immunocompromised NOD/SCID mice. Each tumor tested was either untreated (open circles, grey lines) or administered 1e7 VACVluc virus (filled squares, black lines). Each primary patient-derived tumor includes 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.

FIGS. 25A-F show 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 matrices isolated from xenograft models derived from patients with bladder, head and neck, liver, colon, lung and ovarian cancer at day 0 and 48 hours after day 14, from VACV-luciferase treated mice.

FIGS. 26A-E show the efficiency of VACV-IL12 in infecting human tumors in vitro, as measured by detecting IL-12p70 (A) in supernatants from dissociated tumor cell cultures, IL-12p70 expression (B) in supernatants from tumor section cultures, IFNγ mRNA levels (C), IFNγ protein levels (D), and B8R mRNA levels (E). * <0.0001, one-way analysis of variance corrected using Tukey multiple comparisons. Each point is a single slice with 1-4 replicates per condition. FIG. 26F is a schematic showing the timing of VACV infection, transgene production and immune activation.

FIGS. 27A-C show COPTG19673 selective replication in tumor cells relative to normal human cells. There was no observable expansion in PBMC relative to cancer cells (SW 780), and minimal replication in normal hepatocytes (HUCPG and HUCPI) and Normal Human Dermal Fibroblasts (NHDF).

Fig. 28A-C show experimental protocol (a), oncolytic VACV efficacy and overall response across multiple tumor indications (B), and replication kinetics observed 48 hours after dose 1 and 3 (C). CRC, colorectal cancer, HN, head and neck squamous cell carcinoma.

FIGS. 29A-E show oncolytic activity of VACV-Luc and VACVIL-12 (COPTG 1673) 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 oncolytic effects of VACV Luc and VACV-IL12 on human tumor cell lines. Panels D and E show VACV-IL12 transgene production and replication in tumor cell lines 5 days post infection.

FIGS. 30A-F show the antitumor efficacy of VACVIL-12 (COPTG 1673) in a human xenograft tumor model. Panels a-C show the mean tumor volume change over time for different doses of VACV-IL12 compared to VACV-Luc in mice bearing SW780 tumor, NCI-H292 tumor and HCT-116 tumor. Panels D and E show viral load and recovered intratumoral IL-12 over time for each dose in SW780 tumor-transplanted mice. Panel 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.

FIGS. 31A-D show analysis of TSC culture gene expression following infection with VACV IL-12 (COPTG 1673). Panel A shows the production of IL-12p70 in VACV-IL12 infected cells compared to mock-infected or VACV GFP-infected groups. Panels B-D show production of IFN proteins (ifnγ in panel B, ifnα2a in panel C, and ifnβ in panel D) in VACV-IL 12-infected cells compared to mock-infected or VACVGFP-infected groups.

FIGS. 32A-H show that VACV-muIL exhibits similar oncolytic activity and similar IL-12 bioactivity as VACV IL-12 (COPTG 1673) in human tumor cells. Panel A shows the dose-dependent relationship between culture supernatant dilutions of VACV-IL12 infected cells and neutralization of rat IFNγ. Panels B-C show viral (panel B) and IL12 transgene production across 3 tumor cell lines (panel C) for VACV-GFP and VACV-muIL. Panels D-F show the percent survival of the complex number of infection with VACV-GFP and VACV-muIL for each cancer cell line. Panels G-H show IL12p70 (panel G) and ifnγ (panel H) plasma levels over time in rats transplanted with F98 rat glioma and intravenously treated with vehicle, VACV-luc, or three doses of VACV-muIL.

FIGS. 33A-E show that VACV-muIL exhibits similar oncolytic activity and similar IL-12 bioactivity as VACVIL-12 (COPTG 1673) in cultured human tumor cells. Panels a-C show the percent survival with respect to the complex number of infections for VACV-LUC, VACV-muIL and VACV-huIL12 using the cultured SW780 bladder, NCI-H292 lung and HCT-116 colorectal tumor cell lines. Panel D shows the IL-12p70 concentrations produced by VACV-Luc, VACV-muIL12 and VACV-huIL12 in human and murine cells for the SW780, NCI-H292 and HCT-116 tumor cell lines. Panel E shows the IL-12 activity of recombinant human IL-12 (rIL-12) at various concentrations and IL-12 measured in cell culture supernatants from cultured SW780 tumor cells treated with VACV-muIL, VACV-huIL12, or VACV-Luc.

FIGS. 34A-F show that VACV-muIL shows similar oncolytic activity to VACV-LUC in murine tumor cells and that IL-12 with moderate viral replication is produced across tumor cell lines. Panels a-D show the percent survival of the complex number of infection with VACV-Luc and VACV-muIL in murine tumor cell lines. Panel E shows the IL-12 concentration produced in multiple murine tumor cell lines over a period of 5 days for VACV-muIL-12 and VACV-Luc. Panel F shows the amount of virus recovered in each murine tumor cell line on day 5 for VACV-luc and VACV-muIL.

FIGS. 35A-G show that VACV-muIL12 enhances anti-tumor immune response in murine isogenic CT26 tumor models. Fig. a shows a general procedure. Panel B shows survival curves for vehicle control, VACV-Luc (1X 10 ⁷ PFU) and VACV-muIL12 (1X 10 ⁷ PFU). * P <0.0001. Panels C-E show tumor volume changes for vehicle control, VACV-Luc, and VACV-muIL. Panel F shows plasma IL12p70 concentrations at 4 and 24 hours post-injection for vehicle control, VACV-Luc and VACV-muIL. Panel G shows the mouse plasma IFN-gamma concentrations at 4 and 24 hours post injection for vehicle control, VACV-Luc and VACV-muIL. * P <0.01 =p <0.001.

FIGS. 36A-C show tumor volumes over time in CT26 tumor-transplanted mice after administration of vehicle (A), VACV-LUC (B) or VACV-muIL (C). The Y-axis shows tumor volume and the X-axis shows time in days.

FIGS. 37A-B show the results of ex vivo splenocyte re-stimulation assays following VACV administration and stimulated with VACV specific peptide A52L (A) or tumor associated peptide antigen AH-1 (B).

Detailed Description

For easier understanding of the present disclosure, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings set forth below, unless the context clearly dictates otherwise. Additional definitions are set forth throughout the application.

Definition of the definition

Generally, the terms and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry as well as hybridization described herein are well known and commonly used in the art. Amino acids herein may be referred to by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee (IUPAC-IUB BiochemicalNomenclatureCommission). Likewise, nucleotides may be referred to by their commonly accepted single letter codes.

Throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In certain aspects, the term "a/an" means "single. In other aspects, the term "a/an" includes "two or more" or "a plurality of".

The term "about" includes the recited numbers + -10%. Thus, "about 10" means 9 to 11. Reference herein to "about" a value or parameter includes (and describes) aspects that relate to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

The term "or" is used to mean "and/or" unless explicitly indicated to mean only alternatives or that the alternatives are mutually exclusive, although the disclosure supports definitions of only alternatives and "and/or". Furthermore, "and/or" as used herein is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the terms "and/or" as used in phrases such as "a and/or B" herein are intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of A, B and C, A, B or C, A or B, B or C, A and B, B and C, A (alone), B (alone), and C (alone).

As used in this specification and in one or more claims, the terms "comprises" (and any form of comprising), such as "comprises" and "comprising"), having (and any form of having, such as "having" and "having"), including (and any form of comprising, such as "including" and "comprising") or containing (and any form of containing, such as "contain" and "contain") are inclusive or open-ended and do not exclude other 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 of the disclosure.

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

"Nucleic acid", "nucleic acid molecule", "nucleotide sequence", "oligonucleotide" or "polynucleotide" refers to a polymeric compound that includes covalently linked nucleotides. The term "nucleic acid" includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), both of which may 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.

"Gene" refers to the assembly of nucleotides encoding a gene product (RNA or protein). Genes include cDNA and genomic DNA molecules.

As used herein, "functional" gene (including functional transgenes) refers to a gene capable of expressing an RNA or protein product, wherein the RNA or protein product retains at least one functional activity. As used herein, a "non-functional" gene (including non-functional transgenes) refers to a gene that is incapable of expressing an RNA or protein product that retains any functional activity. A non-functional gene may refer to a gene that has been completely removed or replaced. A non-functional gene may also refer to a gene that has been partially removed or replaced such that the remainder of the gene is not capable of expressing an active RNA or protein product.

A "coding sequence" is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, can be transcribed and translated into a polypeptide in a cell in vitro or in vivo. "regulatory sequences" include nucleotide sequences that are located upstream (5 'non-coding sequences), internal or downstream (3' non-coding sequences) of the coding sequence and affect transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem loop structures. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences include, but are 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 eukaryotic cells, the polyadenylation signal and transcription termination sequence may be located 3' of the coding sequence.

The abbreviation ORF for "open reading frame" refers to a stretch of nucleic acid sequence (DNA, cDNA or RNA) that includes a translation initiation signal or initiation codon (such as ATG or AUG) and a stop codon and that may be translated into a polypeptide sequence.

"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 typically contains a region of homology sufficiently long to the sequence of the target DNA sequence to allow complementary binding of the inserted DNA sequence and incorporation into the target DNA sequence. Longer regions of homology and greater degrees of sequence similarity generally increase the efficiency of homologous recombination.

"Heterologous" describes the relationship of one nucleic acid or amino acid sequence to one or more different nucleic acid or amino acid sequences and indicates that the sequences are not found linked together in nature in the same position, structure, and orientation. Ligation of heterologous sequences results in juxtaposition of non-naturally occurring sequences. This connection is an engineered product made in the laboratory. The product of such ligation may be referred to as a "recombinant".

The two heterologous nucleic acid or amino acid sequences may be directly linked (fused) or may be linked by a "linker". In certain aspects, the linker is a chemical linker. In certain aspects, the linker comprises one or more amino acids. Glycine-serine linkers are linkers that contain both glycine and serine amino acids in any proportion, such as GGGS.

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

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

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

An "late promoter" is a promoter naturally associated with expression of a late gene. A "metaphase promoter" is a promoter that controls gene expression following early gene expression but is not under the control of a late promoter. Genes expressed early in the viral life cycle prior to mid-and late-stage gene expression are referred to as "early promoters". Expression of the late and mid genes, controlled by mid/late promoters, is dependent on viral replication (unlike expression of the early genes, which is independent of viral replication). The expression timeframe of early, mid and late promoters is a distinguishing factor, as discussed in Yang et al J.Virol. [ J.Virol., vol.85, 19, pages 9899-9908 (2011), reference Baldick et al, J.Virol. [ J.Virol., 67:3515-3527, (1993), each of which is incorporated herein by reference. Baldick discloses that early, medium and late mRNA can be detected within 20min, 100min and 140min, respectively, after simultaneous infection of HeLa cells with VACV. Yang prepared full genome early, mid and late transcriptional maps revealing the unique features of mid and late promoters. As used herein, the terms "mid-term promoter" and "late promoter" may refer to any of the mid-term promoter and late promoter discussed in Yang, particularly the promoters shown in fig. 8 therein.

Exemplary late promoters include, but are not limited to, pA10L, pA11R, pA13L, pA14L, pA L, pG L and pF17R. Exemplary metaphase promoters include, but are not limited to, pI1L, pA12L, pA19L, pA42R, pD 3513L, pA L or pA27L.

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

"Transfection" refers to the introduction of an exogenous nucleic acid molecule into a cell. "transfected" cells include an exogenous nucleic acid molecule within a cell, while "transformed" cells are cells in which the exogenous nucleic acid molecule within the cell induces a phenotypic change in the cell.

The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

The starting point of a protein or polypeptide is referred to as the "N-terminus" (or amino-terminus, NH ₂ -terminus, N-terminus, or amine-terminus) and refers to the free amine (-NH ₂) group of the first amino acid residue of the protein or polypeptide. The terminus of a protein or polypeptide is referred to as the "C-terminus" (or carboxyl-terminus (carboxy-terminus), carboxyl-terminus (carboxyl-terminus), C-terminus or COOH-terminus) and refers to the free carboxyl (-COOH) group of the last amino acid residue of a protein or peptide.

As used herein, "amino acid" refers to a compound that includes both carboxyl (-COOH) and amino (-NH ₂) groups. "amino acid" refers to natural and unnatural amino acids, e.g., synthetic amino acids. Natural amino acids (using their three-letter and one-letter abbreviations) include alanine (Ala; a), arginine (Arg, R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (gin; 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), valine (Val; V).

"Amino acid substitution" in a polypeptide or protein refers to a polypeptide or protein that includes substitution of one or more wild-type or naturally occurring amino acids at the amino acid residue with an amino acid that differs from the wild-type or naturally occurring amino acid. The substituted amino acid may 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 (5 th) amino acid residue is substituted may be abbreviated as "X5Y", where "X" is a wild-type or naturally occurring amino acid to be substituted, "5" is the position of the amino acid residue within the amino acid sequence of a protein or polypeptide, and "Y" is a substituted or non-wild-type or non-naturally occurring amino acid.

An "isolated" polypeptide, protein, peptide, or nucleic acid has been removed from its natural environment. It will also be appreciated that an "isolated" polypeptide, protein, peptide or nucleic acid may be formulated with an excipient (such as a diluent) or adjuvant, and still be considered to be isolated.

When used in reference to a nucleic acid molecule, peptide, polypeptide, or protein, the term "recombinant" means a new combination of genetic material or production thereof that is known to be absent in nature. Recombinant molecules can be produced by any well known technique known in the recombinant arts, 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.

"Poxvirus" refers to a virus of the family Poxviridae, including, for example, viruses of the genus orthopoxvirus. The "genome" of a recombinant poxvirus provided herein includes the poxvirus genome, which 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 a recombinant poxvirus may refer to the genome of an attenuated poxvirus.

"Oncolytic virus" refers to a DNA or RNA virus that preferentially infects and kills cancer cells over normal cells. Oncolytic viruses can kill cancer cells by 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.

"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 may establish persistent immunity in addition to destroying existing cancer cells). Oncolytic activity may be detected by known methods including, but not limited to, detection of cell death or apoptosis, inhibition of cell proliferation, and/or by detection of a decrease in tumor size.

A virus is considered "cytotoxic" if it reduces the cell viability in the treated target cells relative to untreated target cells. Methods for determining cytotoxicity of viruses are known and include, for example, cytotoxicity assays that measure cell necrosis and/or apoptosis after virus infection, such as MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assays 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 fragment assays, DNA step assays, and cytochrome C release assays. Another method of determining cytotoxicity is to monitor tumor size and location before and after treatment. In some cases, it may be desirable to monitor the size at several time points to obtain information about the increase or decrease in size of the tumor or metastasis.

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

"Pathogenic virus" refers to a virus that produces a disease. In some aspects, the recombinant poxviruses provided herein are not pathogenic viruses.

"Replicable" refers to the ability of a virus to replicate in a cell or cell line and produce infectious progeny virions. Viruses that are capable of producing infectious progeny virions in a cell or cell line are considered "replicable", while viruses that are incapable of producing infectious progeny virions in a cell or cell line are considered "replication defective". Viral replication can be expressed by the ratio of the amount of virus produced by an infected cell to the amount used to infect the cell, referred to as the "amplification rate". An amplification ratio of 1 or greater means that the amount of virus produced from the infected cells is the same or greater than the amount used to infect the cells, indicating that replication has occurred, while an amplification ratio of less than 1 means that the amount of virus produced from the infected cells is less than the amount used to infect the cells, indicating a lack of replication in the cells.

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

The terms "cell proliferation disorder" and "proliferation disorder" refer to disorders associated with a degree of abnormal cell proliferation. Cell proliferation disorders may include cancer.

"Cancer" or "cancerous" refers to a physiological condition in a mammal characterized by unregulated cell growth, lack of differentiation, localized 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.

An "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.

"PD-1 inhibitor" refers to an agent that reduces the amount or activity of PD-1. For example, a PD-1 inhibitor may be an agent that binds to PD-1 protein and inhibits its interaction with PD-L1. The PD-1 inhibitor may 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, the 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, palivizumab (Keystuda), nal Wu Liyou mab (Opdivo) and cimipro Li Shan mab (Libtayo).

"PD-L1 inhibitor" refers to an agent that decreases the amount or activity of PD-L1. For example, a PD-L1 inhibitor may be an agent that binds to the PD-L1 protein and inhibits its interaction with PD-1. The PD-L1 inhibitor may be, for example, an antibody or antigen-binding fragment thereof that binds 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 atilizumab (TECENTRIQ), avilamab (Bavencio), and Dewaruzumab (Imfinzi).

As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to an antibody molecule that recognizes and specifically binds a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing (e.g., 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 molecule, so long as the antibodies exhibit the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins, igA, igD, igE, igG and IgM, or subclasses (isotypes) thereof (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), referred to as α, δ, epsilon, γ, and μ, respectively, based on the characteristics of their heavy chain constant domains. Antibodies of different classes have different and well known subunit structures and three-dimensional configurations. For the structure and properties of different classes of antibodies, see for example Basic AND CLINICAL Immunology [ Basic Immunology and clinical Immunology ], 8 th edition, daniel P.Stites, abbaI.Terr and tristram g. Parslow (ed.), alston and Lang company, norwalk, CT., 1994, pages 71 and chapter 6, norwalk, conn.

The term "antibody fragment" refers to a portion of an antibody. An "antigen-binding fragment" of an antibody refers to a portion of an antibody that binds to an antigen. An antigen binding fragment of an antibody may comprise an antigen-determining region (e.g., a Complementarity Determining Region (CDR)) of the antibody. Examples of antigen binding fragments of antibodies include, but are not limited to, fab ', F (ab') 2, and Fv fragments, linear antibodies, and single chain antibodies. The antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., bivalent). Antigen binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

An "antigen binding domain" or "antigen binding region" refers to a monovalent portion of an antibody that binds an antigen. An "antigen binding domain" may comprise an antigen-determining region (e.g., a Complementarity Determining Region (CDR)) of an antibody. Antibodies or antigen-binding fragments thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof) may comprise an antigen-binding domain.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the 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 the "framework" region, exhibit less intermolecular variability. The framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops connecting the β -sheet structure, and in some cases a portion of the β -sheet structure. Thus, the framework regions act to form a scaffold that provides for positioning of the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen binding domain formed by the localized CDRs defines a surface complementary to an epitope on the immunoreactive antigen. Such complementary surfaces facilitate non-covalent binding of the 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 CDRs and framework regions, respectively, as they have been precisely defined (see, "Sequences of Proteins ofImmunological Interest [ immunologically significant protein sequences ]", kabat, e. Et al, U.S. device ofHealth andHuman Services [ U.S. health and public service ], (1983); and Chothia and Lesk, j. Mol. Biol. [ journal of molecular biology ],196:901-917 (1987), which is incorporated herein by reference in its entirety).

The term "Kabat numbering" and similar terms are well known in the art and refer to the system by which amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding fragment thereof are numbered. In certain aspects, CDRs may be determined according to the Kabat numbering system (see, e.g., kabat EA and Wu TT (1971) ANN NY ACAD SCI [ New York Proc. Natl. Acad. Sci. Year. ]190:382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest [ protein sequences of immunological significance ], fifth edition, U.S. Pat. No. HEALTH AND Human Services [ U.S. department of health and public service ], NIH publication No. 91-3242). CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (optionally one or two additional amino acids after position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) using the Kabat numbering system (CDR 1), amino acid positions 50 to 65 (CDR 2) and amino acid positions 95 to 102 (CDR 3). CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR 1), amino acid positions 50 to 56 (CDR 2) and amino acid positions 89 to 97 (CDR 3) using the Kabat numbering system. In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

Chothia refers to the position of the structural ring (Chothia and Lesk, J.mol.biol. [ 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 (since the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop end point is at 32; if only 35A is present, the loop end point is at 33; if both 35A and 35B are present, the loop end point is at 34). The AbM hypervariable region represents a compromise between kabat cdrs and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software.

As used herein, the term "heavy chain" when used in reference to an antibody, the constant domain-based amino acid sequence may refer to any of the different types, e.g., α (a), δ (d), ε (e), γ (g), and μ (m), which produce the IgA, igD, igE, igG and IgM classes of antibody, respectively, including subclasses of IgG, e.g., igG1, igG2, igG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In particular aspects provided herein, the heavy chain is a human heavy chain.

As used herein, the term "light chain" when used in reference to an antibody may refer to any of the different types of amino acid sequences based on constant domains, e.g., kappa or lambda. The light chain amino acid sequences are well known in the art. In particular aspects provided herein, the light chain is a human light chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are functionally used. In this regard, it is understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the constant domains of the light Chain (CL) and the heavy chain (CH 1, CH2 or CH 3) confer important biological properties such as secretion, transplacental mobility, fc receptor binding, complement fixation, etc. Conventionally, the numbering of constant region domains increases as they become farther from the antigen binding site or amino terminus of an antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region, and the CH3 and CL domains comprise the carboxy-terminal ends of the heavy and light chains, respectively.

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 VL domain and VH domain of a binding molecule (e.g., an antibody) or subclasses of Complementarity Determining Regions (CDRs) 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 Y. More particularly, the antigen binding site is defined by three CDRs on each of the VH and VL chains.

A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homologous antibodies or antigen-binding fragments that are involved in the highly specific recognition and binding of a single epitope or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen binding fragment thereof encompasses whole and full length monoclonal antibodies as well as antibody fragments (e.g., fab ', F (ab') 2, fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. In addition, "monoclonal" antibodies or antigen-binding fragments thereof refer to antibodies and antigen-binding fragments thereof prepared in any number of ways, including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from a library of human immunoglobulins or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins, as described below and in, for example, U.S. patent No. 5,939,598 to kucherlpati et al.

"Patient," "subject," and "individual" are used interchangeably and refer to animals, including humans and non-human animals, including, for example, primates, cows, pigs, sheep, goats, dogs, cats, rabbits, and rodents, as well as non-mammals such as chickens, amphibians, and reptiles, to be treated. In one aspect, the subject is a human. In one aspect, the subject is a human suffering from cancer. In another aspect, the subject is a laboratory animal or animal disease model.

The term "treatment" or "treatment" refers to a therapeutic treatment in which the aim is to reduce or eliminate one or more symptoms. Beneficial or desired results include, but are not limited to, elimination of symptoms, alleviation of symptoms, diminishment of extent of disorder, stabilized (e.g., not worsening) state of disorder, delay or slowing of progression of disorder. Treating cancer may include inducing cell death of cancer cells or intratumoral cells.

"Tumor progression" refers to the stage of a tumor, including tumorigenesis, tumor growth and proliferation, invasion and metastasis. By "inhibiting tumor progression" is meant 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 infiltration of cancer cells into adjacent peripheral organs and/or tissues, inhibiting or reducing metastasis, increasing the length of survival of a patient or patient population after treatment, and/or decreasing the mortality of a patient or patient population at a given point in time after treatment.

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 of parts or instructions for combined administration, wherein the recombinant poxvirus and PD-1/PD-L1 inhibitor may be administered simultaneously, independently or separately at intervals. The combined pharmaceutical compositions may be adapted 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.

Recombinant poxviruses, methods of making recombinant poxviruses, and compositions comprising the same

Provided herein is 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 or mid-term 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.

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 strains WESTERNRESERVE (WR), elstree, wyeth, lister, tianTan, LIVP, and Copenhagen (Cop). In some aspects, the oncolytic vaccinia virus is selected from the Copenhagen (Cop) strains.

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

In some aspects, the recombinant poxvirus is attenuated.

As provided herein, a variety of late promoters can be used in the recombinant poxviruses. In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxviruses comprise a late promoter selected from pA10L, pA11R, pA13L, pA L, pA26L, pG L and pF17R. In some aspects, the late promoter is selected from pA14L, pA L 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.

The sequences of the late promoters pA10L, pA, R, pA13L, pA L, pA26L, pG L and pF17R are provided in Table 1 below.

Table 1.

As provided herein, a variety of metaphase promoters can be used in the recombinant poxviruses. In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxviruses comprise a metaphase promoter selected from pI1L, pA12L, pA19L, pA42R, pD13L, pA L or pA 27L.

The sequences of the metaphase promoters pI1L, pA, L, pA19L, pA42R, pD, 13L, pA L or pA27L are provided in table 2 below.

Table 2.

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.

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.

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 to the nucleotide sequence of SEQ ID NO. 13. In some aspects, the late promoter comprises the nucleotide sequence of SEQ ID NO. 13.

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.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 25. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 25.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 26. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 26.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 27. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 27.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 28. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 28.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 29. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 29.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 30. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 30.

In some aspects, the metaphase 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 to the nucleotide sequence of SEQ ID NO. 31. In some aspects, the metaphase promoter comprises the nucleotide sequence of SEQ ID NO. 31.

As described above, the recombinant poxviruses provided herein comprise in their genome a heterologous nucleic acid sequence encoding IL-12.IL-12 can be human IL-12.IL-12 can be murine IL-12.

In some aspects of the recombinant poxviruses 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 N-terminally to 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 linker) or may be fused via a linker. The linker may be, for example, a chemical linker or an amino acid linker. The amino acid linker may be a glycine-serine linker. In some aspects, the linker is about 5 to about 10 amino acids in length. In some aspects, the linker is 7 amino acids in length. In some aspects, the linker comprises the amino acid sequence of SEQ ID NO. 18.

Widely used vaccinia virus vectors include highly attenuated strains, such as new york vaccinia virus (NYVAC). In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxviruses are not NYVAC.

In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit comprises 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 of the recombinant poxviruses disclosed herein, the IL-12p40 subunit has the amino acid sequence of SEQ ID NO. 17.

In some aspects of the recombinant poxviruses disclosed herein, the IL-12p35 subunit comprises 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 of the recombinant poxviruses disclosed herein, the IL-12p35 subunit comprises the amino acid sequence of SEQ ID NO. 19.

In some aspects of the recombinant poxviruses disclosed herein, the IL-12p40 subunit comprises 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, and the IL-12p35 subunit comprises 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 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.

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.

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 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 of the recombinant poxviruses disclosed herein, the Thymidine Kinase (TK) of the poxvirus is deficient. In some aspects of the recombinant poxviruses disclosed herein, the poxviruses lack a functional J2R gene

In some aspects of the recombinant poxviruses disclosed herein, the poxviruses have defective Ribonucleotide Reductase (RR) activity. In some aspects of the recombinant poxviruses disclosed herein, the poxviruses lack a functional I4L gene. In some aspects of the recombinant poxviruses disclosed herein, the poxviruses lack a functional F4L gene. In some aspects of the recombinant poxviruses disclosed herein, the poxviruses lack a functional I4L gene and lack a functional F4L gene.

In some aspects of the recombinant poxviruses disclosed herein, the Thymidine Kinase (TK) and/or Ribonucleotide Reductase (RR) activity of the poxviruses is defective. In some aspects of the recombinant poxviruses disclosed herein, the poxviruses lack a functional J2R gene and lack 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.

In some aspects of the recombinant poxviruses 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 nonfunctional. In some aspects, the JR2 locus is completely deleted by insertion. In some aspects, the JR2 locus is not completely deleted by insertion.

In some aspects of the recombinant poxviruses 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 nonfunctional. In some aspects, the I4L locus is completely deleted by insertion. In some aspects, the I4L locus is not completely deleted by insertion.

In some aspects of the recombinant poxviruses 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 nonfunctional. In some aspects, the F4L locus is completely deleted by insertion. In some aspects, the F4L locus is not completely deleted by insertion.

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

In some aspects of the recombinant poxviruses disclosed herein, the recombinant poxviruses are capable of lysing one or more cancer cells.

In some aspects, the recombinant poxvirus is capable of expressing at least 50ng/mL, at least 100ng/mL, at least 300ng/mL, at least 500ng/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 100ng/mL, at least 300ng/mL, at least 500ng/mL, at least 1.0 μg/mL, at least 2.0 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 in cancer cells 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2, at least 8.0 μg/mL, or about 8.3 μg/mL IL-12 (e.g., A549 cells). In some aspects, the recombinant poxvirus is capable of expressing from about 50ng/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. In some aspects, the recombinant poxvirus is capable of expressing from about 1 μg/mL to about 50 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 2 μg/mL to about 50 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 3 μg/mL to about 50 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 1 μg/mL to about 40 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 2 μg/mL to about 40 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 3 μg/mL to about 40 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 1 μg/mL to about 30 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 2 μg/mL to about 30 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 3 μg/mL to about 30 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 1 μg/mL to about 25 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 2 μg/mL to about 25 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2. In some aspects, the recombinant poxvirus is capable of expressing from about 3 μg/mL to about 25 μg/mL of IL-12 in a cancer cell 72 hours after infection at a multiplicity of infection (MOI) of 10 ^-2.

In some aspects, the cancer cells that are capable of being lysed by and/or capable of expressing IL-12 from the recombinant poxviruses provided herein include, but are not limited to, kidney cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, stomach 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.

In some aspects of the recombinant poxviruses disclosed herein, the virus is in Chicken Embryo Fibroblasts (CEF), heLa cells,Cells, vero cells, HEK293 cells, perC6 cells, BHK21 cells, or MRC5 cells. In some aspects of the recombinant poxviruses disclosed herein, the virus is produced in Chicken Embryo Fibroblasts (CEF).

In some aspects, the recombinant poxvirus is capable of up-regulating Interferon (IFN) - γ.

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

Also provided herein are methods for producing 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 or mid-term promoter. In some aspects, a method for producing a recombinant poxvirus comprises the steps of a) obtaining or preparing a producer cell, b) infecting the obtained or prepared producer cell with the recombinant poxvirus, c) culturing the infected producer cell under suitable conditions to allow production of the recombinant poxvirus. In some aspects, such methods further comprise d) recovering the recombinant poxvirus produced from the culture of the producer cells. In some aspects, such methods further comprise e) purifying the recovered recombinant poxvirus. In some aspects, the producer cell is a Chicken Embryo Fibroblast (CEF), heLa,Vero, HEK 293, perC6, BHK21 or MRC5 cells. In some aspects, the producer cell is a Chicken Embryo Fibroblast (CEF). Also provided herein are recombinant poxviruses produced by such methods.

In some aspects, the production cells may be cultured in step a) in a suitable medium, which may or may not be supplemented with serum and/or with suitable one or more growth factors, if desired (e.g. a chemically defined medium free of animal-derived or human-derived products may be used). The person skilled in the art can choose a suitable medium according to the production cells. Such media are commercially available. Prior to infection, the producer cells are cultured at a temperature between +30 ℃ and +38 ℃ (e.g., about 37 ℃) for 1 to 8 days. If desired, multiple passages can be made within 1 to 8 days to increase the total number of cells.

Also provided herein are pharmaceutical compositions comprising 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 or mid-term promoter. In some aspects, the present disclosure provides pharmaceutical compositions comprising a 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, a therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 1x 10 ³ pfu to 1x 10 ¹² pfu. In some aspects, a therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 1x 10 ⁴ pfu to 1x 10 ¹¹ pfu. In some aspects, a therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 1x 10 ⁵ pfu to 1x 10 ¹⁰ pfu. In some aspects, a therapeutically effective amount of an individual dose of a recombinant poxvirus described herein comprises 5x 10 ⁷ pfu to 4x 10 ⁹ pfu.

In some aspects, the disclosure provides a pharmaceutical composition comprising 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 or mid-term promoter, for use in the treatment or prevention of a proliferative disease, such as cancer. 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, cholangiocarcinoma, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, and malignant glioma.

PD-1 and PD-L1 inhibitors

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, are expressed on antigen presenting cells as well as many human cancers and have been demonstrated to down regulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1/PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. patent nos. 8,008,449 and 7,943,743), and six immune checkpoint inhibitors for the PD-1/PD-L1 pathway have been approved by the FDA since month 5, including three for PD-1 (pamelizumab, nano Wu Liyou mab and cimiput Li Shan antibody), and three for PD-L1 (atilizumab, avilamab and avilamab) (Ai et al Drug DES DEVEL THER. [ Drug design, development and treatment ],14:3625-3649,2020).

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

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, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-1. In some aspects, the PD-1 inhibitor is a peptide-based inhibitor. In some aspects, the PD-1 inhibitor is a small molecule. In some aspects, the PD-1 inhibitor is an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to PD-1.

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 CD 80. In some aspects, the PD-L1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-L1. In some aspects, the PD-L1 inhibitor is a peptide-based inhibitor. In some aspects, the PD-L1 inhibitor is a small molecule. In some aspects, the PD-L1 inhibitor is an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to PD-L1.

In some aspects, the anti-PD-l therapy is an antibody selected from the group consisting of Na Wu Liyou mab (also referred to as5C4, BMS-936558, MDX-1106 and ONO-4538), palivizumab (Merck, inc.; also known asLanlo Li Zhushan antibody and MK-3475; see WO 2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (Aspirikang (AstraZeneca)), also known as AMP-514; see WO 2012/145493), simipre Li Shan antibody (Regeneron), also known as REGN-2810; see WO 2015/112800), JS001 (Tauzhou jun solid pharmaceutical company (TAIZHOU JUNSHI PHARMA); see Si-Yang Liu et al J.Hematol.Oncol. [ J.hematology & oncology ]70:136 (2017)), BGB-A317 (Beigene; see WO 2015/35606 and US 2015/0079209), INCSHR1210 (Jiangsu Hengrui medical Co., ltd. (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; si-Yang Liu et al, J Hematol Oncol. J. Hematol. 70:136 (2017)), TSR-042 (Tesaro biopharmaceutical Co., also known as ANB011; see WO 2014/179664)), A. The use of the compositions for the preparation of drugs for treating cancer is described in the specification, Pittuzumab (Medivation/CureTech company; see U.S. Pat. No. 8,686,119B2 or WO 2013/014668 Al), GLS-010 (tin-free/Harbin Yu He Jiu pharmaceutical Co., ltd (Harbin Gloria Pharmaceuticals), also known as WBP3055; see Si-Yang Liu et al J.Hematol Oncol. [ J.Hematol. J.70:136 (2017)), AM-0001 (Armo Co.), A. Sum. Of the formula (I.S. Pat. No.), STI-1110 (Soren Torr medical Co., ltd. Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Ai Jina Sitting Co., ltd. (Agenus)), see WO 2017/040790), MGA012 (Macrogenics Co., ltd., see WO 2017/19846) and IBI308 (Xinda Co., ltd. (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 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.

In some aspects, the anti-PD-L1 therapy is an antibody selected from Dewaruzumab (Ab Likang Corp.; 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. Nos. 7,943,743 and WO 2013/173223), abilizumab (Roche); also known as Roche Corp.)MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al (2013) J Clin Oncol [ journal of clinical oncology ]3l (journal): 3000), averment (Pfizer); also known asMSB-0010718C; see WO 2013/079174), STI-1014 (Soren Torr; see WO 2013/181634), CX-072 (Cytomx Inc; see WO 2016/14971), KN035 (3 DMed/Alphamab Inc.; see Zhang et al, cell discovery [ Cell discovery ]7:3 (3 months 2017)), LY3300054 (Gift company (ElilillyCo.); see, e.g., WO 2017/034916) and CK-301 (Checkpoint Therapeutics Inc.; see Gorelik et al, AACR: abstract 4606 (4 months) each of these references is incorporated herein by reference in its entirety).

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

TABLE 3 Dewaruzumab antibody sequences

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, an 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, an anti-PD-L1 antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain (e.g., a human IgG1 heavy chain). In some aspects, an 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 IgG1 kappa 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 IgG1 kappa 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 IgG1 kappa monoclonal antibody produced in CHO cells comprising variant Fc comprising amino acid substitutions 234F, 235F and 331S (as numbered by the EU index as set forth in Kabat), VH comprising the amino acid sequence of SEQ ID NO:38, and 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 provided in 10mL of solution, wherein each mL of solution comprises the antibody or antigen-binding fragment thereof (e.g., dewaruzumab) (50 mg), L-histidine (2 mg), L-histidine hydrochloride monohydrate (2.7 mg), α -trehalose dihydrate (104 mg), polysorbate 80 (0.2 mg), and water for injection, USP. The solution may comprise, for example, 120mg of the antibody or antigen-binding fragment thereof (e.g., dewaruzumab) in 2.4mL or 500mg of the antibody or antigen-binding fragment thereof (e.g., dewaruzumab) in 10 mL.

In some aspects of the therapeutic combinations, methods and uses described herein, the PD-L1 inhibitor is dewaruzumab @African Corp.). Dewaruzumab 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. Pat. No. 9,493,565, incorporated herein by reference in its entirety. The fragment crystallizable (Fc) domain of dewaruzumab contains triple mutations in the constant domain of the IgG1 heavy chain that reduce binding to complement component C1q and fcγ receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

Kit for detecting a substance in a sample

Provided herein are kits comprising 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 or mid-promoter described herein, or a pharmaceutical composition comprising a recombinant poxvirus described herein. In certain aspects, the kit comprises a unit dose of such recombinant virus or pharmaceutical composition. In certain aspects, provided herein is a kit comprising one or more containers filled with one or more components of the compositions described herein (recombinant poxviruses as described herein), optionally together with instructions for use.

In some aspects, provided herein is a kit comprising one or more containers filled with one or more components of the compositions described herein (such as the recombinant poxviruses described herein), and one or more PD-1 or PD-L1 inhibitors described herein, optionally together with instructions for use. In some aspects, the kits described herein comprise a recombinant poxvirus described herein and an anti-PD 1 antibody (e.g., palbockiumab or nal Wu Liyou mab). In some aspects, the kits described herein comprise a recombinant poxvirus described herein and an anti-PD-L1 antibody (e.g., dewaruzumab).

Therapeutic uses and methods

Provided herein is a method of inducing apoptosis in a cancer cell, the method comprising contacting the cancer cell with a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) or a pharmaceutical composition comprising a recombinant poxvirus described herein under conditions that induce apoptosis, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late or mid-term promoter. In some aspects, the cancer cells may include, but are not limited to, kidney cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, stomach 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.

Also provided herein is a method of inhibiting growth or promoting death of a cancer cell, the method comprising contacting the cancer cell with a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) or a pharmaceutical composition comprising a recombinant poxvirus described herein under conditions that inhibit growth or promote death of the cancer cell, wherein the heterologous nucleic acid sequence encoding IL-12 is operably linked to a late or mid-stage promoter. In some aspects, the cancer cells may include, but are not limited to, kidney cancer cells, prostate cancer cells, breast cancer cells, bladder cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, stomach 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 of the disclosure, the method of inducing apoptosis in a cancer cell is performed in vitro. In some aspects of the disclosure, the methods of inhibiting cancer cell growth or promoting cancer cell death are performed in vitro. In some aspects of the disclosure, the method of inducing apoptosis in a cancer cell is performed in vivo. In some aspects of the disclosure, the methods of inhibiting cancer cell growth or promoting cancer cell death are performed in vivo.

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

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 in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) or a pharmaceutical composition comprising a 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 or mid-term promoter.

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

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.

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

In some aspects, the disclosure provides a method of 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) an inhibitor of apoptosis protein 1 (PD-1) or an inhibitor of apoptosis ligand 1 (PD-L1). In some aspects, the disclosure provides 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. In some aspects, the disclosure provides a method of 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.

In some aspects, administration of an immune checkpoint inhibitor in combination with a recombinant poxvirus comprising in its genome a heterologous nucleic acid sequence encoding interleukin-12 (IL-12) as described herein results in an enhanced therapeutic effect (e.g., more significant reduction in tumor growth, increased infiltration of tumor by lymphocytes, increased length of progression free survival, etc.) compared to that observed following treatment with the recombinant poxvirus alone or with the immune checkpoint inhibitor alone. The enhanced therapeutic effect may for example be an enhancement of a tumor-specific immune response. This can be characterized, for example, by an increase in IFN-gamma production and/or an increase in the number of IFN-gamma secreting cells.

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

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 concurrently 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.

In some aspects, the 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 or mid-term promoter, or a pharmaceutical composition comprising a recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or PD-L1 inhibitor, or a pharmaceutical composition comprising a PD-1 inhibitor or PD-L1 inhibitor, for use in a method of inducing apoptosis in a cancer cell described herein.

In some aspects, the 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 or mid-term promoter, or a pharmaceutical composition comprising a recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or PD-L1 inhibitor, or a pharmaceutical composition comprising a PD-1 inhibitor or PD-L1 inhibitor, for use in a method of inhibiting the growth of or promoting the death of a cancer cell described herein.

In some aspects, the 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 or mid-term promoter, or a pharmaceutical composition comprising a recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or PD-L1 inhibitor, or a pharmaceutical composition comprising a PD-1 inhibitor or PD-L1 inhibitor, for use in a method of treating cancer in a subject as described herein.

In some aspects, the 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 or mid-term promoter, or a pharmaceutical composition comprising a recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or PD-L1 inhibitor, or a pharmaceutical composition comprising a PD-1 inhibitor or PD-L1 inhibitor, for use in a method of reducing the amount of cancer cells in a subject described herein.

In some aspects, the 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 or mid-term promoter, or a pharmaceutical composition comprising a recombinant poxvirus described herein, and (ii) a PD-1 inhibitor or PD-L1 inhibitor, or a pharmaceutical composition comprising a PD-1 inhibitor or PD-L1 inhibitor, for use in a method of eliciting an anti-cancer immune response in a subject as described herein.

Examples

The examples in this example section are provided by way of illustration and not by way of limitation.

COPTG19673 and COPTG19674 vectors were generated as described below, which are recombinant vaccinia viruses (VACVs) expressing human IL-12 (hIL-12) under the control of two different promoters pF17R and pA14L, respectively. Experiments and analyses related to the selection of the pF17R and pA14L promoters are also described.

COPTG19673 and COPTG19674 vectors encode human IL-12 (hIL-12) as a fusion of the p40 and p35 subunits linked by glycine-serine (GS) -linkers. The same hIL-12 coding sequence was inserted into both viruses but was under the control of two different late promoters COPTG19673 comprising the pF17R promoter and COPTG19674 comprising the pA14L promoter. IL-12 transgenes were carried in the vaccinia virus Copenhagen strain, which lacks both the thymidine kinase gene (J2R) and the ribonucleotide reductase gene (I4L) as in WO 2009/065546, which is incorporated herein by reference in its entirety. These two deletions limit viral replication into highly proliferating cells (containing high concentrations of nucleotides), such as tumor cells. Thus, transgene expression that is directly dependent on viral genome replication is limited to tumor cells (see, e.g., foloppe et al, 2019, molThermoOncolytics [ molecular therapy oncology ]14:1-14; and KLEINPETER et al, 2016, oncoimmunography [ oncology ]5:e 1220467). An expression cassette containing a promoter and an IL-12 transgene was inserted into the J2R locus of the double deletion vaccinia virus Copenhagen strain.

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

Material

Virus (virus)

VVTG18058 (empty VACV, VACV control or unarmed control VACV) is a vaccinia virus (Copenhagen strain) with deletions of the J2R and I4L genes. VVTG18058 was used as an unarmed control virus. VVTG18058 are produced in Chick Embryo Fibroblasts (CEF). Vero cells were titrated by plaque assay.

COPTG19104 is a poxvirus (Copenhagen strain) expressing the fluorescent protein mCherry under the control of the pH5R promoter at the J2R locus. It lacks the I4L gene. It is used as the starting parent virus for the production of recombinant viruses.

VACVwt (also known as COPwt) is a wild-type vaccinia virus (Copenhagen strain) without deletions. VACVwt is produced in CEF.

Cells and cell lines

Chick Embryo Fibroblasts (CEF) CEF cells were isolated from 11 day old embryonated eggs (Charles river Co. (CHARLES RIVER)) free of Specific Pathogen (SPF).

Vero cells African green monkey (Cercopithecus aethiops) (African green) kidney cell line VeroCCL-81 ^TM) was grown in DMEM (Ji Boke company (Gibco)) 4.5g/L glucose supplemented with 10% FBS, 2mM L-glutamine and containing gentamicin at a final concentration of 40 mg/L. The growth conditions were 37℃to 5% CO ₂.

Human tumor cell lines

Human lung cancer cell line A549 ]CCL-185 ^TM) was grown in DMEM (Ji Boke company) 4.5g/L glucose supplemented with 10% FBS, 2mM L-glutamine and containing gentamicin at a final concentration of 40 mg/L. The growth conditions were 37℃to 5% CO ₂.

HeLa of human cervical tumor cell lineCCL-2 ^TM) was grown in DMEM (Ji Boke company) supplemented with 10% fbs and 40mg/L gentamicin at 37 ℃ with 5% co ₂.

Human pancreatic tumor cell line MIAPaCa-2%CCL-1420 ^TM) in DMEM supplemented with 10% FBS and containing gentamicin at a final concentration of 40mg/L at 37℃in 5% CO ₂ And (3) growing in the middle.

HCT116 of human colorectal tumor cell lineCCL-247 ^TM) under 5% CO ₂ at 37℃in Mc Coys'5A supplemented with 10% FBS and containing gentamicin at a final concentration of 40mg/LAnd (3) growing in the middle.

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

HEK-Blue ^TM IL-12 cells (InvivoGen, reference hkb-IL 12) were grown in DMEM (Ji Boke) supplemented with 10% inactivated FBS, 100 μg/mL Normocin ^TM (Invitrogen), HEK Blue ^TM Selection (Enitrogen) and containing penicillin and streptomycin at final concentrations of 100U/mL and 100 μg/mL, respectively.

Natural killer cell line NK-92%CRL-2407 ^TM) was grown in alpha-limiting essential medium (Ji Boke) containing 1. 1XGlutamax (Ji Boke), 1.5g/L sodium bicarbonate (Ji Boke), 0.2mM inositol (Sigma), 0.1mM beta-mercaptoethanol (Sigma), 0.02mM folic acid (Sigma), 150U/ml recombinant IL-2, and 25% fetal bovine serum.

Chicken fibroblasts and human cell lines

Chicken embryo fibroblast DF-1%CRL-12203 ^TM) was grown in DMEM (Ji Boke company) supplemented with 10% FBS, 2mM L-glutamine and 40mg/L gentamicin at 39℃in 10% CO ₂.

From healthy donors (national blood service (EFS: etablissement)Du sang) were prepared on a polysucrose gradient and in RPMI supplemented with 10% FBS and containing gentamicin and glutamine at final concentrations of 40mg/L and 2mM, respectivelyAnd (3) growing in the middle.

Bacteria and method for producing same

Coli DH 5. Alpha. Strain (genotype: F-. Phi.80 lacZΔM15Δ (lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 thi-1gyrA96 relA1 lambda-; engineer, 1826312) was used for cloning and plasmid amplification in LB medium supplemented with 100. Mu.g/mL ampicillin.

Cloning of DNA

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

DNA sequencing

The DNA sequencing adopts the Mulberry method.

Example 1 comparative evaluation of vaccinia Virus promoters

Construction of reporter transfer plasmids

The reporter transfer plasmid is intended to evaluate the strength of the different poxvirus promoters to be evaluated. The reporter gene encodes a fusion between Renilla luciferase (RLuc) separated by linker (Ala) 5-Thr and jellyfish Green Fluorescent Protein (GFP) (GenBank: ABZ 79968.1). A different poxvirus promoter was inserted upstream of the RLuc-GFP fusion (SEQ ID NO: 1). The expression cassette is inserted into a poxvirus transfer plasmid designed to allow insertion of the nucleotide sequence into the J2R locus of the vaccinia virus genome by homologous recombination. This plasmid was derived from plasmid pUC18, in which the left (L) and right (R) arms of the flanking sequences surrounding the J2R locus were cloned.

Nucleic acid sequence of SEQ ID NO. 1:RLuc/GFP

ATGACAAGCAAGGTGTACGACCCCGAGCAGCGGAAGCGGATGATTACAGGACCTCAGTGGTGGGCCAGATGCAAGCAGATGAACGTGCTGGACAGCTTCATCAACTACTACGACAGCGAGAAGCACGCCGAGAACGCCGTGATCTTCCTGCATGGAAATGCCGCCAGCAGCTACCTTTGGAGACACGTGGTGCCTCACATCGAGCCTGTGGCCAGGTGCATCATCCCTGACCTGATCGGCATGGGCAAGAGCGGCAAGTCTGGCAACGGCAGCTACAGACTGCTGGACCACTACAAGTACCTGACCGCTTGGTTTGAGCTGCTGAACCTGCCTAAGAAGATCATCTTCGTCGGCCACGATTGGGGCGCCTGTCTGGCCTTTCACTACAGCTACGAGCACCAGGACAAGATCAAGGCCATCGTGCACGCCGAAAGCGTGGTGGATGTGATCGAGAGCTGGGACGAGTGGCCCGACATCGAGGAAGATATCGCCCTGATCAAGAGCGAAGAGGGCGAGAAGATGGTGCTGGAAAACAACTTCTTCGTGGAAACCATGCTGCCCAGCAAGATCATGCGGAAGCTGGAACCCGAGGAATTCGCCGCCTACCTGGAACCTTTCAAAGAAAAGGGCGAAGTGCGGAGGCCCACACTGTCCTGGCCTAGAGAGATCCCTCTGGTCAAAGGCGGCAAGCCCGATGTGGTGCAGATCGTGCGGAACTACAATGCCTACCTGCGGGCCTCCGATGATCTGCCCAAGATGTTCATCGAGAGCGACCCCGGCTTCTTCAGCAACGCCATAGTGGAAGGCGCCAAGAAGTTCCCCAACACCGAGTTCGTGAAAGTGAAGGGCCTGCACTTCAGCCAAGAGGACGCCCCTGATGAGATGGGCAAGTACATCAAGAGCTTTGTGGAACGGGTGCTCAAGAACGAGCAGGCCGCTGCCGCCACAATGAGCAAAGGCGAGGAACTGTTTACCGGCGTGGTGCCCATTCTGGTGGAACTGGATGGGGATGTGAACGGCCACAAGTTCAGCGTTAGCGGAGAAGGCGAAGGCGACGCCACATACGGAAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCTTGGCCTACACTGGTCACAACCTTTACCTACGGCGTGCAGTGCTTCAGCAGATACCCCGACCATATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACGTGCAAGAGAGAACCATCTTTTTCAAGGACGACGGCAACTACAAGACCAGGGCCGAAGTGAAGTTCGAGGGCGACACCCTGGTCAACCGGATCGAGCTGAAGGGCATCGACTTCAAAGAGGACGGCAATATCCTGGGCCACAAGCTTGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAAAAGAACGGCATCAAAGTGAACTTCAAGATCCGGCACAATATCGAGGACGGCTCCGTGCAGCTGGCCGATCACTATCAGCAGAACACCCCTATCGGCGACGGACCTGTGCTGCTGCCCGATAATCACTACCTGAGCACACAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGAGGGATCACATGGTGCTGCTGGAATTCGTGACCGCCGCTGGCATCACACACGGCATGGATGAGCTGTACAAGTGA

Synthetic fragments designated "RLuc-GFP" were generated synthetically, which contained fusion genes encoding the RLuc-GFP protein. It was inserted by homologous recombination into a set of transfer plasmids containing poxvirus early/late promoters, which were restriction digested with PvuII, yielding the plasmids described in table 4. FIG. 1 shows a schematic representation of plasmid pTG19409 containing the gene encoding RLuc-GFP under the expression control of the pH5R promoter.

TABLE 4 nucleic acid sequences of early and early/late promoters and the corresponding plasmid names containing the early and early/late promoters

To evaluate late promoters, the coding sequence of the RLuc-GFP fusion was cloned downstream of the promoter using its natural start codon. The synthetic fragment containing the late promoter and the RLuc-GFP fusion initiation point was generated synthetically and inserted into pTG19409 restriction-digested with SmaI-MscI by homologous recombination, yielding the plasmids described in table 5.

TABLE 5 nucleic acid sequences of late promoters and corresponding plasmid names containing the late promoters

The resulting reporter transfer plasmids were first tested in transient infection/transfection expression studies to identify the best candidates. In the second stage, some of them are used to produce recombinant poxviruses, as described below.

Transient infection/transfection with luciferase reporter plasmid

The reporter transfer plasmid using the RLuc-GFP reporter allows for assessment of cell infection while accurately measuring the level of expression of the reporter.

To better control expression levels and minimize off-target expression of different transgenes, they can be used depending on the strength or expression time of several VACV promoters during poxvirus infection (early, mid or late). Some poxvirus promoters (e.g., p7.5k or pH 5R) have both early and late elements, allowing transgene expression early after viral infection and late after viral genome replication, respectively. Thus, to further minimize off-target expression of potentially toxic transgenes (e.g., IL-12), transgene expression may be driven by late promoters that are active only after replication of the viral genome.

Six late promoters were selected for preliminary evaluation of 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. Plasmids encoding Firefly luciferase under the control of the p11K7.5 promoter were co-transfected to normalize for transfection variability. Two luciferases were measured after 24 hours (h).

Briefly, DF-1 cells were cultured in 24-well plates and then infected with empty vaccinia virus (VVTG 18058) without any transgene at MOI 1. After 2 hours, 0.5ng of a different Renilla reporter plasmid (see Table 2) complexed with 0.625. Mu.L of Liposome transfection 2000 (England Corp.) in opti-MEM medium, as well as 0.5ng of a control reporter plasmid encoding firefly luciferase under the control of p11K7.5 and 250ng of a control plasmid pTG15839 encoding GFP under the control of CMV promoter were added to each well. Transfection was performed in triplicate. Plates were then incubated at 37 ℃ and 5% co ₂ for 24 hours. For luciferase measurements, the supernatant was removed, cells lysed and treated according to the "dual luciferase reporter assay system" (Promega).

FIG. 2 illustrates the results obtained using six late promoters. 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-fold stronger than the pA14L and pA10L promoters. The pA11R, pA L and pG7L promoters are 8-to 10-fold weaker than the pF17R promoter.

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

Recombinant poxviruses were generated for comparative evaluation of vaccinia promoters

A transfer plasmid containing the fusion reporter gene RLuc-GFP under the control of various promoters was 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, pB2R (SEQ ID NO: 5), pA35R (SEQ ID NO: 7) and pC11R (SEQ ID NO: 6), were also evaluated. Three late promoters (pF 17R (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.

Ten different VACV-RLuc-GFP vectors were generated by homologous recombination in CEF by inserting an RLuc-GFP expression cassette into the J2R locus of the double deleted vaccinia virus Copenhagen strain under the transcriptional control of the different poxvirus promoters described herein. Thymidine kinase (TK, J2R locus) and ribonucleotide reductase (RR, I4L locus) activity is defective in all of these viruses.

Recombinant vaccinia virus was generated by homologous recombination in CEF using COPTG19104 as the starting parent virus, and a shuttle plasmid containing an expression cassette to be integrated with flanking sequences around the J2R locus (L-arm and R-arm). Homologous recombination between the shuttle plasmid and the parental vaccinia virus (copenhagen strain) can produce a recombinant vaccinia virus that lost the mCherry expression cassette and yielded an expression cassette that produced white (non-fluorescent) plaques. More particularly, CEF in F175 flasks was infected with COPTG19104 at MOI 0.05 for 1 hour at room temperature. The virus suspension was then discarded and the infected cells were incubated in mbe+5% FBS in 37 ℃ +5% CO ₂ for 2 hours, followed by trypsinization and counting. Ten million infected cells were then transfected with 2 μ g I-SceI restriction transfer plasmid by nuclear transfection. Transfected cells were then transferred to wells of a 6-well plate, incubated at 37 ℃ for 48 hours, and then frozen. After sonication, CEF was infected with serial dilutions of the transfer mixture to select for recombinant virus. Non-fluorescent white plaques were picked and used for the second round of plaque purification. Selected non-fluorescent white plaques were picked and amplified in 6-well plates at 37 ℃ at 5% co ₂ for 72 hours. The amplified products were used for PCR analysis, followed by selection of recombinant vaccinia virus.

Primary stock was generated by infection with CEF grown 72 hours prior to infection with 100. Mu.L of selected clones. Viral amplification was performed in MBE supplemented with 5% fbs at 37 ℃ 5% co ₂ for 72 hours. The infected cells and medium were subjected to a freeze/thaw cycle and then homogenized by sonication. This so-called primary stock solution is then characterized and stored aliquoted until use. After virus amplification in CEF-inoculated F500 flasks, purified material was produced. The infected cells and medium were harvested to produce a crude harvest and stored at-80 ℃. The virus was purified according to the procedure described in WO 2007/147528, which is incorporated herein by reference in its entirety.

Identification was made with reference to recombinant vaccinia viruses COPTG19409 (VACV containing promoter pH 5R), COPTG19410 (VACV containing promoter p11k7.5), COPTG19411 (VACV containing promoter p7.5k), COPTG19412 (VACV containing promoter pB 2R), COPTG19415 (VACV containing promoter pA 14L), COPTG19416 (VACV containing promoter pA 26L), COPTG19417 (VACV containing promoter pF 17R), COPTG19431 (VACV containing promoter pC 11R), COPTG19436 (VACV containing promoter pA 35R), and COPTG19437 (VACV containing promoter psel).

Comparative evaluation of vaccinia Virus promoter in human tumor cell lines and human PBMC

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

For these human tumor cell lines, cells were seeded at 1E+05 cells/well/200. Mu.L in 96-well plates the previous day. 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 MOI of 1.

For human PBMCs, cells were seeded into 96-well plates at 2e+05 cells/well/125 μl the day before. For infection, 50 μl of virus dilution in FBS-containing medium was added per well and infection was performed at MOI of 1. Infection was performed in triplicate and in two separate plates (one for luciferase measurement and the other for GFP analysis). For luciferase measurements, the supernatant was removed, cells lysed and treated according to the "Renilla reporter assay System" (Promega).

For GFP quantification, the supernatant was removed, and the cells were then trypsinized, centrifuged, washed with 100. Mu.L of PBS, stained with 100. Mu.L of 100-fold diluted live/dead IR, and incubated in the dark at room temperature for 15 minutes. 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 (meitian gentle biotechnology company (MiltenyiBiotec)) and analyzed using Kaluza software (beckmann coulter). Results are expressed as a percentage of live GFP positive cells (infected cells). FACS (fluorescence activated cell sorting) analysis showed that about 70% to 90% of cells were GFP positive for all MOI and cell lines 24 hours after infection (data not shown). All viruses produced the same results, indicating that their level of infectivity was similar.

Renilla luciferase was measured after 6 hours and 24 hours, and the results obtained in MIAPaCa-2 cells are shown in FIG. 3A and FIG. 3B, respectively. Expression was normalized to the weakest promoter pA 26L. 6 hours after infection, the late promoters pF17R, pA L and pA26L produced very low levels of expression. The highest expression level was obtained for the early/late promoter pH5R, followed by the early promoters pB2R and pC11R and the early/late promoters pSE/L and p11K7.5. Similar results were obtained with the other two cell lines (Hela and HCT 116) regardless of infection MOI (data not shown). The results for the early promoters pB2R, pC R and pA35R, which resulted in low expression levels, were different 24 hours after infection. Higher expression levels (11-fold to 21-fold higher than pA 26L) were detected using promoters p11K7.5, pSE/L, pF R, and pH5R, whereas p7.5K and pA14L produced moderate levels of expression (7-fold to 11-fold higher than pA 26L).

The results obtained 24 hours after infection of HeLa and HCT-116 cells are shown in FIGS. 4 and 5, respectively. The results were similar to those obtained in MIAPaca-2 cells. The intensity differences between promoters were more pronounced at MOI 0.1 than at MOI 1 (up to 14-fold, and up to 7-fold relative to pA26L, respectively).

The results obtained in the three cell lines allow to divide the promoters into three intensity groups. The weak promoters include three early promoters (pB 2R, pA R and pC 11R) and the late promoter A26L. The efficiency of the medium promoter is 2-to 5-fold higher than that of the weak promoter. They correspond to the early/late promoter p7.5K and the pH5R and the late pA14L promoter. The strongest promoters were 2-to 3-fold more potent than the medium promoters. They correspond to the early/late promoters p11K7.5 and pSE/L and the late pF17R promoter.

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

The results of the flow cytometry analysis are shown in fig. 6. After 6 hours, about 12% of cells were detected as GFP positive cells after infection with viruses containing early or early/late promoters. In contrast, very few GFP positive cell numbers were detected (less than 4% for pA26L and less than 1% for pF17R and pA 26L) after infection with viruses containing late promoters. The percentage of GFP positive cells decreased 24 hours after infection due to cell death of the infection and lack of replication of the recombinant vaccinia virus. The percentage of infected cells was about 5% for viruses containing early and early/late promoters, while the percentage of infected cells was negligible for viruses containing late promoters.

Renilla luciferase was measured after 6 hours and 24 hours, and the results are shown in FIG. 7. Expression was normalized to the weakest promoter pA 26L. Luciferase levels were lower (approximately 100-fold lower) than those detected after infection with human tumor cells. Furthermore, expression decreased between 6 and 24 hours due to death of infected cells. Cells infected with viruses containing the late promoters (pF 17R, pA L and pA 26L) expressed very low levels of luciferase, about 100-fold to 300-fold less than the expression detected with the early or early/late promoters.

The vaccinia virus used in this study was derived from the Copenhagen strain, and the thymidine kinase gene (J2R) and ribonucleotide reductase gene (I4L) were deleted. These two deletions limit viral replication into highly proliferating cells (containing high concentrations of nucleotides), such as tumor cells. These viruses do not replicate efficiently in primary human PBMCs, and therefore late promoters are not active in these cells.

Conclusion(s)

This study allowed the identification of two late promoters, pF17R and pA14L, which driven strong or moderate expression in human tumor cells, while little expression was detected in primary human cells. Thus, these promoters are selected to minimize off-target expression of potentially toxic transgenes (e.g., IL-12).

EXAMPLE 2 Generation and production of recombinant vaccinia Virus encoding IL-12 by homologous recombination

Construction of transfer plasmids pTG19673 and pTG19674

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

Endogenous human IL-12 is unique in cytokines in that it is a disulfide-linked heterodimer of two separately encoded subunits (p 35 and p 40). Single chain IL-12 protein is expressed from vaccinia constructs in which the full length p40 subunit is fused via a G6S linker to a p35 subunit whose leader sequence is truncated (i.e., IL-12.p40. Delta. P35) (see Lieschke et al, 1997,Nat Biotechnol [ Nature Biotechnology ]1997, month 1; 15 (1): 35-40).

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

Fusion IL-12.p40.delta.p35 (SEQ ID NO: 15):

underlined sequence is the signal peptide (SEQ ID NO: 16)

Bold and underlined sequence IL-12-p40 subunit (SEQ ID NO: 17)

Underlined and bolded sequence: linker (SEQ ID NO: 18)

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

The mature IL-12 fusion protein comprises amino acids 23-532 of SEQ ID NO. 15, as shown below in SEQ ID NO. 20.

Mature fusion IL-12.p40.delta.p35 (SEQ ID NO: 20):

The nucleotide sequence of fusion IL-12.p40.delta.p35 was optimized for human codon usage and optimal gene expression using the GeneOptimezer algorithm of Geneart. The sequence of the expression cassette present in pTG19673 is shown in FIG. 8 (SEQ ID NO: 21). SEQ ID NO. 21 is provided below.

AAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATAGAGCTCGGTAACCGCCACCATGTGCCACCAGCAGCTGGTCATCAGCTGGTTCAGCCTGGTGTTCCTGGCCTCTCCTCTGGTGGCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTGGAACTGGACTGGTATCCCGATGCTCCTGGCGAGATGGTGGTGCTGACCTGCGATACCCCTGAAGAGGACGGCATCACCTGGACACTGGATCAGTCTAGCGAGGTGCTCGGCAGCGGCAAGACCCTGACCATCCAAGTGAAAGAGTTTGGCGACGCCGGCCAGTACACCTGTCACAAAGGCGGAGAAGTGCTGAGCCACAGCCTGCTGCTGCTCCACAAGAAAGAGGATGGCATTTGGAGCACCGACATCCTGAAGGACCAGAAAGAGCCCAAGAACAAGACCTTCCTGAGATGCGAGGCCAAGAACTACAGCGGCCGGTTCACATGTTGGTGGCTGACCACCATCAGCACCGACCTGACCTTCAGCGTGAAGTCCAGCAGAGGCAGCAGTGATCCTCAGGGCGTTACATGTGGCGCCGCTACACTGTCTGCCGAAAGAGTGCGGGGCGACAACAAAGAATACGAGTACAGCGTGGAATGCCAAGAGGACAGCGCCTGTCCAGCCGCCGAAGAGTCTCTGCCTATCGAAGTGATGGTGGACGCCGTGCACAAGCTGAAGTACGAGAACTACACCTCCAGCTTTTTCATCCGGGACATCATCAAGCCCGATCCTCCAAAGAACCTGCAGCTGAAGCCTCTGAAGAACAGCAGACAGGTGGAAGTGTCCTGGGAGTACCCCGACACCTGGTCTACACCCCACAGCTACTTCAGCCTGACCTTTTGCGTGCAAGTGCAGGGCAAGTCCAAGCGCGAGAAGAAAGATCGGGTGTTCACCGACAAGACCAGCGCCACCGTGATCTGCAGAAAGAACGCCAGCATCAGCGTCAGAGCCCAGGACCGGTACTACAGCAGCTCTTGGAGCGAATGGGCCAGCGTGCCATGTTCTGGTGGCGGAGGCGGAGGCTCTAGAAATCTGCCTGTGGCCACTCCTGATCCTGGCATGTTCCCTTGTCTGCACCACAGCCAGAACCTGCTGAGAGCCGTGTCCAACATGCTGCAGAAGGCCAGACAGACCCTGGAATTCTACCCCTGCACCAGCGAGGAAATCGACCACGAGGACATCACCAAGGATAAGACCAGCACCGTGGAAGCCTGCCTGCCTCTGGAACTGACCAAGAACGAGAGCTGCCTGAACAGCCGGGAAACCAGCTTCATCACCAACGGCTCTTGCCTGGCCAGCAGAAAGACCTCCTTCATGATGGCCCTGTGCCTGAGCAGCATCTACGAGGACCTGAAGATGTACCAGGTGGAATTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCAAGCGGCAGATCTTCCTGGACCAGAATATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTCAACAGCGAGACAGTGCCCCAGAAGTCTAGCCTGGAAGAACCCGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCCGGATCAGAGCCGTGACCATCGACAGAGTGATGAGCTACCTGAACGCCTCCTGATTTTTCT

Vaccinia virus transfer plasmids pTG19535 and pTG19537 were 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.

Fragments containing IL-12 fusion were generated synthetically by Geneart and inserted into plasmids. The corresponding plasmid was restriction by SnaB1, and the resulting fragment "hIL-12" was inserted by homologous recombination into pTG19535 (pF 17R promoter) or pTG19537 (pA 14L promoter) restriction by PvuII, resulting in pTG19673 (FIG. 9) or pTG19674 (FIG. 10), respectively. In these plasmids, the expression cassette is inserted between the recombination arms, allowing homologous recombination in the J2R locus of the vaccinia virus genome. The largest preparations of both plasmids were generated (Maxipreparations) and analyzed by sequencing the recombinant arms and the expression cassette inserted between them. The alignment of the analyzed sequences with the theoretical sequences showed 100% homology between the two plasmids.

Two different late poxvirus promoters were used. The natural initiation codons of the pA14L and pF17R genes were not used for IL-12 expression. Thus, the sequence ATA was added downstream of the promoter instead of native ATG.

The sequences of the two promoters pA14L and pF17R and downstream ATA are shown below as pA14L (SEQ ID NO: 22)

TTTGTTCATTCGGCGATTTAAAATTTTTATTAGTTAAATApF17R(SEQ ID NO:23)

AAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAAATA production of recombinant vaccinia Virus by homologous recombination

Recombinant COPTG19673 and COPTG19674 were generated by homologous recombination in CEF using COPTG19104 as the starting parent virus and shuttle plasmids pTG19673 and pTG19674 encoding hIL-12 under the control of pF17R and pA14L, respectively. COPTG19104 contains in its J2R locus the expression cassette of mCherry. Homologous recombination between the shuttle plasmid and the parental vaccinia virus was able to generate a recombinant vaccinia virus (COPTG 19673 and COPTG 19674) that lost the mCherry expression cassette and yielded the hll-12 expression cassette (see example 1 for methods of recombinant vaccinia virus generation).

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

The primary stock of the recombinant virus COPTG19674 was obtained from homologous recombination between the transfer plasmid pTG19674 and the parent COPTG19104, hereinafter referred to as COPTG19674 primary stock. Expression of IL-12 in supernatants of a549 cells infected with COPTG19673 and COPTG19674 as measured by ELISA is shown in figure 11.

These primary study stock solutions were used for in vitro characterization in example 3.

Example 3 recombinant oncolytic vaccinia Virus COPTG19673 and expressing interleukin-12

COPTG19674 in vitro characterization

Vaccinia viruses COPTG19673, COPTG19674, and VVTG18058 described in example 2 herein were used in the following studies.

VACVwt (also known as COPwt) is a wild-type vaccinia virus (Copenhagen strain) without deletions. VACVwt is produced in CEF. The plaque assay on Vero cells was used to determine the infectious titer in the following assay.

Virus replication assay

Replication in human tumor cell lines

Replication of COPTG19673 and COPTG19674 was assessed in three human tumor cell lines (A549, HT-29, and MIA PaCa-2) and compared to replication of the reference unarmed control VACVVVTG 18058. The A549, HT-29 and MIAPaCa-2 tumor cell lines were infected with each virus at MOI 10 ^-3. The infected cells were then plated into 6-well plates and incubated at 37 ℃ under 5% co ₂ 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.

Figures 12A-C show that COPTG19673, COPTG19674, and VVTG18058 replicates in three human tumor cell lines were similar.

Replication in producer cells

Replication of COPTG19673 and COPTG19674 was assessed in CEF and in the human cell line (HeLa) (as cells for virus production) and compared to replication of the reference unarmed control VACV. In 6-well plates, heLa or CEF were infected with each virus at MOI 0.05. The plates were then incubated for 72 hours in a 37 ° C, CO ₂ atmosphere. The production of virus in each cell type was determined by titration of Vero cells using a plaque assay method. Fig. 13 shows that the replication of COPTG19673, COPTG19674, and VVTG18058 in each of the two cells (CEF and HeLa) is similar (i.e., the log difference at 72h is less than 0.7).

Oncolytic Activity assay

The oncolytic activity of COPTG19673 and COPTG19674 was evaluated on three human tumor cell lines (A549, HT-29, and MIA PaCa-2) and compared to the activity of the unarmed control VACV (VVTG 18058). Oncolytic activity was assessed by quantifying cell viability after 5 days of incubation.

The comparison of 2 viruses is that tumor cell lines are infected with each virus at 10 different MOI (i.e.A549 and HT-29:3.10 ^-5 to 1, MIA PaCa-2:10 ^-5 to 3.10 ^-1) depending on the cell line used. The infected tumor cells were then incubated in 96-well plates for 5 days in a CO ₂ environment at 37 ℃. Cell viability was determined using a cell titer blue cell viability assay according to the protocol provided by the manufacturer.

Oncolytic activity expressed as cell viability represents the lytic activity of the tested viral sample on tumor cells. The oncolytic activity of each sample is expressed as a percentage of the viability of the mock-infected cells. FIGS. 14A-C show the oncolytic activity of COPTG19673, COPTG19674, and VVTG18058 at different MOI. From these results, EC50 values (MOI at which 50% of the cells are killed) were calculated for each virus in each cell line. Comparison of EC 50's shows that in miappa ca-2, the oncolytic activity of COPTG19673, COPTG19674 and the unarmed control VACV are very similar. In A549 and HT-29, COPTG19673 had an EC50 of greater than VVTG18058, but at high MOI both VACVs exhibited comparable strong oncolytic activity, with less than 20% of viable cells remaining. EC50 s for both viruses were calculated using GraphPadPrism. As a result of the fact that as shown in the figures,

EC50 for a549: COPTG19673 is 6.3.10 ^-3, EC50 for COPTG19674 is 1.9.10 ^-3, EC50 for VVTG18058 is 3.8.10 ^-3;

The EC50 for Mia PaCa-2:COPTGG19673 is 4.8.10 ^-3, the EC50 for COPTG19674 is 2.7.10 ^-3, the EC50 for VVTG18058 is 4.4.10 ^-3, and

The EC50 for HT29: COPTG19673 is 1.2.10: ^-2, the EC50 for COPTG19674 is 7.6.10 ^-3, and the EC50 for VVTG18058 is 4.9.10 ^-3

Expression level of vIL-12 as determined by ELISA

The expression level of cytokine vhIL-12 was measured in the supernatants of 3 infected tumor cell lines 3 days after infection with MOI 0.01. The terms vIL-12 and vhIL-12 refer to IL-12 expressed with VACV according to the present disclosure.

Preparation of supernatant

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, infected at MOI 10 ^-2, and incubated in 3mL of sufficient medium without fetal bovine serum for 72 hours. The supernatant was collected and then filtered to remove the virus.

Determination of IL-12 expression in supernatants by ELISA

UsingELISA development System human IL-12 (R & D systems) reference DY 1270-05) the concentration of IL-12 in supernatants of infected tumor cell lines was determined. VVTG18058, COPTG19673 or COPTG19674 were infected with three tumor cell lines A549, HT-29 and MIA PaCa-272 hours at MOI 0.01. The IL-12 concentration in the supernatant of the infected cells was then measured by ELISA. The supernatant of VVTG18058 infected cells served as a negative control. The results are shown in fig. 15 as the mean and Standard Deviation (SD) of duplicate measurements of three samples. Both the cell line and the promoter controlling the transcription of the transgene affect the expression level of the transgene. The highest expression was obtained in COPTG19673 infected a549 cells. However, high levels of expression of vIL-12 were obtained for both viruses and the three tumor cell lines tested at concentrations ranging from 0.3 to 8.3. Mu.g/mL.

Protein vIL-12 function assay in supernatant of infected tumor cells

The function of IL-12 produced by the virus during the transgene expression assay in the "supernatant preparation" section of this example was assessed using HEK-Blue ^TM IL-12 reporter cells and by cell proliferation assay using NK-92 cells.

HEKBlue IL-12 reporter cell vIL-12 bioactivity

The biological activity of vhIL-12 produced in the supernatant of a human tumor cell line was determined using HEK-Blue ^TM IL-12 reporter cells. The biological activity of vIL-12 produced in COPTG19673 and COPTG 19674-infected 3 human tumor cell lines was measured and compared with the biological activity of human recombinant hIL-12 (hereinafter referred to as rhIL-12).

HEK-Blue ^TM IL-12 cells were designed to detect biologically active human and mouse IL-12 by monitoring activation of the STAT-4 pathway. Binding of IL-12 to the IL-12 receptor on the surface of HEK-Blue ^TM IL-12 cells triggers a signaling cascade that activates STAT-4, which in turn produces a secreted alkaline phosphatase (SEAP) marker protein. Detection of SEAP in HEK-Blue ^TM IL-12 cell supernatants can be readily assessed using QUANTI-Blue ^TM.

Briefly, HEK-Blue ^TM IL-12 cells were plated at 5E+04 cells/well into 96-well flat bottom microtiter plates. Culture supernatants at different dilutions and rhIL-12 standard (ranging in concentration from 10pg/mL to 100 ng/mL) were added to 96-well cell culture microplates. Plates were incubated at 37 ℃. After 24 hours of incubation, detection of SEAP in supernatants of HEK-Blue ^TM IL-12 cells was determined using QUANTI-Blue ^TM (Inje, inc., reference rep-qbs) according to the supplier protocol. Supernatants from mock-infected cells and unarmed control VACV-infected cells were used as negative controls.

The results presented in FIGS. 16A-F show that all 3 different samples tested contained biological activities vhIL-12. Media without rIL-12, supernatant (SN) from mock-infected cells, and SN from an unarmed control VACV-infected cell line were added as controls, which did not show SEAP expression. After administration of all samples, a comparison of vhIL-12 to rhIL-12 bioactivity was performed using the same ELISA. At equal concentrations, vhIL-12 and rhIL-12 produced by the three infected tumor cell lines induced relatively close absorbance levels. The results indicate that vIL-12 produced by two VACV-IL12 activates HEK-Blue IL-12 cell lines with an EC50 that is superior to rhIL-12. These results indicate that IL-12 produced by VACV-infected tumor cells retains its cytokine activity.

Bioactivity of vIL-12 on NK-92 cells

Cytokine dependent cell proliferation assays of NK-92 cell lines were used to determine the biological activity of vIL-12 produced in supernatants of infected human tumor cell lines. IL-12 can induce NK-92 cell proliferation. The biological activity of vhIL-12 produced by COPTG19673 and COPTG19674 infected 3 human tumor cell lines was measured and compared to the biological activity of human recombinant hIL-12.

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

Briefly, NK-92 cells were plated at 1E+04 cells/well into 96 well flat bottom microtiter plates. Culture supernatants at different dilutions and rhIL-12 standard (ranging in concentration from 1pg/mL to 100 ng/mL) were added to 96-well cell culture microplates. Plates were incubated at 37 ℃. After 48 hours incubation, add according to the provider protocolSupernatants from mock-infected cells and unarmed control VACV-infected cells were used as negative controls.

The results presented in figures 17A-E show that all 3 different samples tested against both viruses COPTG19673 and COPTG19674 contained biological activity 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 proliferation of NK-92. After administration of all samples, a comparison of vhIL-12 to rhIL-12 bioactivity was performed using the same ELISA. At equal concentrations vhIL-12 from the three tumor cell lines infected with virus and rhIL-12 induced similar levels of NK-92 proliferation. As with the HEK-Blue IL12 cell line, the EC50 value of vIL-12 is lower than that of rhIL-12. These results also demonstrate the functionality and powerful efficiency of IL-12 produced by VACV.

In vitro safety assay

Replication rate of normal human hepatocytes

Normal healthy human hepatocytes were selected to monitor the safety profile of COPTG19673 and COPTG19674, as these primary cells can be obtained periodically directly from the donor.

Hepatocytes were provided by Biopredic in 6-well plates. Hepatocytes were infected with each virus at MOI 10 ^-3 and incubated for 72 hours in a 37 ° C, CO ₂ atmosphere. The amount of virus produced within 72 hours of culture was determined by virus titration per well by plaque assay in Vero cells. The results are expressed as replication yields corresponding to the ratio between the input/output virus numbers. The results are the average of three wells.

VACVwt spread well, replication yield 837 (ratio between output virus and input virus). In the case of the unarmed control virus VVTG18058, replication rate was drastically reduced to 1 (fig. 18). Also, the results showed that COPTG19673 and COPTG19674 had no replication in human hepatocytes, with replication rates below 1. These results indicate that the attenuated replication provided by the two deletions (TK and RR) against normal cells is conserved between the unarmed control VACV (VVTG 18058) and the IL-12 expressing VACV (COPTG 19673 and COPTG 19674).

Virus replication in hBMC

Human PBMCs were selected as second normal primary cells to evaluate the safety profile of the newly generated VACVs. Wild-type VACVs or unarmed VACVs are not normally replicated in hpbmcs. The presence of cytokines or immunostimulatory molecules expressed by VACV can activate or stimulate immune cells, thereby altering replication of VACV in these cells. Thus, the replication of COPTG19673 and COPTG19674 in these cells (i.e., hPBMC) was evaluated and compared to benchmarks VACVwt and VVTG 18058. After infection with MOI 1 and incubation for 3 days, virus production was measured by plaque assay in Vero cells.

Viral replication yield was determined as the ratio between total infectious particles detected 72 hours after infection (output) and viral particles used for PBMC infection (input).

FIG. 19 shows that two IL-12 expressing VACVs, COPTG19673 and COPTG19674, as well as VACVwt and the unarmed control VACV were not replicated in the hBMC. In other words, vectorization of human IL-12 does not alter the replication behavior of VACV at hBMC. In addition, figures 27A-C show that COPTG19673 expansion was not observed in human PBMC and minimal replication was observed in normal human hepatocytes and dermal fibroblasts, indicating that replication is relatively specific for tumor cells.

Example 4 efficacy of COPTG19673 ("VACV IL-12") across human tumor cell lines

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

Cultured human tumor cells were grown on tissue culture plastic and incubated with VACVIL-12 at MOI ranging from 6.4E-06 to 10 to determine virus-mediated oncolytic efficacy. Seven days after incubation of the cells with the virus, CELL TITER was usedCell viability assays measure cell viability in culture. The data were analyzed using GRAPHPAD PRISM software version 9.0.0 to determine the viral MOI (PFU) required for half maximal cell killing (EC 50 value) inferred from the sigmoidal dose response curve. In the graph of fig. 20, the y-axis represents the mean and standard deviation of EC50 values for each cell line determined in independent experiments. Tumor types from which each cell line was derived are indicated below the x-axis. The dotted line was arbitrarily set to a MOI of 0.1PFU, which represents the viral load of one tenth of the Vero cells of cynomolgus monkeys infected and lysed in the plaque formation assay for measuring PFU. These results, as well as the results shown in FIGS. 29B and 29C, demonstrate the efficacy of VACV IL-12 across various human tumor cell lines.

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 MOI and cell killing was assessed on day 5 (FIG. 29A-B; see also FIG. 20). IL-12 receptor is expressed predominantly on immune cells and not on tumor cells, and thus, cell killing differences between VACV-LUC and VACV IL-12 are not 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 indicating that encoding IL-12 did not interfere with VACV replication (fig. 29C). VACVIL-12 mediated oncolytic effects are widely observed across tumor cell lines of different cancer types, with 27/30 tumor cell lines exhibiting average EC50 values of 0.1MOI or less. Transgene production and viral replication were also assessed in tumor cell lines 5 days after infection (MOI 0.004) (FIGS. 29D-E).

Example 5 VACV recovered from human bladder tumor after IV administration to tumor-bearing mice

Amounts of IL12 Virus and human IL-12

Tumors derived from SW780 human bladder cancer cells were transplanted onto the flank of immunodeficient NOD/SCID mice. After a single dose of 10 ⁵、10⁶ or 10 ⁷ PFU of VACV IL-12 was administered by intravenous route, tumors were removed and analyzed for viral infiltration (viral PFU/gram tumor tissue) by plaque formation assay and for IL-12 transgene (ngIL-12/gram tumor) produced by virus-infected cells using a human IL12 specific ELISA (fig. 21A). In fig. 21B, the X-axis represents the time point after virus administration. Asterisks indicate time points at which no virus and transgene were determined. Zero above the X-axis indicates no measurable virus or transgene recovered. The results in fig. 21A show 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).

Additional experiments were also performed to assess transgene production and replication in SW780 tumor-bearing mice. Dose and time dependent increases in viral replication were observed in the treated tumors. 96 hours after dosing, 2.5X10 ⁴±4.3×10⁴ PFU/g was recovered from mice treated with 1X 10 ⁵ PFU, 9.5X10 ⁷±1.0×10⁸ PFU/g was recovered from mice treated with 1X 10 ⁶ PFU, and 1.3X10 ⁸±1.2×10⁸ PFU/g was recovered from mice treated with 1X 10 ⁷ PFU (FIG. 30A). Similarly, IL-12 production increased over time in tumors (FIG. 30E), whereas IL-12 was detectable around the periphery, but at a lower concentration (FIG. 30F).

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, mice were treated with single intravenous doses (10 ⁵、10⁶ and 10 ⁷ PFU) of either VACV-LUC or VACV IL-12 (FIGS. 30A-C). In the NCI-H292 and HCT-116 models, significant tumor control was observed by administration of a single dose of 10 ⁵ PFU (p-value < 0.001). In the SW780 model, significant tumor control was observed after a single dose of VACV IL-12 of 10 ⁶ PFU (p-value < 0.001). Due to the lack of an intact immune system, VACV-LUC and VACV IL-12 exhibit similar tumor control, indicating that the oncolytic activity of VACV is a key factor in tumor control.

EXAMPLE 6 Activity of VACV mul-12 on murine syngeneic tumors

After intratumoral administration of multiple doses to C57BL/6 mice implanted with subcutaneous MC38 colorectal tumors, activity of VACV encoding murine IL-12 was assessed (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 were randomized to treatment groups after growing to a median size of about 80mm ³. A total of 5 doses of 1e ⁷ PFU virus were administered twice a week over a 14 day period. Blood samples were collected 4 hours and 24 hours after the first dose administration, followed by tumor volume recordings. Tumor growth was assessed in vehicle-treated controls, mice administered with a non-transgenic VACV (null VACV), and mice administered with a VACV encoding muIL-12 (VACV muIL-12). Tumor growth is shown in individual tumor spider plots in fig. 22B-D, and survival of tumor-bearing mice is shown in Kaplan-Meier plot in fig. 22E (CR, complete response of tumor to therapy, where tumor volume is undetectable), by log rank (Mantel-Cox) comparison of three or more groups, p=0.0024, performed in GRAPHPAD PRISM. These results indicate that VACV muIL-12 is effective against murine syngeneic tumors.

Example 7 expression of murine IL-12 by VACV muIL-12 and induction of proinflammatory cytokines

Expression of murine IL-12 was detected in the peripheral blood of mice 4 and 24 hours after intratumoral administration VACV muIL-12, but not after administration of empty VACV or vehicle. (FIG. 23A). Also, peripheral blood ifnγ levels were significantly higher in VACV muIL-12 treated mice compared to mice administered either null VACV or vehicle (fig. 23B). Other pro-inflammatory cytokines detected in peripheral blood tended to be higher in VACVmuIL-12 treated mice than the control, and included ifnγ -inducible cytokine CXCL10 (fig. 23C) as well as pro-inflammatory cytokines IL6 (fig. 23D) and tnfα (fig. 23E).

EXAMPLE 8 Activity of VACV luc in Primary human patient derived tumor xenografts

The antitumor activity of luciferase-expressing VACV (VACVluc) was determined using primary tumors (Table 6) (PDX: patient-derived xenografts) transplanted into immunocompromised NOD/SCID mice from 47 cancer patients (FIGS. 24A-H). Each tumor tested was either untreated (open circles, grey lines) or a total of three doses of 1x 10 ⁷ pfuVACVluc virus (filled squares, black lines) was administered by the Intravenous (IV) route weekly starting when the tumor reached an average volume of about 200mm ³ (protocol shown in fig. 28A). Each primary patient-derived tumor includes 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. Responses (complete responders (CR) were determined to have no measurable tumor, partial Responders (PR) were determined to have a decrease in tumor volume of >30%, disease Stabilization (DS) were determined to have a tumor increase of less than 100%, and disease Progression (PD) were determined to have progressive growth, relative to the tumor volume of the untreated control. Overall, VACV Luc virus treated animals showed anti-tumor activity across tumor types compared to untreated controls. (FIG. 28B and Table 6).

To evaluate VACV replication kinetics, tumors were assessed for viral replication 48 hours after dose 1 and 3. Infectious virus was recovered from all tumor models and demonstrated a significant increase in viral replication between dose 1 and dose 3 (p-value=0.0079), indicating that VACV is accumulating and replicating in the treated tumor (fig. 28C).

TABLE 6 tumor types represented by implanted PDX

Tumor type	Number of PDX tumors assessed
		Bladder cancer	5
Colorectal cancer (CRC)	7
		Head and neck cancer (HNSCC)	7
Hepatocellular (liver) carcinoma	1
		Malignant melanoma	7
Mercker 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

EXAMPLE 9 IL-12R, NK cells, PDL1, CXCL9 and 10 upregulation after VACV infection

MRNA expression levels of the IL12RB1, IL12RB2, NKp46, PD-L1, CXCL9, and CXCL10 genes in mouse matrices isolated from xenograft (PDX) models derived from bladder, head and neck, liver, colon, lung and ovarian cancer patients were measured 48 hours after day 0 and day 14, and these models were derived from VACV-luciferase treated mice as shown in FIGS. 25A-F. Total RNA was isolated from freshly frozen PDX tissue and ribosomal RNA and globulin transcripts were removed. A library of linked RNAseq was created and paired-end sequencing was performed.

The double-ended reads were aligned with the mouse genome reference construct mm10 using a STAR aligner and gene-level read counts were generated using Salmon. The read counts were further normalized to sequencing depth and gene length using tximport to generate transcript (transcriptsperkilobasemillion, TPM) values of millions of kilobases per kilobase. All plots were generated using R version 4.1. These results indicate that IL-12R, NK cells, PDL1 and CXCL9/10 are upregulated following VACV infection, thereby initiating Tumor Microenvironment (TME) and eliciting effective anti-tumor immunity.

Example 10 VACV-IL-12 effectively infects human tumors in vitro, resulting in IL-12 and early B8R-dependent IFN-gamma blocking.

The efficiency of VACV-IL12 infection in human tumors in vitro was investigated. In particular, the efficacy of VACV IL-12 is evaluated in tumor Tissue Section Cultures (TSCs) derived from melanoma, bladder, colorectal, lung and ovarian cancer, as well as dissociated human tumor cells (DTCs) derived from patients with melanoma, head and neck squamous cell carcinoma and ovarian cancer. DTCs from different tumor types were thawed in medium supplemented with 1X CTL anti-aggregation wash. 50,000 cells were plated in 96-well u-bottom flasks and incubated with PBS (control), VACV-luciferase (MOI 1) or VACV-IL-12 (MOI 1). Supernatants were harvested from cultured DTCs 72 hours after treatment. IL-12 (i.e., IL12p 70) concentrations were measured using either a custom U-Plex human IL-12p70 assay or a U-Plex mouse IL-12p70 assay (catalog number K151UAK; K152UAK; meso Scale Diagnostics company), respectively, according to the manufacturer's protocol. Expression of human IL-12 was detected 3 days after infection VACVIL-12 in supernatants from tumor Tissue Section Cultures (TSCs), but not after infection with VACV-GFP or in mock-infected tissues (fig. 26A), and the same was true for DTCs (fig. 26B). An increase in ifnγ mRNA levels was detected in VACV IL-12 treated tissue slice cultures compared to 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).

Vaccinia virus encodes several immunomodulatory genes that inhibit immune cell activation, such as B8R, which is capable of sequestering and neutralizing ifnγ. VACV-IL-12 infection of TSC resulted in B8R expression, which has been demonstrated to sequester ifnγ in supernatant. Significantly higher B8R levels were detected in TSCs infected with VACV-GFP or VACV-IL-12 compared to mock-infected tissues. (FIG. 26E). In fig. 26E, each point is a single slice, with 1-4 replicates per condition. Data was analyzed using GRAPHPAD PRISM software version 9.0.0. Schematic of VACV infection, transgene production, and tumor cell lysis is shown in fig. 26F.

VACVIL-12 (COPTG 1673) infection resulted in the production of IL-12p70 (17984 pg/mL.+ -. SD 49523) in the 19/19 tested DTC sample. In contrast, the IL-12 concentration in supernatants from (6.15 pg/mL.+ -. SD 9.13) or VACVGFP treated (4.99 pg/mL.+ -. SD 6.36) DTCs from mock infection (FIGS. 31A-D) was negligible. Although the ifnγ RNA transcripts were significantly increased after VACV IL-12 infection compared to mock infection in TSC (1.5-fold; p-value < 0.001) and VACV GFP (1.4-fold; p < 0.001), evaluation of supernatants of both DTC and TSC indicated that ifnγ protein was not produced after VACV IL-12 treatment (fig. 26B, 26D and 31A-D).

EXAMPLE 11 VACV encoding murine IL-12 in vivo rat model overcomes the inhibition of B8R, leading to IFN-gamma induction

Since IFNγ signaling is critical for the efficacy of IL-12, the effect of B8R on downstream IL-12 and IFNγ activation was evaluated. VACV-encoded B8R does not bind murine ifnγ, however, B8R binds and neutralizes ifnγ from humans and rats. To confirm binding of B8R to rat ifnγ, a competition 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 ℃. Next, ELISA was performed to measure available rat ifnγ. Incubation with rB8R resulted in greater than 50% neutralization, and supernatants from infected cells were able to inhibit detection of ifnγ in a dose-dependent manner, confirming that B8R bound to rat ifnγ (fig. 32A).

Vacv IL-12 was evaluated in an immunocompetent model using a syngeneic rat tumor model based on the ability of B8R to bind to rat IFNγ (COPTG 1673). An alternative VACV-muIL virus was used because muIL12 has been shown to cross-react in rats. In addition, VACV-muIL was efficiently replicated in rat tumor cells and produced muIL-12 transgene compared to murine tumor cells (FIGS. 32B-C). Likewise, VACV-muIL12 mediated potent oncolytic effects in the rat tumor cell line, while cell killing activity in the mouse tumor cell line was lower (fig. 32D-F). Rats were implanted with F98 tumor cells and treated with VACV-LUC (1 x 10 ⁷ PFU) or VACV-muIL (1 x 10 ⁵、1x 10⁶ or 1x 10 ⁷) by intravenous administration on days 0, 4 and 7. Cytokine analysis in plasma demonstrated a modest increase in IL12p70 production between rats treated with 1x 10 ⁵ and 1x 10 ⁶ (fig. 32G). In addition, IFN-gamma was detected in the plasma of VACV-muIL treated rats, but not in the VACV-LUC control (FIG. 32H). These results indicate that VACV-encoded IL12 can induce production of peripheral ifnγ, whereas B8R does not inhibit such production.

Interferon-gamma competitive ELISA

Recombinant rat IFN-. Gamma.s (1 ng/ml) (R & D company) were incubated with PBS, recombinant B8R (50 ng/ml) (supplier) or 0.2um filtered supernatant of HeLa cells infected with VACV IL-12 (MOI 1). The mixture was incubated at 37 ℃ for 1 hour. Since B8R binds IFN-gamma and prevents antibodies from binding and being detected by ELISA, ELISA was performed to determine the percent recovery of IFN-gamma as described below. Percent recovery was calculated relative to IFN-gamma alone.

ELISA

Rat interferon-gamma was measured in plasma of rats treated with VACV at the indicated time points. Plasma was diluted and measured using rat IFN-. Gamma. DuoSetElisa according to the manufacturer's (R & D systems company) protocol. This demonstrates that recombinant poxviruses are therein capable of up-regulating Interferon (IFN) - γ. Example 12 VACV expressing IL12 improves therapeutic efficacy in murine isogenic tumor models

To evaluate the immunomodulatory effects of IL-12, murine alternative VACV-muIL virus was tested in a murine tumor cell line and tumor model. VACV-muIL12 exhibited replication, oncolysis and transgenic bioactivity similar to VACV IL-12 in human and mouse tumor cell lines (fig. 33A-E and 34A-F). Mice bearing subcutaneous CT26 tumors were treated with 5 intratumoral (i.t.) doses (10 ⁷ PFU) of vehicle control, VACV-LUC or alternative VACV-muIL (fig. 35A-D). Treatment with VACV-LUC did not lead to control of CT26 tumors (0/10 complete responders) and was similar to vehicle groups (fig. 35B-D). However, treatment with VACV-muIL12 resulted in tumor regression of CT26, with 6/10 of the complete responders (fig. 35E).

Vacv-LUC and Vacv-muIL showed similar oncolytic activity and replication in murine tumor cells (FIGS. 34A-F). Thus, it is hypothesized that the observed differences in tumor control may be due to the immunomodulatory effects of muIL. Thus, animals were treated according to the protocol shown in fig. 35A. Evidence of IL12 production in serum was detected 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 hours and 24 hours post injection (fig. 35G). Similar results were observed in MC38 isogenic tumor models (FIGS. 22A-E, 23A-B, 23E). Overall, these results indicate that VACV encoding IL-12 is involved in an adaptive immune response to control tumor growth, whereas the oncolytic activity of VACV-LUC alone is insufficient to control tumor burden.

Example 13 in vivo and ex vivo methods of investigation

In vivo study

Cell line derived xenografts were established by injecting 5×10 ⁶ cells/200 μl subcutaneous (sc) suspended in PBS into the right flank of 8 to 12 week old animals. Tumors reached 150-250mm ³ before randomization. Animal body weight was measured 2 times per week for 4 weeks, then once per week, and any body weight loss was monitored for health purposes.

To establish MC38 murine syngeneic tumor models, cells were removed from tissue culture plastic using accutase solution. Harvested cells were kept on ice (not more than 3 hours) from harvest to implantation. The implantation site of each animal was shaved at least 24 hours prior to cell injection. Syngeneic tumors were established by subcutaneously injecting 5X 10 ⁵ cells suspended in 100. Mu.L of Phosphate Buffered Saline (PBS) into the right flank of 7 to 9 week old C57BL/6J females. When the tumor volume reached 200mm ³, the mice were randomized. Mice body weight was monitored throughout the study.

To establish CT26 tumors, cells were removed from T-150 flasks using trypsin solution and trypsin was neutralized by the addition of rpmi+10% fbs prior to implantation. Harvested cells were kept on ice (not more than 3 hours) from harvest to implantation. The implantation site of each animal was shaved at least 24 hours prior to cell injection. Syngeneic tumors were established by subcutaneously injecting 5X 10 ⁵ cells suspended in 200. Mu.L of PBS into the right flank of 9-to 12-week-old Balb/c females. When tumor volumes reached 150-250mm ³, mice were randomized. Mice body weight was monitored throughout the study.

To establish 47 PDX models, cryovials containing tumor cells were thawed and prepared for injection into mice. The thawed cells were washed, counted in RPMI medium and resuspended in cold RPMI at a concentration of 50,000-100,000 viable cells per 50 μl. The cell suspension was mixed with an equal volume of CULTUREX ^TM extracellular matrix (ECM) and kept on ice during transport to the animal facility. Cells were prepared for injection by drawing the ECM-cell mixture into a chilled 1ml slide tip syringe. The filled syringe was kept on ice to avoid ECM clotting. Animals were shaved prior to injection. One mouse was fixed at a time and the injection site was sterilized with an alcohol swab. 100mL of the 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. Up to 5 animals were injected with 100ml of cell suspension per syringe.

For tumor mass inoculation, frozen tumor fragments were thawed and cut into tumor masses of approximately 2-3mm in diameter. Each mouse received a single buprenorphine injection 30 minutes prior to tumor mass implantation. A tumor mass was placed 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 tumor growth was observed. Tumors were allowed to reach approximately 130-230mm ³ before distribution to the treatment group. The anticancer effect of VACV Luc was determined by comparing the growth curve of each treated tumor to the size-matched untreated tumor. In this way, the optimal overall response of the treated tumor compared to the untreated tumor is designated as Complete Response (CR), partial Response (PR), disease Stabilization (DS) or disease Progression (PD). Tumors that do not meet the classification are considered non-evaluable (NE).

To establish F98 tumors in rats, cells were removed from 10 stacked flasks using trypsin solution and trypsin was neutralized by addition of dmem+10% fbs. Harvested cells were kept on ice (not more than 3 hours) from harvest to implantation. The F98 cell line was transplanted by Subcutaneous (SC) injection of 5.0x10 ⁶ cells suspended in 0.2mL PBS into the right flank of 7 to 9 week old animals. Tumors were allowed to reach approximately 300-350mm ³ prior to randomization.

Ex vivo analysis

To assess viral replication in tumors, mice were euthanized and blood was collected, tumor and normal tissue excised and snap frozen. Frozen tumors and normal tissues were weighed and suspended in ice-cold homogenization buffer (PBS, supplemented with 1x antibiotic-antifungal agent and 1x HALT ^TM protease-phosphatase inhibitor). Tissues containing homogenization buffer were transferred to MatrixA tubes and homogenized using Fast-prep-24 lysis system at a rate of 4 m/sec for 20 seconds. The tissue homogenates were then subjected to two freeze-thaw cycles, aliquoted and stored at-80 ℃. Two additional aliquots were prepared without freeze thawing for DNA isolation or cytokine analysis by MSD.

Cytokine analysis

Aliquots from tumor and spleen homogenates were centrifuged at 1,500rpm for 5 minutes at 4 ℃. The supernatant was collected and stored at-80 ℃ to measure cytokines. Blood was collected from mice at the indicated time points and centrifuged at 13,000rpm for 10 minutes at 4 ℃. Plasma was collected and stored at-80 ℃. Plasma, tumor and spleen lysates were diluted and measured using murine and human IL12p 70U-Plex assays (mesoscale diagnostics company (Meso Scale Diagnostics)) or custom U-Plex murine cytokine sets according to manufacturer's protocols.

Statistical analysis

The differences in tumor volume size at the indicated times during the experiment were assessed using GRAPHPAD PRISM software (san diego, california, usa) with one-way analysis of variance (ANOVA) and multiple comparison Tukey corrections. Statistical differences in the calculated tumor growth rates were assessed using a two-sided Mann-WhitneyU test (also known as the Wilcoxon rank sum test). Growth rate averages and growth rate inhibition statistics are reported with Mann-Whitney U test p-values. Differences in virus recovery (PFU) of tumors, human IL-12 transgene detected in tumors (pg/mL), and IL-12 transgene detected in mouse plasma (pg/mL) were determined using ANOVA and multiple comparison Tukey corrections. Significance p-values were obtained from ANOVA analysis.

Example 14 combination therapy with VACV-muIL and an immune checkpoint inhibitor

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

The anti-tumor immune response was further characterized by an ex vivo splenocyte re-stimulation assay. Splenocytes were collected on day 7 post VACV administration and stimulated with tumor-associated peptide antigen (AH-1) or VACV-specific peptide (a 52L). Combining VACV-muIL12 with anti-PD-L1 resulted in a significant increase in the number of IFN- γ secreting cells following 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 other treatment groups. In addition, 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 PD-L1 inhibitors in a tumor model that is resistant to anti-PD-L1 therapy. These results also indicate that the combination of VACV-muIL and an anti-PD-L1 blocking antibody is involved in 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 331S 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 331S 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 ^1x103 pfu to ^1x1012 pfu, optionally ^1x104 pfu to ^1x1011 pfu, optionally ^1x105 pfu to ^1x1010 pfu, optionally ^5x107 pfu to ^4x109 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.