Recombinant poxviruses for cancer immunotherapy

This application is a US National Phase Application under 37 U.S.C. § 371 of International Application No. PCT/US2019/051343, filed on Sep. 16, 2019, which claims the benefit of and priority to U.S. Provisional Application No. 62/731,876, filed Sep. 15, 2018, U.S. Provisional Application No. 62/767,485, filed Nov. 14, 2018, and U.S. Provisional Application No. 62/828,975, filed Apr. 3, 2019, the disclosures of which are incorporated by reference herein in their entireties.

This invention was made with government support under AI073736, AI095692, AR068118, and CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 23, 2023, is named 115872-2159_SL.txt and is 688,356 bytes in size.

The technology of the present disclosure relates generally to the fields of oncology, virology, and immunotherapy. In particular, the present technology relates to the use of poxviruses, including a recombinant modified vaccinia Ankara (MVA) virus comprising a deletion of E3L (MVAΔE3L) genetically engineered to express OX40L (MVAΔE3L-OX40L); a recombinant MVA virus comprising a deletion of C7L (MVAΔC7L) genetically engineered to express OX40L (MVAΔC7L-OX40L); a recombinant MVAΔC7L engineered to express OX40L and hFlt3L (MVAΔC7L-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of C7L, a deletion of E5R, and to express hFlt3L and OX40L (MVAΔC7LΔE5R-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of E5R (MVAΔE5R); a recombinant MVA genetically engineered to comprise a deletion of E5R and to express hFlt3L and OX40L (MVAΔE5R-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of E3L, a deletion of E5R, and to express hFtl3L and OX40L (MVAΔE3LΔE5R-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of E5R, a deletion of C11R, and to express hFlt3L and OX40L (MVAΔE5R-hFlt3L-OX40L-ΔC11R); a recombinant MVA genetically engineered to comprise a deletion of E3L, a deletion of E5R, a deletion of C11R, and to express hFlt3L and OX40L (MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R); a recombinant vaccinia virus comprising a deletion of C7L (VACVΔC7L) genetically engineered to express OX40L (VACVΔC7L-OX40L); a recombinant VACVΔC7L genetically engineered to express both OX40L and hFlt3L (VACVΔC7L-hFlt3L-OX40L); a VACV genetically engineered to comprise a deletion of E5R (VACVΔE5R); a recombinant VACV genetically engineered to comprise a deletion of E5R, a deletion of thymidine kinase (TK), and to express anti-CTLA-4, hFlt3L, and OX40L (VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L); a VACV genetically engineered to comprise a deletion of B2R (VACVΔB2R); a VACV genetically engineered to comprise an E3LΔ83N deletion and a B2R deletion (VACVE3LΔ83NΔB2R); a VACV genetically engineered to comprise an E5R deletion and a B2R deletion (VACVΔE5RΔB2R); a VACV genetically engineered to comprise an E3LΔ83N deletion, an E5R deletion, and a B2R deletion (VACVE3LΔ83NΔE5RΔB2R); a VACV genetically engineered to comprise an E3LΔ83N deletion, a deletion of thymidine kinase (TK), and an E5R deletion, and expressing anti-CTLA-4, hFlt3L, OX40L, and IL-12 (VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12); a VACV genetically engineered to comprise an E3LΔ83N deletion, a deletion of thymidine kinase (TK), an E5R deletion, and a B2R deletion, and expressing anti-CTLA-4, hFlt3L, OX40L, and IL-12 (VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R); a MYXV genetically engineered to comprise a deletion of M31R (MYXVΔM31R); a recombinant MYXV genetically engineered to comprise a deletion of M31R and to express hF 1 3L and OX40L (MYXVΔM31R-hFlt3L-OX40L); a MYXV genetically engineered to comprise a deletion of M63R (MYXVΔM63R); a MYXV genetically engineered to comprise a deletion of M64R (MYXVΔM64R); an MVA genetically engineered to comprise a deletion of WR199 (MVAΔWR199); an MVA genetically engineered to comprise a deletion of E5R, a deletion of WR199, and expressing hFlt3L and OX40L (MVAΔE5R-hFlt3L-OX40L-ΔWR199); or combinations thereof, alone or in combination with immune checkpoint blocking agents, immunomodulatory agents, and/or anti-cancer drugs as an immunotherapeutic and/or oncolytic composition. In some embodiments, the technology of the present disclosure relates to any one of the foregoing viruses further modified to express a specific gene of interest (SG), such as genes encoding any one or more of the following immunomodulatory proteins, including but not limited to hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L. In some embodiments, the virus backbones are further modified to comprise deletions or mutations of genes, including but not limited to thymidine kinase (TK), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), E5R, K7R, C12L (IL18BP), B8R, B14R, N1L, C11R, K1L, M1L, N2L, and/or WR199. In some embodiments, the technology of the present disclosure relates to the use of any one of the foregoing viruses as a vaccine adjuvant. In particular, the present technology relates to the use of MVAΔC7L-hFlt3L-TK(−)-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, and Heat-inactivated MVAΔE5R as a vaccine adjuvant for tumor antigens in cancer vaccines alone or in combination with immune checkpoint blockade (ICB) antibodies for use as a cancer immunotherapeutic. In some embodiments, the technology of the present disclosure relates to the use of any one of the foregoing viruses as a vaccine vector. In particular, the present technology relates to the use of MVAΔE5R or MVAΔE5R-hFlt3L-OX40L as vaccine vectors for cancer vaccines. In some embodiments, the present technology relates to a recombinant poxvirus selected from MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-CTLA-4, and MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-CTLA-4, or combinations thereof, alone or in combination with immune checkpoint blocking agents, immunomodulatory agents, and/or anti-cancer drugs as an immunotherapeutic and/or oncolytic composition.

The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art.

Malignant tumors such as melanoma are inherently resistant to conventional therapies and present significant therapeutic challenges. Immunotherapy is an evolving area of research and an additional option for the treatment of certain types of cancers. The immunotherapy approach rests on the rationale that the immune system may be stimulated to identify tumor cells and target them for destruction. Despite presentation of antigens by cancer cells and the presence of immune cells that could potentially react against tumor cells, in many cases, the immune system is not activated or is affirmatively suppressed. Key to this phenomenon is the ability of tumors to protect themselves from immune response by coercing cells of the immune system to inhibit other cells of the immune system. Tumors develop a number of immunomodulatory mechanisms to evade antitumor immune responses. Thus, improved immunotherapeutic approaches are needed to enhance host antitumor immunity and target tumor cells for destruction.

In one aspect, the present disclosure provides a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant C7 gene and a heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-OX40L). In some embodiments, the recombinant MVAΔC7L-OX40L virus further comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔC7L-hFlt3L-OX40L). In some embodiments, the recombinant MVAΔC7L-OX40L virus of the present technology further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the recombinant MVAΔC7L-OX40L virus comprising the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a MVA viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15RΔ, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the recombinant MVA virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔC7L virus; induction of increased splenic production of effector T-cells as compared to the corresponding MVAΔC7L virus; and reduction of tumor volume in tumor cells contacted with the recombinant MVAΔC7L-OX40L virus as compared to tumor cells contacted with the corresponding MVAΔC7L virus. In some embodiments, the tumor cells comprise melanoma cells.

In another aspect, the present disclosure provides, an immunogenic composition comprising the recombinant MVAΔC7L-OX40L virus of the present technology. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant MVAΔC7L-the present technology. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the method for treating a solid tumor in a subject in need thereof comprises the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔC7L virus; and increased splenic production of effector T-cells as compared to the corresponding MVAΔC7L virus. In some embodiments, the method of treating a solid tumor in a subject in need thereof the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the composition comprises one or more immune checkpoint blocking agents. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agent is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the combination of the MVAΔC7L-OX40L or MVAΔC7L-hFlt3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either the MVAΔC7L-OX40L or MVAΔC7L-hFlt3L-OX40L or of the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus of the present technology (e.g., MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody.

In another aspect, the present disclosure provides, a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant E3 gene and a heterologous nucleic acid molecule encoding OX40L (MVAΔE3L-OX40L). In some embodiments, the recombinant MVAΔE3L-OX40L virus comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments of the virus of the present technology, the OX40L is expressed from within a MVA viral gene. In some embodiments of the virus of the present technology, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, of the virus of the present technology, the OX40L is expressed from within the TK gene. In some embodiments, of the virus of the present technology, the virus comprises a heterologous nucleic acid molecule encoding one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), E5R, K7R, C12L (IL18BP), B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, of the virus of the present technology, the recombinant MVA virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔE3L virus; induction of increased splenic production of effector T-cells as compared to the corresponding MVAΔE3L virus; and reduction of tumor volume in tumor cells contacted with the recombinant MVAΔE3L-OX40L virus as compared to tumor cells contacted with the corresponding MVAΔE3L virus. In some embodiments, of the virus of the present technology, the tumor cells comprise melanoma cells. In some embodiments, the mutant E3 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein.

In another aspect, the present disclosure provides an immunogenic composition comprising the recombinant MVAΔE3L-OX40L virus of the present technology. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provided a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant MVAΔE3L-OX40L virus of the present technology or an immunogenic composition of the present technology. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔE3L virus; and increased splenic production of effector T-cells as compared to the corresponding MVAΔE3L virus. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition further comprises one or more immune checkpoint blocking agents. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agent is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the combination of the MVAΔE3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either MVAΔE3L-OX40L or the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of a virus of the present technology (e.g., MVAΔE3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering one or more immune checkpoint blocking agents to the subject. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of MVAΔE3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either MVAΔE3L-OX40L or the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a recombinant vaccinia virus (VACV) comprising a mutant C7 gene and a heterologous nucleic acid molecule encoding OX40L (VACVΔC7L-OX40L). In some embodiments, the virus comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔC7L-hFlt3L-OX40L). In some embodiments, the virus comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (VACVΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a vaccinia viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; AWR200), E5R, K7R, C12L (IL18BP), B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the virus further comprises a heterologous nucleic acid encoding hIL-12 and a heterologous nucleic acid encoding anti-huCTLA-4 (VACVΔC7L-anti-huCTLA-4-hFlt3L-OX40L-hIL-12). In some embodiments, the recombinant VACVΔC7L-OX40L virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding VACVΔC7L virus; induction of increased splenic production of effector T-cells as compared to the corresponding VACVΔC7L virus; and reduction of tumor volume in tumor cells contacted with the recombinant VACVΔC7L-OX40L virus as compared to tumor cells contacted with the corresponding VACVΔC7L virus. In some embodiments, the tumor cells comprise melanoma cells.

In another aspect, the present disclosure provides, an immunogenic composition comprising the recombinant VACVΔC7L-OX40L virus of the present technology. In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant VACVΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding VACVΔC7Lvirus; and increased splenic production of effector T-cells as compared to the corresponding VACVΔC7L virus. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition further comprises one or more immune checkpoint blocking agents. In some embodiments, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises the one or more immune checkpoint blocking agent comprises anti-PD-1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises the one or more immune checkpoint blocking agent comprises anti-PD-L1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, comprises the one or more immune checkpoint blocking agent comprises anti-CTLA-4 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the combination of the VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L or of the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides, a method for stimulating an immune response comprising administering to a subject an effective amount of the virus of the present technology (e.g., VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering one or more immune checkpoint blocking agents. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the immune checkpoint blocking agent comprises anti-PD-1 antibody. In some embodiments, the immune checkpoint blocking agent comprises anti-PD-L1 antibody. In some embodiments, the immune checkpoint blocking agent comprises anti-CTLA-4 antibody.

In another aspect, the present disclosure provides, a recombinant modified vaccinia Ankara (MVA) virus nucleic acid sequence, wherein the nucleic acid sequence between position 75,560 and 76,093 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L, and wherein the MVA further comprises a C7 mutant. In some embodiments of the MVA virus of the present technology, the nucleic acid sequence between position 18,407 and 18,859 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L).

In another aspect, the present disclosure provides a recombinant modified vaccinia Ankara (MVA) virus nucleic acid sequence, wherein the nucleic acid sequence between position 75,798 to 75,868 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L or encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L), and wherein the MVA further comprises an E3 mutant.

In another aspect, the present disclosure provides a recombinant vaccinia virus (VACV) nucleic acid sequence, wherein the nucleic acid sequence between position 80,962 and 81,032 of SEQ ID NO: 2 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L or, and wherein the VACV further comprises a C7 mutant. In some embodiments the recombinant VACV of the present technology the nucleic acid sequence between position 15,716 and 16,168 of SEQ ID NO: 2 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L).

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant MVAΔC7L-OX40L virus of the present technology.

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant MVAΔE3L-OX40L virus of the present technology.

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant VACVΔC7L-OX40L virus of any one of the present technology.

In another aspect, the present disclosure provides a kit comprising the recombinant MVAΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology, and instructions for use.

In another aspect, the present disclosure provides a kit comprising the recombinant MVAΔE3L-OX40L virus of any one of the present technology or the immunogenic composition of the present technology, and instructions for use.

In another aspect, the present disclosure provides a kit comprising the recombinant VACVΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology, and instructions for use.

In some embodiments, the present disclosure provides a recombinant MVAΔC7L-OX40L virus wherein the mutant C7 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔC7L-OX40L virus, wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔE3L-OX40L virus wherein the mutant E3 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔE3L-OX40L virus wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present technology provides a recombinant VACVΔC7L-OX40L virus wherein the mutant C7 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant VACVΔC7L-OX40L virus wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an antigen and a therapeutically effective amount of an adjuvant comprising a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant C7 gene and a heterologous nucleic acid encoding OX40L (MVAΔC7L-OX40L). In some embodiments, the MVAΔC7L-OX40L virus further comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔC7L-hFlt3L-OX40L). In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a MVA viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199.

In some embodiments, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof.

In some embodiments, the administration step comprises administering the antigen and adjuvant in one or more doses and/or wherein the antigen and adjuvant are administered separately, sequentially, or simultaneously.

In some embodiments, the method further comprises administering to the subject an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In some embodiments, the antigen and adjuvant are delivered to the subject separately, sequentially, or simultaneously with the administration of the immune checkpoint blockade agent.

In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of the tumor cells, or prolonging survival of the subject. In some embodiments, the induction, enhancement, or promotion of the immune response comprises one or more of the following: (i) increased levels of interferon gamma (IFN-γ) expression in T-cells in the spleen, draining lymph nodes, and/or serum as compared to an untreated control sample; (ii) increased levels of antigen-specific T-cells in the spleen, draining lymph nodes, and/or serum as compared to an untreated control sample; and (iii) increased levels of antigen-specific immunoglobulin in serum as compared to an untreated control sample. In some embodiments, the antigen-specific immunoglobulin is IgG1 or IgG2.

In some embodiments, the antigen and adjuvant are formulated to be administered intratumorally, intramuscularly, intradermally, or subcutaneously.

In some embodiments, the tumor is selected from the group consisting of melanoma, colorectal cancer, breast cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, squamous cell carcinoma of the skin, Merkel cell carcinoma, gastric cancer, liver cancer, and sarcoma.

In some embodiments, the MVAΔC7L-OX40L virus is administered at a dosage per administration of about 10 5 to about 10 10 plaque-forming units (pfu).

In some embodiments of the method, the subject is human.

In one aspect, the present disclosure provides an immunogenic composition comprising the antigen and the adjuvant of the present technology. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof. In some embodiments, the immunogenic composition further comprises an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In one aspect, the present disclosure provides a kit comprising instructions for use, a container means, and a separate portion of each of: (a) an antigen; and (b) an adjuvant of the present technology. In some embodiments of the kit, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof.

In some embodiments, the kit further comprises (c) an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In some embodiments of the methods of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSL VIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In some embodiments of the immunogenic compositions of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSL VIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In some embodiments of the kit of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In one aspect, the present disclosure provides a modified vaccinia Ankara (MVA) virus genetically engineered to comprise a mutant E5R gene (MVAΔE5R). In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant E5R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (MVAΔE5R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔE5R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔE5R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule.

In one aspect, the present disclosure provides an immunogenic composition comprising the MVAΔE5R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the MVAΔE5R virus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof.

In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MVAΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of either the MVAΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition. In som e embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MVAΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the MVAΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered MVAΔE5R viruses described herein.

In one aspect, the present disclosure provides a kit comprising the engineered MVAΔE5R viruses described herein, and instructions for use.

In one aspect, the present disclosure provides a vaccinia virus (VACV) genetically engineered to comprise a mutant E5R gene (VACVΔE5R). In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant E5R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (VACVΔE5R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔE5R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔE5R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule.

In one aspect, the present disclosure provides an immunogenic composition comprising the VACVΔE5R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the VACVΔE5R virus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof.

In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the VACVΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of the VACVΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition of. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the VACVΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the VACVΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered VACVΔE5R viruses of the present technology.

In one aspect, the present disclosure provides a kit comprising the engineered VACVΔE5R viruses of the present technology, and instructions for use.

In one aspect, the present disclosure provides a myxoma virus (MYXV) genetically engineered to comprise a mutant M31R gene (MYXVΔM31R). In some embodiments, tthe virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of myxoma orthologs of vaccinia virus thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; AWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant M31R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (MYXVΔM31R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MYXVΔM31R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MYXVΔM31R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a myxoma ortholog of a vaccinia viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant myxoma ortholog of vaccinia virus thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant myxoma ortholog of vaccinia virus C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule.

In one aspect, the present disclosure provides an immunogenic composition comprising the MYXVΔM31R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the MYXVΔM31R virus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma.

In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXVΔM31R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of either the MYXVΔM31R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition of. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG,and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXVΔM31R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the MYXVΔM31R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered MYXVΔM31R viruses of the present technology.

In one aspect, the present disclosure provides a kit comprising the engineered MYXVΔM31R viruses of the present technology, and instructions for use.

In one aspect, the present disclosure provides a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant C7 gene and a heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-OX40L). In some embodiments, the recombinant MVAΔC7L-OX40L virus further comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔC7L-hFlt3L-OX40L). In some embodiments, the recombinant MVAΔC7L-OX40L virus of the present technology further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the recombinant MVAΔC7L-OX40L virus comprising the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a MVA viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the recombinant MVA virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔC7L virus; induction of increased splenic production of effector T-cells as compared to the corresponding MVAΔC7L virus; and reduction of tumor volume in tumor cells contacted with the recombinant MVAΔC7L-OX40L virus as compared to tumor cells contacted with the corresponding MVAΔC7L virus. In some embodiments, the tumor cells comprise melanoma cells.

In another aspect, the present disclosure provides, an immunogenic composition comprising the recombinant MVAΔC7L-OX40L virus of the present technology. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant MVAΔC7L-the present technology. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the method for treating a solid tumor in a subject in need thereof comprises the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔC7L virus; and increased splenic production of effector T-cells as compared to the corresponding MVAΔC7L virus. In some embodiments, the method of treating a solid tumor in a subject in need thereof the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the composition comprises one or more immune checkpoint blocking agents. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agent is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments of the method of treating a solid tumor in a subject in need thereof, the combination of the MVAΔC7L-OX40L or MVAΔC7L-hFlt3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either the MVAΔC7L-OX40L or MVAΔC7L-hFlt3L-OX40L or of the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus of the present technology (e.g., MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody.

In another aspect, the present disclosure provides, a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant E3 gene and a heterologous nucleic acid molecule encoding OX40L (MVAΔE3L-OX40L). In some embodiments, the recombinant MVAΔE3L-OX40L virus comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments of the virus of the present technology, the OX40L is expressed from within a MVA viral gene. In some embodiments of the virus of the present technology, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, of the virus of the present technology, the OX40L is expressed from within the TK gene. In some embodiments, of the virus of the present technology, the virus comprises a heterologous nucleic acid molecule encoding one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), E5R, K7R, C12L (IL18BP), B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, of the virus of the present technology, the recombinant MVA virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔE3L virus; induction of increased splenic production of effector T-cells as compared to the corresponding MVAΔE3L virus; and reduction of tumor volume in tumor cells contacted with the recombinant MVAΔE3L-OX40L virus as compared to tumor cells contacted with the corresponding MVAΔE3L virus. In some embodiments, of the virus of the present technology, the tumor cells comprise melanoma cells. In some embodiments, the mutant E3 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein.

In another aspect, the present disclosure provides an immunogenic composition comprising the recombinant MVAΔE3L-OX40L virus of the present technology. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present technology comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provided a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant MVAΔE3L-OX40L virus of the present technology or an immunogenic composition of the present technology. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVAΔE3L virus; and increased splenic production of effector T-cells as compared to the corresponding MVAΔE3L virus. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition further comprises one or more immune checkpoint blocking agents. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agent is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the combination of the MVAΔE3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either MVAΔE3L-OX40L or the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of a virus of the present technology (e.g., MVAΔE3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering one or more immune checkpoint blocking agents to the subject. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of MVAΔE3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of either MVAΔE3L-OX40L or the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides a recombinant vaccinia virus (VACV) comprising a mutant C7 gene and a heterologous nucleic acid molecule encoding OX40L (VACVΔC7L-OX40L). In some embodiments, the virus comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔC7L-hFlt3L-OX40L). In some embodiments, the virus comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (VACVΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a vaccinia viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), E5R, K7R, C12L (IL18BP), B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the virus further comprises a heterologous nucleic acid encoding hIL-12 and a heterologous nucleic acid encoding anti-huCTLA-4 (VACVΔC7L-anti-huCTLA-4-hFlt3L-OX40L-hIL-12). In some embodiments, the recombinant VACVΔC7L-OX40L virus exhibits one or more of the following characteristics: induction of increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding VACVΔC7L virus; induction of increased splenic production of effector T-cells as compared to the corresponding VACVΔC7L virus; and reduction of tumor volume in tumor cells contacted with the recombinant VACVΔC7L-OX40L virus as compared to tumor cells contacted with the corresponding VACVΔC7L virus. In some embodiments, the tumor cells comprise melanoma cells.

In another aspect, the present disclosure provides, an immunogenic composition comprising the recombinant VACVΔC7L-OX40L virus of the present technology. In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant VACVΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the treatment comprises the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding VACVΔC7Lvirus; and increased splenic production of effector T-cells as compared to the corresponding VACVΔC7L virus. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the composition further comprises one or more immune checkpoint blocking agents. In some embodiments, the method further comprises administering to the subject one or more immune checkpoint blocking agents. In some embodiments the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises the one or more immune checkpoint blocking agent comprises anti-PD-1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the one or more immune checkpoint blocking agents comprises the one or more immune checkpoint blocking agent comprises anti-PD-L1 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, comprises the one or more immune checkpoint blocking agent comprises anti-CTLA-4 antibody. In some embodiments of the method for treating a solid tumor in a subject in need thereof, the combination of the VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L and the immune checkpoint blocking agent has a synergistic effect in the treatment of the tumor as compared to administration of VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L or of the immune checkpoint blocking agent alone.

In another aspect, the present disclosure provides, a method for stimulating an immune response comprising administering to a subject an effective amount of the virus of the present technology (e.g., VACVΔC7L-OX40L or VACVΔC7L-hFlt3L-OX40L) or an immunogenic composition of the present technology. In some embodiments, the method further comprises administering one or more immune checkpoint blocking agents. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof. In some embodiments, the immune checkpoint blocking agent comprises anti-PD-1 antibody. In some embodiments, the immune checkpoint blocking agent comprises anti-PD-L1 antibody. In some embodiments, the immune checkpoint blocking agent comprises anti-CTLA-4 antibody.

In another aspect, the present disclosure provides, a recombinant modified vaccinia Ankara (MVA) virus nucleic acid sequence, wherein the nucleic acid sequence between position 75,560 and 76,093 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L, and wherein the MVA further comprises a C7 mutant. In some embodiments of the MVA virus of the present technology, the nucleic acid sequence between position 18,407 and 18,859 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L).

In another aspect, the present disclosure provides a recombinant modified vaccinia Ankara (MVA) virus nucleic acid sequence, wherein the nucleic acid sequence between position 75,798 to 75,868 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L or encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L), and wherein the MVA further comprises an E3 mutant.

In another aspect, the present disclosure provides a recombinant vaccinia virus (VACV) nucleic acid sequence, wherein the nucleic acid sequence between position 80,962 and 81,032 of SEQ ID NO: 2 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes OX40L or, and wherein the VACV further comprises a C7 mutant. In some embodiments the recombinant VACV of the present technology the nucleic acid sequence between position 15,716 and 16,168 of SEQ ID NO: 2 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes human Fms-like tyrosine kinase 3 ligand (hFlt3L).

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant MVAΔC7L-OX40L virus of the present technology.

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant MVAΔE3L-OX40L virus of the present technology.

In another aspect, the present disclosure provides a nucleic acid sequence encoding the recombinant VACVΔC7L-OX40L virus of any one of the present technology.

In another aspect, the present disclosure provides a kit comprising the recombinant MVAΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology, and instructions for use.

In another aspect, the present disclosure provides a kit comprising the recombinant MVAΔE3L-OX40L virus of any one of the present technology or the immunogenic composition of the present technology, and instructions for use.

In another aspect, the present disclosure provides a kit comprising the recombinant VACVΔC7L-OX40L virus of the present technology or the immunogenic composition of the present technology, and instructions for use.

In some embodiments, the present disclosure provides a recombinant MVAΔC7L-OX40L virus wherein the mutant C7 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔC7L-OX40L virus, wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔE3L-OX40L virus wherein the mutant E3 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant MVAΔE3L-OX40L virus wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present technology provides a recombinant VACVΔC7L-OX40L virus wherein the mutant C7 gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein. In some embodiments, the present disclosure provides a recombinant VACVΔC7L-OX40L virus wherein the mutant TK gene is at least partially deleted, is not expressed, is expressed at levels so low as to have no effect, or expressed as a non-functional protein.

In another aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an antigen and a therapeutically effective amount of an adjuvant comprising a recombinant modified vaccinia Ankara (MVA) virus comprising a mutant C7 gene and a heterologous nucleic acid encoding OX40L (MVAΔC7L-OX40L). In some embodiments, the MVAΔC7L-OX40L virus further comprises a heterologous nucleic acid encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔC7L-hFlt3L-OX40L). In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprise the heterologous nucleic acid molecule encoding OX40L. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid. In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding hFlt3L. In some embodiments, the mutant C7 gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding hFlt3L, and wherein the virus further comprises a mutant TK gene comprising replacement of at least a portion of the TK gene with one or more gene cassettes comprising the heterologous nucleic acid molecule encoding OX40L (MVAΔC7L-hFlt3L-TK(−)-OX40L). In some embodiments, the OX40L is expressed from within a MVA viral gene. In some embodiments, the OX40L is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the OX40L is expressed from within the TK gene. In some embodiments, the OX40L is expressed from within the TK gene and the hFlt3L is expressed from within the C7 gene. In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199.

In some embodiments, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof.

In some embodiments, the administration step comprises administering the antigen and adjuvant in one or more doses and/or wherein the antigen and adjuvant are administered separately, sequentially, or simultaneously.

In some embodiments, the method further comprises administering to the subject an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In some embodiments, the antigen and adjuvant are delivered to the subject separately, sequentially, or simultaneously with the administration of the immune checkpoint blockade agent.

In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of the tumor cells, or prolonging survival of the subject. In some embodiments, the induction, enhancement, or promotion of the immune response comprises one or more of the following: (i) increased levels of interferon gamma (IFN-γ) expression in T-cells in the spleen, draining lymph nodes, and/or serum as compared to an untreated control sample; (ii) increased levels of antigen-specific T-cells in the spleen, draining lymph nodes, and/or serum as compared to an untreated control sample; and (iii) increased levels of antigen-specific immunoglobulin in serum as compared to an untreated control sample. In some embodiments, the antigen-specific immunoglobulin is IgG1 or IgG2.

In some embodiments, the antigen and adjuvant are formulated to be administered intratumorally, intramuscularly, intradermally, or subcutaneously.

In some embodiments, the tumor is selected from the group consisting of melanoma, colorectal cancer, breast cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, squamous cell carcinoma of the skin, Merkel cell carcinoma, gastric cancer, liver cancer, and sarcoma.

In some embodiments, the MVAΔC7L-OX40L virus is administered at a dosage per administration of about 10 5 to about 10 10 plaque-forming units (pfu).

In some embodiments of the method, the subject is human.

In one aspect, the present disclosure provides an immunogenic composition comprising the antigen and the adjuvant of the present technology. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof. In some embodiments, the immunogenic composition further comprises an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In one aspect, the present disclosure provides a kit comprising instructions for use, a container means, and a separate portion of each of: (a) an antigen; and (b) an adjuvant of the present technology. In some embodiments of the kit, the antigen is selected from the group consisting of tumor differentiation antigens, cancer testis antigens, neoantigens, viral antigens in the case of tumors associated with oncogenic virus infection, GPA33, HER2/neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, p53, kras, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), NY-ESO-1, human papilloma virus E6 and E7, and any combination thereof.

In some embodiments, the kit further comprises (c) an immune checkpoint blockade agent selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof.

In some embodiments of the methods of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSL VIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In some embodiments of the immunogenic compositions of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSL VIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In some embodiments of the kit of the present technology, the antigen is a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSL VIHD) (SEQ ID NO: 17), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 18), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 19), and combinations thereof.

In one aspect, the present disclosure provides a modified vaccinia Ankara (MVA) virus genetically engineered to comprise a mutant E5R gene (MVAΔE5R). In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant E5R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (MVAΔE5R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔE5R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MVAΔE5R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the MVAΔE5R-OX40L-hFlt3L virus further comprises a mutant C11R gene (MVAΔE5R-OX40L-hFlt3L-ΔC11R). In some embodiments, the mutant C11R gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C11R gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the MVAΔE5R-OX40L-hFlt3L virus further comprises a mutant WR199 gene (MVAΔE5R-OX40L-hFlt3L-ΔWR199). In some embodiments, the mutant WR199 gene comprises an insertion or one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant WR199 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the MVAΔE5R virus further comprises a mutant E3L gene (ΔE3L). In some embodiments, the mutant E3L gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant E3L gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L. In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid encoding human Fms-like typrsine kinase 3 ligand (hFlt3L).

In one aspect, the present disclosure provides an immunogenic composition comprising the MVAΔE5R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the MVAΔE5R virus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof.

In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MVAΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of either the MVAΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition. In som e embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MVAΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the MVAΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered MVAΔE5R viruses described herein.

In one aspect, the present disclosure provides a kit comprising the engineered MVAΔE5R viruses described herein, and instructions for use.

In one aspect, the present disclosure provides a vaccinia virus (VACV) genetically engineered to comprise a mutant E5R gene (VACVΔE5R). In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant E5R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (VACVΔE5R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔE5R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔE5R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule.

In one aspect, the present disclosure provides an immunogenic composition comprising the VACVΔE5R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the VACVΔE5R virus or the immunogenic composition.

In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma. In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof.

In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the VACVΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of the VACVΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition of. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the VACVΔE5R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the VACVΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered VACVΔE5R viruses of the present technology.

In one aspect, the present disclosure provides a kit comprising the engineered VACVΔE5R viruses of the present technology, and instructions for use.

In one aspect, the present disclosure provides a myxoma virus (MYXV) genetically engineered to comprise a mutant M31R gene (MYXVΔM31R). In some embodiments, tthe virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of myxoma orthologs of vaccinia virus thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the mutant M31R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (MYXVΔM31R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MYXVΔM31R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (MYXVΔM31R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a myxoma ortholog of a vaccinia viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R gene (WR200), the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene. In some embodiments, the virus further comprises a mutant myxoma ortholog of vaccinia virus thymidine kinase (TK) gene. In some embodiments, the mutant TK gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the virus further comprises a mutant myxoma ortholog of vaccinia virus C7 gene. In some embodiments, the mutant C7 gene comprises an insertion of one or more gene cassettes comprising a heterologous nucleic acid molecule. In some embodiments, the mutant C7 gene comprises replacement of all or at least a portion of the gene with one or more gene cassettes comprising a heterologous nucleic acid molecule.

In one aspect, the present disclosure provides an immunogenic composition comprising the MYXVΔM31R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the MYXVΔM31R virus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma.

In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXVΔM31R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of either the MYXVΔM31R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition of. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXVΔM31R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the MYXVΔM31R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In one aspect, the present disclosure provides a nucleic acid encoding the engineered MYXVΔM31R viruses of the present technology.

In one aspect, the present disclosure provides a kit comprising the engineered MYXVΔM31R viruses of the present technology, and instructions for use.

In one aspect, the present disclosure a vaccinia virus (VACV) genetically engineered to comprise a mutant B2R gene (VACVΔB2R).

In some embodiments, theVACVΔB2R virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199. In some embodiments, the VACVΔB2R virus is selected from one or more of VACVΔE3L83NΔB2R, VACVΔE5RΔB2R, VACVΔE3L83NΔE5RΔB2R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-OX40L-hIL-12-ΔB2R. In some embodiments, the mutant B2R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the one or more gene cassettes comprises a heterologous nucleic acid molecule encoding OX40L (VACVΔB2R-OX40L). In some embodiments, the one or more gene cassettes further comprises a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔB2R-OX40L-hFlt3L). In some embodiments, the one or more gene cassettes comprise a heterologous nucleic acid molecule encoding human Fms-like tyrosine kinase 3 ligand (hFlt3L) (VACVΔB2R-hFlt3L). In some embodiments, the heterologous nucleic acid is expressed from within a viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B18R (WR200) gene, the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene.

In some embodiments, the present disclosure provides an immunogenic composition comprising the VACVΔB2R virus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In some embodiments, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the VACVΔB2R virus or the immunogenic composition.

In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject.

In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection.

In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma.

In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, in the combination of the VACVΔB2R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of the VACVΔE5R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In some embodiments, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the VACVΔB2R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the VACVΔB2R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In some embodiments, the present disclosure provides a nucleic acid encoding the VACVΔB2R virus of the present technology.

In some embodiments, the present disclosure provides a kit comprising the VACVΔB2R virus of the present technology, and instructions for use.

In one aspect, the present disclosure provides a myxoma virus (MYXV) genetically engineered to comprise one or more mutants selected from (i) a mutant M63R gene (MYXVΔM63R); (ii) a mutant M64R gene (MYXVΔM64R); and (iii) a mutant M62R gene (MYXVΔM62R). In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding one or more of OX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or a deletion of any one or more of myxoma orthologs of vaccinia virus thymidine kinase (TK), C7 (ΔC7L), E3L (ΔE3L), E3LΔ83N, B2R (ΔB2R), B19R (B18R; ΔWR200), IL18BP, K7R, C12L, B8R, B14R, N1L, C11R, K1L, M1L, N2L, or WR199 (ΔWR199), or of myxoma M31R (ΔM31R). In some embodiments, the mutant M63R gene,M64R gene, and/or M62R gene comprises replacement of at least a portion of the gene with one or more gene cassettes comprising the heterologous nucleic acid molecule. In some embodiments, the heterologous nucleic acid is expressed from within a myxoma ortholog of a vaccinia viral gene selected from the group consisting of the thymidine kinase (TK) gene, the C7 gene, the C11 gene, the K3 gene, the F1 gene, the F2 gene, the F4 gene, the F6 gene, the F8 gene, the F9 gene, the F11 gene, the F14.5 gene, the J2 gene, the A46 gene, the E3L gene, the B2R gene, the B18R (WR200) gene, the E5R gene, the K7R gene, the C12L gene, the B8R gene, the B14R gene, the N1L gene, the K1L gene, the C16 gene, the M1L gene, the N2L gene, and the WR199 gene.

In some embodiments, the present disclosure provides an immunogenic composition comprising the MYXV virus of the present technology. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In some embodiments, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the MYXV virus of the present technology or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection. In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma.

In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXVΔM62R, MYXVΔM63R, and/or MYXVΔM64R virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of either the MYXVΔM62R, MYXVΔM63R, and/or MYXVΔM64R virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In some embodiments, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus of or the immunogenic composition. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the MYXV virus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the MYXV virus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In some embodiments, the present disclosure provides a nucleic acid encoding the MYXV virus.

In some embodiments, the virus further comprises a heterologous nucleic acid molecule encoding hIL-12. In some embodiments, the virus comprises MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-hIL-12. In some embodiments, the virus further comprises a mutant C11R gene (MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-hIL-12ΔC11R). In some embodiments, the virus further comprises a nucleic acid molecule encoding hIL-15/IL-15Rα. In some embodiments, the virus further comprises a mutant ΔE3L83N, a mutant thymidine kinase (ΔTK), a mutant B2R (ΔB2R), a mutant WR199 (ΔWR199), and a mutant WR200 (ΔSR200), and comprising a nucleic acid molecule encoding anti-CTLA-4 and a nucleic acid molecule encoding IL-12 (VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200). In some embodiments, the VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200 virus further comprises a nucleic acid molecule encoding hIL-15/IL-15Rα (VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα). In some embodiments, the VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200 virus further comprises a mutant C11R gene (ΔC11R) (VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200ΔC11R). In some embodiments, the VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200ΔC11R virus further comprises a nucleic acid molecule encoding hIL-15/IL-15Rα (VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα ΔC11R).

In some embodiments, MYXV viruses of the present technology are genetically engineered to comprise a mutant M62R gene (ΔM62R), a mutant M63R gene (ΔM63R), and a mutant M64R gene (ΔM64R) (MYXVΔM62RΔM63RΔM64R).

In one aspect, the present disclosure provides a recombinant poxvirus selected from the group consisting of: MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-CTLA-4, and MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-CTLA-4.

In some embodiments, the present disclosure provides a nucleic acid sequence encoding the recombinant poxvirus.

In some embodiments, the present disclosure provides a kit comprising the recombinant poxvirus.

In some embodiments, the present disclosure provides an immunogenic composition comprising the recombinant poxvirus. In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, theimmunogenic composition further comprises a pharmaceutically acceptable adjuvant.

In some embodiments, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor a composition comprising an effective amount of the recombinant poxvirus or the immunogenic composition. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting the growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject.

In some embodiments, the composition is administered by intratumoral or intravenous injection or a simultaneous or sequential combination of intratumoral and intravenous injection.

In some embodiments, the tumor is melanoma, colon, breast, bladder, or prostate carcinoma.

In some embodiments, the composition further comprises one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs, fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises. anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the recombinant poxvirus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the treatment of the tumor as compared to administration of the recombinant poxvirus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

In some embodiments, the present disclosure provides a method of stimulating an immune response comprising administering to a subject an effective amount of the virus or the immunogenic composition. In some embodiments, the method further comprises separately, sequentially, or simultaneously administering to the subject one or more agents selected from: one or more immune checkpoint blocking agents; one or more anti-cancer drugs; fingolimod (FTY720); and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, LAG-3, TIM3, B7-H3, B7-H4, TIGIT, AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, PDR001, and any combination thereof; and/or the one or more anti-cancer drugs is selected from the group consisting of a Mek inhibitor (U0126, selumitinib (AZD6244), PD98059, trametinib, cobimetinib), an EGFR inhibitor (lapatinib (LPN), erlotinib (ERL)), a HER2 inhibitor (lapatinib (LPN), Trastuzumab), a Raf inhibitor (sorafenib (SFN)), a BRAF inhibitor (dabrafenib, vemurafenib), an anti-OX40 antibody, a GITR agonist antibody, an anti-CSFR antibody, a CSFR inhibitor, paclitaxel, TLR9 agonist CpG, and a VEGF inhibitor (Bevacizumab), and any combination thereof. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-L1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-PD-1 antibody. In some embodiments, the one or more immune checkpoint blocking agents comprises anti-CTLA-4 antibody. In some embodiments, the combination of the recombinant poxvirus with the immune checkpoint blocking agent, anti-cancer drug, and/or fingolimod (FTY720) has a synergistic effect in the stimulation of an immune response as compared to administration of the recombinant poxvirus or of the immune checkpoint blocking agent, anti-cancer drug, or fingolimod (FTY720) alone.

FIG. 1 is a schematic diagram of homologous recombination between plasmid DNA pCB vector and MVAΔE3L viral genomic DNA at the thymidine kinase gene (TK; J2R) locus. pCB-gpt plasmid was used to insert murine OX40L gene under the control of the vaccinia synthetic early and late promoter (PsE/L) into the TK locus. In this case, drug selection marker (gpt) is under the control of the vaccinia p7.5 promoter. The expression cassette was flanked by partial sequence of TK gene flank regions (TK-L and TK-R) on each side.

FIGS. 2 A- 2 B show the verification of OX40L expression from recombinant virus MVAΔE3L-TK(−)-mOX40L. FIG. 2 A is an image of PCR amplification of mOX40L gene and TK gene in MVAΔE3L and MVAΔE3L-TK(−)-mOX40L viral genome. FIG. 2 B : Representative FACS plots showing the expression of mOX40L in B16-F10 cells infected with MVAΔE3L-TK(−)-mOX40L. Briefly, B16-F10 murine melanoma cells were infected at a MOI of 10 for 24 hours. Cells were then stained with PE-conjugated anti-mOX40L antibody.

FIGS. 3 A- 3 H are a series of graphical representations of data showing that intratumoral injection of MVAΔE3L-OX40L generated more activated tumor-infiltrating effector T cells in distant tumors compared with MVAΔE3L in B16-F10 bilateral tumor model. B16-F10 murine melanoma bilateral tumor implantation model was used. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, 2×10 7 pfu of either MVAΔE3L, MVAΔE3L-OX40L, or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice, three days apart. Tumors were harvested at 2 days post second injection and tumor infiltrating lymphocytes were analyzed by FACS. FIGS. 3 A- 3 C : Representative dot plots of Granzyme B + CD8 + T cells in none-injected tumors after treatment with either MVAΔE3L, MVAΔE3L-OX40L, or PBS. FIG. 3 D : Graph of percentages of Granzyme B + CD8 30 T cells out of CD8 + cells. Data are means ±SEM (n=3 or 4). (**P<0.01; t test). FIGS. 3 E- 3 G : Representative dot plots of Granzyme B + CD4 + T cells in non-injected tumors after treatment with MVAΔE3L, MVAΔE3L-OX40L, or PBS. FIG. 3 H : Graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=3 or 4). (**P<0.01; ***P<0.001, t test).

FIGS. 4 A and 4 B are representative ELISPOT blots and graph showing that IT injection of MVAAE3L-OX40L generated more antitumor CD8+ T cells in the spleens compared with MVA. B16-F10-bearing mice were treated with IT injection of either MVAΔE3L, MVAΔE3L-hFlt3L at 2×10 7 pfu, or PBS twice, three days apart. Spleens were collected at 2 days after second injection. ELISPOT assay was performed by co-culturing irradiated B16-F10 cells (150,000) and purified CD8 + T cells (300,000) in a 96-well plate. FIG. 4 A : Image of ELISPOT of triplicate samples from left to right. FIG. 4 B : Graph of IFN-γ + spots per 300,000 purified CD8 + T cells. Each bar represents spleen sample from individual mouse (n=3 or 4).

FIGS. 5 A and 5 B show a schematic diagram of two-step homologous recombination to generate MVAΔC7L-hFlt3L-TK(−)-muOX40L. FIG. 5 A : First step: homologous recombination between plasmid DNA pUC57 vector and MVA viral genomic DNA at the C6 and C8 gene flanking C7 locus to insert hFlt3L and GFP expression cassette into the C7 locus (replacing C7 gene). The human Flt3L gene is under the control of the vaccinia synthetic early and late promoter (PsE/L). GFP is under the control of the vaccinia P7.5 promoter. FIG. 5 B : Second step: homologous recombination between plasmid DNA pCB vector and MVAΔC7L-hFlt3L viral genomic DNA at the TK (J2R) gene locus to insert muOX40L and drug selection marker expression cassette into the TK (J2R) gene locus. The murine OX40L gene is under the control of the vaccinia synthetic early and late promoter (PsE/L). The drug selection marker gpt is under the control of the vaccinia P7.5 promoter. FIG. 5 C shows that viral genomic DNAs were analyzed by PCR to verify the expression of OX40L and hFlt3L and confirm the insertion of the transgenes.

FIG. 6 are a series of dot plots from FACS analysis demonstrating hFl3L expression in B16-F10 and SK-MEL-28 cell lines infected with either MVAΔC7L-hFl3L or MVAΔC7L-hFlt3L-TK(−)-muOX40L. Cells were infected at a MOI of 10 for 24 hours prior to antibody staining and FACS analysis. MVAΔC7L, MVAΔC7L-hFlt3L or MVAΔC7L-hFlt3L-TK(−)-muOX40L-infected cells expressed GFP marker.

FIG. 7 are a series of dot plots from FACS analysis demonstrating murine OX40L expression in B16-F10 and SK-MEL-28 cell lines infected with MVAΔC7L-hFlt3L-TK(−)-muOX40L. Cells were infected at a MOI of 10 for 24 hours prior to antibody staining and FACS analysis.

FIGS. 8 A- 8 C are a series of graphical representations of data showing that intratumoral injection of MVAΔC7L-hFlt3L-TK(−)-muOX40L generated more activated tumor-infiltrating effector CD8 + T cells in distant tumors compared with MVAΔC7L, MVAΔC7L-hFlt3L, or Heat-inactivated MVAΔC7L-hFlt3L in a B16-F10 bilateral murine melanoma model. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, intratumoral (IT) injections (2×10 7 pfu) of either MVAΔC7L-hFlt3L-TK(−)-muOX40L, MVAΔC7L, MVAΔC7L-hFlt3L, or Heat-inactivated MVAΔC7L-hFlt3L were performed to the larger tumors on the right flank twice, three days apart. The non-injected distant tumors were harvested at 2 days post second injection and tumor-infiltrating lymphocytes were analyzed by FACS. FIG. 8 A : Representative dot plots of Granzyme B + CD8 + T cells in none-injected tumors after treatment with either PBS, MVAΔC7L, MVAΔC7L-hFlt3L, MVAΔC7L-hFlt3L-muOX40L, or Heat-iMVAΔC7L-hFlt3L. FIG. 8 B : Graph of the absolute numbers of CD8 + T cells per gram of distant non-injected tumors. Data are means ±SEM (n=4 or 5). (*P<0.05; **P<0.01; t test). FIG. 8 C : Graph of the absolute numbers of Granzyme B + CD8 + T cells per gram of distant non-injected tumors. Data are means ±SEM (n=4 or 5). (*P<0.05; ** P<0.01; t test).

FIGS. 9 A- 9 C are a series of graphical representations of data showing that intratumoral injection of MVAΔC7L-hFlt3L-TK(−)-muOX40L generated more activated tumor-infiltrating effector CD4 + T cells in distant tumors compared with MVAΔC7L, MVAΔC7L-hFlt3L, or Heat-inactivated MVAΔC7L-hFlt3L in a B16-F10 bilateral murine melanoma model. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice ( 5 × 10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, intratumoral (IT) injections (2×10 7 pfu) of either MVAΔC7L-hFlt3L-TK(−)-muOX40L, MVAΔC7L, MVAΔC7L-hFlt3L, or Heat-inactivated MVAΔC7L-hFlt3L were performed to the larger tumors on the right flank twice, three days apart. The distant non-injected tumors were harvested at 2 days post second injection and tumor-infiltrating lymphocytes were analyzed by FACS. FIG. 9 A : Representative dot plots of Granzyme B + CD4 + T cells in none-injected tumors after treatment with either PBS, MVAΔC7L, MVAΔC7L-hFlt3L, MVAΔC7L-hFlt3L-muOX40L, or Heat-iMVAΔC7L-hFlt3L. FIG. 9 B : Graph of the absolute numbers of CD4 + T cells per gram of distant non-injected tumors. Data are means ±SEM (n=4 or 5). (*P<0.05; **P<0.01; t test). FIG. 9 C : Graph of the absolute numbers of Granzyme B + CD4 + T cells per gram of distant non-injected tumors. Data are means ±SEM (n=4 or 5). (*P<0.05; **P<0.01; t test).

FIGS. 10 A and 10 B are representative ELISPOT blots and graph showing that IT injection of MVAΔC7L-hFlt3L-TK(−)-muOX40L generated stronger antitumor CD8 + T cell responses in the spleens compared with MVAΔC7L, MVAΔC7L-hFlt3L, or Heat-inactivated MVAΔC7L-hFlt3L. B16-F10-bearing mice were treated with IT injection of either MVAΔC7L-hFlt3L-TK(−)-muOX40L, MVAΔC7L, MVAΔC7L-hFlt3L at 2×10 7 pfu, or Heat-inactivated MVAΔC7L-hFlt3L twice, three days apart. Spleens were collected at 2 days after second injection. ELISPOT assay was performed by co-culturing irradiated B16-F10 cells (150,000) and purified CD8 + T cells (300,000) in a 96-well plate. FIG. 10 A : Image of ELISPOT of triplicate samples from left to right. FIG. 10 B : Graph of IFN-γ + spots per 300,000 purified CD8 + T cells. Each bar represents spleen sample from individual mouse (n=5).

FIGS. 11 A- 11 G are graphical representations of data showing the combination of IT MVAΔC7L-hFlt3L-TK(−)-muOX40L and systemic delivery immune checkpoint blockade antibody anti-CTLA-4 or anti-PD-L1 delays tumor growth and prolongs survival in murine B16-F10 melanoma bilateral tumor implantation model. FIG. 11 A is a scheme of tumor implantation and treatment for a B16-F10 bilateral tumor implantation model. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 1×10 5 to the left flank). Nine days post tumor implantation, intratumoral injections (2×10 7 pfu) of MVAΔC7L-hFlt3L-TK(−) mOX40L were performed twice weekly to the larger tumors on the right flank. Anti-CTLA-4 or anti-PD-L1 antibody at 250 μg per mouse was given intraperitoneally. The tumor sizes were measured and the survival of mice was monitored. FIGS. 11 B and 11 C are graphical representations of data showing volumes of injected ( FIG. 11 B ) and non-injected ( FIG. 11 C ) tumors over days after PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-CTLA-4 antibody, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. FIGS. 11 D and 11 E are graphical representations of data showing initial volumes of injected ( FIG. 11 D ) and non-injected ( FIG. 11 E ) tumors and at Day 7 and Day 11 post PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-CTLA-4 antibody, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. FIG. 11 F is a graph of the Kaplan-Meier survival curve of tumor-bearing mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-CTLA-4 antibody, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. (n=10, **P<0.01; ***P<0.001; Mantel-Cox test). FIG. 11 G is a table showing median survival of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-CTLA-4 antibody, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody.

FIG. 12 are a series of graphical representations of data showing tumor growth curves in mice treated in a B16-F10 unilateral large tumor model. Briefly, B16-F10 melanoma cells were implanted intradermally to the right flank of C57B/6J mice (5×10 5 cells). Eleven days post implantation viruses were injected intratumorally twice per week, and Anti-CTLA-4 or anti-PD-L1 antibodies were injected twice per week intraperitoneally.

FIGS. 13 A and 13 B are schematic diagrams of two-step homologous recombination to generate MVAΔC7L-hFlt3L-TK(−)-huOX40L. FIG. 13 A : First step: homologous recombination between plasmid DNA pUC57 vector and MVA viral genomic DNA at the C6 and C8 gene flanking C7 locus to insert hFlt3L and GFP expression cassette into the C7 locus (replacing C7 gene). The human Flt3L gene is under the control of the vaccinia synthetic early and late promoter (PsE/L). GFP is under the control of the vaccinia P7.5 promoter. FIG. 13 B : Second step: homologous recombination between plasmid DNA pUC57ΔTK-hOX40L-mCherry and MVAΔC7L-hFlt3L viral genomic DNA at the J1R and J3R (TK-R and TK-L) loci flanking J2R (TK) gene to insert huOX40L and mCherry expression cassette into the TK (J2R) gene locus. The human OX40L gene is under the control of the vaccinia synthetic early and late promoter (PsE/L). mCherry is under the control of the vaccinia P7.5 promoter. FIG. 13 C : PCR verification of three independent clones of recombinant MVAΔC7L-hFlt3L-TK(−)-huOX40L, which contains hOX40L gene and hFlt3L gene insert but lacks TK (J2R) gene. H1, h2 and H3 are individual recombinant MVA. “+”: positive control for the PCR reaction.

FIGS. 14 A and 14 B are two graphs showing a multi-step growth of the parental MVA and recombinant viruses, including MVAΔC7L-hFlt3L, MVAΔC7L-hFlt3L-TK(−)-muOX40L, MVAΔC7L-hFlt3L-TK(−)-hOX40L in primary chicken embryo fibroblasts (CEFs). FIG. 14 A is a multi-step growth curve of these viruses in CEFs. Briefly, CEFs were infected with the above-mentioned viruses at a MOI of 0.05. Cells were collected at 1, 24, 48 and 72 h. Viral titers were determined on BHK21 cells by serial dilution and counting GFP* foci under confocal microscope. FIG. 14 B show the log (fold change) of viral titers at 72 h post infection over 1 h post infection.

FIGS. 15 A and 15 B are a series of representative dot plots of FACS data showing the expression of hOX40L in BHK21 cells and human monocyte-derived dendritic cells (mo-DCs) infected by MVAΔC7L-hFlt3L-TK(−)-hOX40L virus. FIG. 15 A : BHK21 cells were either mock-infected, or infected with MVAΔC7L-hFlt3L or with MVAΔC7L-hFlt3L-TK(−)-hOX40L at a MOI of 10. Cells were collected at 24 h post infection and stained with PE-conjugated anti-hOX40L antibody prior to FACS analyses. FIG. 15 B : human moDCs were either treated with poly I:C at 10 μg/ml, or infected with Heat-iMVA, MVAΔC7L-TK(−), or MVAΔC7L-hFlt3L-TK(−)-hOX40L at MOI of 1. At 24 h post infection, cells were collected and stained with PE-conjugated anti-hOX40L antibody prior to FACS analyses. Untreated murine B16-F10 melanoma cells were used as a negative control.

FIGS. 16 A and 16 B are a series of graphical representations of data showing hOX40L mRNA levels in MVAΔC7L-hFlt3L-TK(−)-hOX40L-infected BHK21 ( FIG. 16 A ) and B16-F10 cells ( FIG. 16 B ). BHK21 or B16-F10 cells were infected with either MVA or MVAΔC7L-hFlt3L-TK(−)-hOX40L at a MOI of 10. At 8 and 16 h post infection, cells were collected and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of viral E3L gene and hOX40L gene.

FIG. 17 is a schematic diagram showing the workflow of constructing vaccinia virus viral early gene expression plasmids. 72 viral early genes were selected. PCR was performed to amplify the gene of interest from vaccinia viral genome. Adaptors were added to both ends of PCR products by a second round of PCR. Then the DNA fragments were cloned into pDONR™/ZEO, and then to pcDNA™3.2-DEST, a mammalian expression vector. The DNA constructs were later verified by sequencing. The plasmid DNAs were then used to transfect into HEK-293T cells, along with other plasmids, which will be described below.

FIG. 18 shows dual luciferase screening strategy. In HEK293T cells, cGAS and STING expression plasmids were co-transfected with IFN-β luciferase plasmid and pRL-TK. Viral gene expression plasmids or vector were transfected together. After 24 h, luciferase signal was measured. The relative luciferase activity was expressed as arbitrary units by normalizing firefly luciferase activity to Renilla luciferase activity.

FIGS. 19 A- 19 C show the dual luciferase screening results of vaccinia virus ORFs that inhibit cGAS/STING-dependent IFNβ-luc activity. FIGS. 19 A- 19 C : HEK293T cells were transfected with plasmids expressing IFNβ-luc reporter, murine cGAS, human STING and vaccinia virus ORFs as indicated. Dual luciferase assays were performed 24 h after transfection. Adenovirus E1A gene was used as a positive control.

FIGS. 20 A- 20 E . Vaccinia virus B18, E5, K7, C11 and B14 inhibits cGAS/STING-induced IFNβ promoter activity. HEK293T cells were transfected with IFNB luciferase reporter, cGAS, STING and expression plasmids as indicated, and luciferase activity was assayed 24 h after transfection. FIG. 20 A : Mouse cGAS was co-transfected with expression plasmids. FIG. 20 B : Human cGAS was co-transfected with expression plasmids. FIG. 20 C : Mouse cGAS was co-transfected with FLAG-tagged vaccinia ORFs of K7R, E5R, B14R, C11R, and B18R. FIGS. 20 D and 20 E are charts showing the induction of IFNB in cells over-expressing E5, B14, K7, or B18 by Heat-iMVA infection ( FIG. 20 D ) or ISD treatment ( FIG. 20 E ).

FIG. 21 shows additional dual luciferase screening results of vaccinia virus ORFs that inhibit cGAS/STING-dependent IFNβ-luc activity. HEK293T cells were transfected with plasmids expressing IFNβ-luc reporter, murine cGAS, human STING and vaccinia virus ORFs as indicated. Dual luciferase assays were performed 24 h after transfection. Adenovirus E1A gene was used as a positive control.

FIG. 22 shows MVA genome sequence as set forth in SEQ ID NO: 1, and given by GenBank Accession No. U94848.1.

FIG. 23 shows the vaccinia virus (Western Reserve strain; WR) genome sequence as set forth in SEQ ID NO: 2, and given by GenBank Accession No. AY243312.1.

FIG. 24 A and 24 B are two graphs showing a multi-step growth of the vaccinia and the recombinant viruses, including E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4, E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -mOX40L, and VAC-TK − -anti-muCTLA-4/C7L − -mOX40L in murine B16-F10 melanoma cells. FIG. 24 A is a multi-step growth of these viruses in B16-F10 cells. Briefly, B16-F10 cells were infected with the above-mentioned viruses at a MOI of 0.1. Cells were collected at 1, 24, 48, and 72 h post infection and viral yields (log pfu) were determined by titrating on BSC40 cells. Viral yields were plotted against hours post infection. FIG. 24 B shows the log (fold change) of viral titers at 72 h post infection over 1 h post infection.

FIG. 25 shows a Western blot analysis of anti-mCTLA-4 antibody, murine OX40L, and human Flt3L expression in E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4 or E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -mOX40L virus-infected murine B16-F10 melanoma cells. B16-F10 cells were infected or mock infected with E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4 or E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -mOX40L viruses at a MOI of 10. Cell lysates were collected at 7, 24 and 48 h post infection, and the polypeptides in cell lysates were separated using 10% SDS-PAGE. HRP-conjugated anti-mouse IgG (heavy and light chain), anti-mOX40L antibody, and anti-human Flt3L antibody was used to detect the anti-mCTLA-4 antibody, murine OX40L, and human Flt3L protein respectively.

FIG. 26 shows the surface expression of murine OX40L protein in E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -mOX40L or VAC-TK − -anti-mCTLA-4/C7L − -mOX40L virus infected murine B16-F10 melanoma cells. Briefly, B16-F10 cells were infected or mock infected with E3LΔ83N-TK − -vector, E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -vector, E3LΔ83N-TK − -hFlt3L-anti-muCTLA-4/C7L − -mOX40L, or VAC-TK − -anti-mCTLA-4/C7L − -mOX40L viruses at a MOI of 5. Cells were collected at 24 h post infection, and stained with PE-conjugated anti-mOX40L antibody, and analyzed by FACS. Data were analyzed with FlowJo software (FlowJo, Becton-Dickinson, Franklin Lakes, NJ).

FIG. 27 shows a scheme of tumor implantation and treatment for a B16-F10 murine melanoma unilateral tumor implantation model. Briefly, 5×10 5 B16-F10 melanoma cells were implanted intradermally to the right flank of C57B/6J mice. Nine days post tumor implantation, 4×10 7 pfu of MVAΔC7L-hFlt3L-TK(−)mOX40L were intratumorally injected twice weekly. Anti-PD-L1 antibody at 250 μg per mouse was given intraperitoneally. The tumor sizes were measured and the survival of mice was monitored.

FIGS. 28 A- 28 C are graphical representations of data showing volumes of tumors over days after PBS ( FIG. 28 A ), MVAΔC7L-hFlt3L-TK(−)-mOX40L ( FIG. 28 B ), or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody ( FIG. 28 C ) treatments.

FIGS. 29 A and 29 B demonstrate survival studies of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody. FIG. 29 A is a graph of the Kaplan-Meier survival curve of tumor-bearing mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. (n=5˜10, **P<0.01; ***P<0.001; Mantel-Cox test). FIG. 29 B is a table showing median survival of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody.

FIG. 30 shows a scheme of tumor implantation and treatment for a MC38 unilateral tumor implantation model. Briefly, 5×10 5 MC38 melanoma cells were implanted intradermally to the right flank of C57B/6J mice. Nine days post tumor implantation, 4×10 7 pfu of MVAΔC7L-hFlt3L-TK(−) mOX40L were intratumorally injected twice weekly. Anti-PD-1 antibody at 250 μg per mouse was given intraperitoneally. The tumor sizes were measured and the survival of mice was monitored.

FIGS. 31 A- 31 C are graphical representations of data showing volumes of tumors over days after PBS ( FIG. 31 A ), MVAΔC7L-hFlt3L-TK(−)-mOX40L ( FIG. 31 B ), or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody ( FIG. 31 C ) treatments.

FIGS. 32 A and 32 B demonstrate survival studies of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody. FIG. 32 A is a graph of the Kaplan-Meier survival curve of tumor-bearing mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAAC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. (n=4˜8, **P<0.01; ***P<0.001; Mantel-Cox test). FIG. 32 B is a table showing median survival of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody.

FIG. 33 shows a scheme of tumor implantation and treatment for a MB49 unilateral tumor implantation model. Briefly, 2.5×10 5 MB49 melanoma cells were implanted intradermally to the right flank of C57B/6J mice. Eight days post tumor implantation, 4×10 7 pfu of MVAΔC7L-hFlt3L-TK(−)mOX40L were intratumorally injected twice weekly. Anti-PD-L1 antibody at 250 μg per mouse was given intraperitoneally. The tumor sizes were measured and the survival of mice was monitored.

FIGS. 34 A- 34 D are graphical representations of data showing volumes of tumors over days after PBS ( FIG. 34 A ), MVAΔC7L-hFlt3L-TK(−)-mOX40L ( FIG. 34 B ), MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody ( FIG. 34 V ), or anti-PD-L1 antibody ( FIG. 34 D ) treatments.

FIGS. 35 A and 35 B demonstrate survival studies of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody, or anti-PD-L1 antibody treatments. FIG. 35 A is a graph of the Kaplan-Meier survival curve of tumor-bearing mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody, or anti-PD-L1 antibody treatments. (n=10, *P<0.05, **P<0.01; ***P<0.001; Mantel-Cox test). FIG. 35 B is a table showing median survival of mice treated with either PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody, or anti-PD-L1 antibody.

FIG. 36 shows a representative graph of tumors isolated from a female MMTV-PyVmT mouse. Briefly, the mice were treated with IT injection of PBS, 4×10 7 pfu of MVAΔC7L-hFlt3L-TK(−)-mOX40L twice weekly after developing palpable mammary tumors with a mean latency of 92 days of age. Anti-PD-L1 antibody at 250 μg per mouse was given intraperitoneally twice weekly. The tumor sizes were measured and the survival of mice was monitored.

FIG. 37 are graphical representations of data showing volumes of tumors over days after PBS, MVAΔC7L-hFlt3L-TK(−)-mOX40L, or MVAΔC7L-hFlt3L-TK(−)-mOX40L plus anti-PD-L1 antibody treatments. (P0-PBS; V1-MVAΔC7L-hFlt3L-muOX40L; C1, C2,C3-MVAΔC7L-hFlt3L-muOX40L+anti-PD-L1).

FIG. 38 shows representative dot plots of CD8 + and CD4 + T cells in injected and non-injected tumors after treatment with either PBS, MVAΔC7L-hFlt3L-TK(−)-muOX40L, or MVAΔC7L-hFlt3L-TK(−)-muOX40L plus anti-PD-L1 antibody.

FIG. 39 shows representative dot plots of CD8 + CD69 + CD103 + T cells in injected and non-injected tumors after treatment with either PBS, MVAΔC7L-hFlt3L-TK(−)-muOX40L, or MVAΔC7L-hFlt3L-TK(−)-muOX40L plus anti-PD-L1 antibody.

FIG. 40 shows representative dot plots of CD4 − CD69 − CD103 + T cells in injected and non-injected tumors after treatment with either PBS, MVAΔC7L-hFlt3L-TK(−)-muOX40L, or MVAΔC7L-hFlt3L-TK(−)-muOX40L plus anti-PD-L1 antibody.

FIGS. 41 A and 41 B are representative FACS plots showing the expression of hFlt3L ( FIG. 41 A ) or mOX40L ( FIG. 41 B ) by B16-F10-hFlt3L or B16-F10-mOX40L stable cell lines.

FIG. 42 is a scheme of tumor implantation and treatment for a B16-F10 bilateral tumor implantation model. Briefly, 5×10 5 B16-F10 melanoma cells were implanted intradermally to right flanks of C57B/6J mice and 5×10 5 B16-F10-hFlt3L melanoma cells were implanted intradermally to left flanks of C57B/6J mice. Nine days post tumor implantation, PBS or 4×10 7 pfu of MVAΔC7L were intratumorally injected twice weekly to the tumors on both flanks. Tumors were harvested 2 days post second injection and tumor infiltrating lymphocytes (TILs) were analyzed by FACS.

FIGS. 43 A- 43 D are graphical representations of data showing volumes of tumors in either the right of left flanks of C57B/6J mice over days after PBS or MVAΔC7L treatments.

FIGS. 44 A- 44 E are graphs of the percentage of tumor infiltrating CD8 + ( FIG. 44 A ), CD8 + GranzymeB + ( FIG. 44 B ), CD4 + ( FIG. 44 C ), CD4 + GranzymeB + ( FIG. 44 D ), and CD4 + FoxP3 + ( FIG. 44 E ) T cells after PBS or MVAΔC7L treatments. (n=5, *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001; One-way ANOVA).

FIGS. 45 A- 45 E are graphs of the absolute numbers of tumor infiltrating CD8 + ( FIG. 45 A ), CD8 + GranzymeB + ( FIG. 45 B ), CD4 + ( FIG. 45 C ), CD4 + GranzymeB + ( FIG. 45 D ), CD4 + FoxP3 + ( FIG. 45 E ) T cells per gram of tumors after PBS, MVAΔC7L treatments. (n=5, *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001; One-way ANOVA).

FIG. 46 is a scheme of tumor implantation and treatment for a B16-F10 bilateral tumor implantation model. Briefly, 5×10 5 B16-F10 melanoma cells were implanted intradermally to right flanks of C57B/6J mice and 5×10 5 B16-F10-OX40L melanoma cells were implanted intradermally to left flanks of C57B/6J mice. Nine days post tumor implantation, PBS or 4×10 7 pfu of MVAΔC7L were intratumorally injected twice weekly to the tumors on both flanks. Tumors were harvested 2 days post second injection and tumor infiltrating lymphocytes (TILs) were analyzed by FACS.

FIGS. 47 A- 47 D are graphical representations of data showing volumes of tumors in either the right of left flanks of C57B/6J mice over days after PBS or MVAΔC7L treatments.

FIGS. 48 A- 48 E are graphs of the percentage of tumor infiltrating CD8 + ( FIG. 48 A ), CD8 + GranzymeB + ( FIG. 48 B ), CD4 + ( FIG. 48 C ), CD4 + GranzymeB + ( FIG. 48 D ), CD4 + FoxP3 + ( FIG. 48 E ) T cells after PBS, MVAΔC7L C7Ltreatments. (n=5, *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001; One-way ANOVA).

FIGS. 49 A- 49 E are graphs of the absolute numbers of tumor infiltrating CD8 + ( FIG. 49 A ), CD8 + GranzymeB + ( FIG. 49 B ), CD4 + ( FIG. 49 C ), CD4 30 GranzymeB + ( FIG. 49 D ), CD4 + FoxP3 + ( FIG. 49 E ) T cells per gram of tumors after PBS, MVAΔC7L C7Ltreatments. (n=5, *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001; One-way ANOVA).

FIGS. 50 A and 50 B show the mechanism of action of FTY720 and its chemical structure. FIG. 50 A is adapted from a drawing in Gong et al. Front. Immunol. (2014), Naïve T cells circulate between lymphoid organs and blood. Upon infection or tumor implantation, antigen-presenting cells present antigen to prime cognate T cells, which then proliferate and differentiate into effector T cells and memory T cells. Effector T cells are recruited to the site of infection or tumors and memory T cells recirculate. FTY720 ( FIG. 50 B ), a sphingosine-1-phosphate receptor modulator, blocks the exit of lymphocytes from lymphoid organs.

FIG. 51 is a scheme of tumor implantation and treatment for a B16-F10 unilateral tumor implantation model. Briefly, 5×10 5 B16-F10 melanoma cells were implanted intradermally to right flanks of C57B/6J mice. Nine days post tumor implantation, PBS or 4×10 7 pfu of MVAΔC7L-hFlt3L-TK(−)-muOX40L were intratumorally injected twice. FTY720 at 25 μg per mouse was given intraperitoneally daily during the treatment, starting 1 day prior to the first MVAΔC7L-hFlt3L-TK(−)-muOX40L injection. The tumor sizes were measured and the survival of mice was monitored.

FIGS. 52 A- 52 D are graphical representations of data showing volumes of tumors in C57B/6J mice over days after PBS or MVAΔC7L C7Ltreatments with or without FTY720 treatment.

FIGS. 53 A and 53 B are graphical representations of data showing volumes of tumors in C57B/6J mice over days after PBS or MVAΔC7L C7Ltreatments with or without FTY720 treatment.

FIGS. 53 C and 53 D are graphs of the Kaplan-Meier survival curve of tumor-bearing mice treated with PBS or MVAΔC7L-hFlt3L-TK(−)-mOX40LC7LhFl3LmOX40Lwith or without FTY720 (n=5˜10, **P<0.01; ***P<0.001; Mantel-Cox test).

FIGS. 54 A and 54 B show domain organization and sequence conservation of vaccinia E5 amongst the poxvirus family. FIG. 54 A is a schematic diagram of vaccinia E5. E5 is 328-aa protein, which is comprised of a N-terminal domain followed by two BEN domains. BEN is named after its presence in BANP/SMAR1, poxvirus E5R, and NAC1. BEN domain containing proteins are involved in chromatin organization, transcription regulation, and possibly viral DNA organization. FIG. 54 B is a schematic diagram that demonstrates that vaccinia E5 is highly conserved in the poxvirus family.

FIGS. 55 A- 55 C show that VACVAE5R is highly attenuated in an intranasal infection model. FIG. 55 A shows a scheme for generating VACVΔE5R virus through homologous recombination at the E4L and E6R loci flanking ER gene of the vaccinia genome. FIG. 55 B shows weight loss over days after intranasal infection with either WT VACV (2×10 6 pfu), VACVΔE5R (2×10 6 pfu), or VACVΔE5R (2×10 7 pfu). FIG. 55 C shows Kaplan-Meier survival curves of mice infected with WT VACV (2×10 6 pfu) or VACVΔE5R (2×10 6 pfu or 2×10 7 pfu).

FIGS. 56 A and 56 B demonstrates that infection with VACVΔE5R of BMDCs induce IFNB gene expression and IFN-β protein secretion. FIG. 56 A shows RT-PCR results of BMDCs that were infected MVA, VACV, or VACVΔE5R at a MOI of 10. Cells were collected at 8 h post infection. RNAs were extracted and RT-PCRs were performed. FIG. 56 B shows that BMDCs were infected MVA, VACV, or VACVΔE5R at a MOI of 10. Supernatants were collected at 21 h post infection. IFN-ß protein levels were determined by ELISA.

FIGS. 57 A- 57 D show IFNB gene induction by MVAΔE5R and MVAΔK7R in BMDCs and BMDMs. FIG. 57 A shows a scheme for generating MVAΔE5R virus through homologous recombination at the E4L and E6R loci flanking E5R gene of the MVA genome. FIG. 57 B shows a scheme for generating MVAΔK7R virus through homologous recombination at the K5,6L and FIL loci flanking K7R gene of the MVA genome. FIG. 57 C show that BMDCs were infected with either MVA, MVAΔE5R, or MVAΔK7R at MOI of 10. Cells were collected at 6 h post infection. IFNB gene expression was measured by RT-PCR. FIG. 57 D shows that BMDMs were infected with either MVA, MVAΔE5R, or MVAΔK7R at MOI of 10. Cells were collected at 6 h post infection. IFNB gene expression was measured by RT-PCR.

FIGS. 58 A- 58 C show that BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 6 h post infection. IFNA ( FIG. 58 A ), CCL4 ( FIG. 58 B ), and CCL5 ( FIG. 58 C ) gene expressions were determined by quantitative RT-PCR.

FIGS. 59 A- 59 C show that MVAΔE5R infection of BMDCs induce high levels of IFNB and viral E3R gene expression and IFN-β protein secretion from BMDCs. FIG. 59 A and 59 B show real-time quantitative PCR (RT-PCR) analyses of IFNB ( FIG. 59 A ) and viral E3R ( FIG. 59 B ) gene expression induced by MVAΔE5R or Heat-inactivated MVAΔE5R (“Heat-iMVAΔE5R”). BMDCs were generated by culturing bone marrow cells in the presence of mGM-CSF. Cells were infected with either MVAΔE5R or Heat-iMVAΔE5R virus at MOIs of 0.25, 1, 3, or 10. Cells were washed after 1 h infection and fresh medium was added. Cells were collected at 14 h post infection. IFNB and E3 gene expressions were determined by RT-PCR. FIG. 59 C BMDCs were infected with either MVAΔE5R or Heat-iMVAΔE5R virus at MOIs of 0.25, 1, 3, or 10. Supernatants were collected at 14 h post infection. The concentrations of IFN-β in the supernatants were measured by ELISA.

FIGS. 60 A- 60 D show that MVAΔE5R-induced IFNB gene expression and IFN-β secretion was dependent on cGAS. FIG. 60 A shows IFNA induction by MVAΔE5R in WT and cGAS −/− BMDCs. FIG. 60 B shows IFNA induction by MVAΔE5R in WT and cGAS −/− BMDCs. FIG. 60 C shows vaccinia E3R gene expression in WT and cGAS −/− BMDCs infected with MVAΔE5R. BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 6 h post infection. RNAs were extracted. Real-time quantitative PCR analysis was performed. FIG. 60 D shows MVAΔE5R induces higher levels of IFN-β protein secretion compared with Heat-iMVA or Heat-iMVAΔE5R, and the induction is completely dependent on cGAS. WT or cGAS −/− BMDCs were infected with either MVA, MVAΔE5R at a MOI of 10, Heat-iMVA, or Heat-iMVAΔE5R at an equivalent of MOI. Supernatants were collected at 8 and 16 h post infection. IFN-protein levels in the supernatants were measured by ELISA.

FIGS. 61 A and 61 B show MVAΔE5R-induced IFNB gene expression and protein secretion from BMDCs is dependent on STING. FIG. 61 A show BMDCs from WT or STING Gt/Gt mice were infected with either MVA, MVAΔE5R, or Heat-iMVAΔE5R. Cells were collected at 8 h post infection and RT-PCR analysis was performed. Fold induction of IFNB gene expression is shown. FIG. 61 B shows bone marrow derived macrophages (BMDMs) were generated by culturing bone marrow cells from WT and STING Gt/Gt mice in the presence of M-CSF (macrophage colony stimulating factor). BMDMs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R. Supernatants were collected at 16 h post infection and IFN-β protein levels were measured by ELISA.

FIGS. 62 A- 62 D show that MVAΔE5R-induced IFN-β protein secretion requires IRF3,IRF7 and IFNAR. FIG. 62 A shows that WT or IRF3 −/− BMDCs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R. Supernatants were collected at 8 and 16 h post infection. IFN-β levels were determined by ELISA. FIG. 62 B shows that WT or IRF3 −/− BMDMs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R. Supernatants were collected at 8 and 16 h post infection. IFN-β levels were determined by ELISA. FIG. 62 C shows that WT or IRF7 −/− BMDCs were infected with MVAΔE5R at a MOI of 10. Supernatants were collected at 21 h post infection. IFN-β levels in the supernatants were determined by ELISA. FIG. 62 D shows that WT, cGAS −/− , or IFNAR −/− BMDCs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-IMVAΔE5R. Supernatants were collected at 16 h post infection. IFN-β levels were determined by ELISA.

FIGS. 63 A- 63 C demonstrate that WT VACV-induced cGAS degradation is mediated through a proteasome-dependent pathway. FIG. 63 A shows that murine embryonic fibroblasts were pretreated with either cycloheximide (CHX), a proteasomal inhibitor, MG132, a pan-caspase inhibitor, Z-VAD, or an AKT1/2 inhibitor VIII for 30 min. MEFs were then infected with WT VACV in the presence of each drug. Cells were collected at 6 h post infection. Western blot analysis was performed with anti-cGAS and anti-GAPDH antibodies. FIG. 63 B shows that MEFs were treated with DMSO or MG132. Cells were collected at 2, 4, and 6 h post treatment. Western blot analysis was performed with anti-cGAS and anti-GAPDH antibodies. FIG. 63 C demonstrates that WT VACV infection of BMDCs resulted in cGAS degradation, whereas VACVΔE5R did not. In the presence of MG132, WT VACV-induced cGAS degradation was blocked. BMDCs were infected with WT VACV or VACV at MOI of 10 in the presence or absence of MG132. Cells were collected at 2, 4, and 6 h post infection. Western blot analyses were performed using anti-cGAS and anti-GAPDH antibodies.

FIG. 64 demonstrates that the E5R gene in MVA is important in mediating cGAS degradation in BMDCs. BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 2, 4, 6, 8, and 12 h post infection. Western blot analysis was performed using anti-cGAS and anti-GAPDH antibodies.

FIG. 65 demonstrates that MVAΔE5R induces higher levels of phosphorylated Stat2 compared with MVA. BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 2, 4, 6, 8, and 12 h post infection. Western blot analysis was performed using anti-phospho-STAT2, anti-STAT2, and anti-GAPDH antibodies.

FIG. 66 shows that MVAΔE5R induces high levels of cGAMP production in infected BMDCs. 2.5×10 6 BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 2, 4, 6 and 8 h post infection. cGAMP concentrations were measured by incubating cell lysates with permeabilized differentiated THP1-Dual™ cells, which were derived from the human THP-1 monocyte cell line by stable integration of two inducible reporter constructs. Supernatants were collected at 24 h, and luciferase activities (as an indication for IRF pathway activation) were measured. cGAMP levels were calculated by comparing with cGAMP standards.

FIGS. 67 A and 67 B show that MVAΔE5R induces IFN-β protein secretion from plasmacytoid dendritic cells. FIG. 67 A shows 1.2×10 5 pDCs (B220 + PDCA-1 + ) sorted from splenocytes were infected with either MVA, Heat-iMVA, or MVAΔE5R. Non-infected splenocytes were included as a control. Supernatants were collected at 18 h post infection. IFN-β levels in the supernatants were measured by ELISA. FIG. 67 B shows 4×10 5 pDCs (B220 + PDCA-1 + ) sorted from Flt3L-BMDCs were infected with either MVA, Heat-iMVA, or MVAΔE5R. Non-infected sorted pDCs were included as a control. Supernatants were collected at 18 h post infection. IFN-levels in the supernatants were measured by ELISA.

FIG. 68 shows that MVAΔE5R-induced IFN-Δ secretion from pDCs is dependent on cGAS. pDCs were sorted from Flt3L-cultured BMDCs (B220 + PDCA-1 + ) obtained from WT, cGAS −/− , or MyD88 −/− -mice. 2×10 5 cells were infected with either MVA or MVAΔE5R. NT control was included. Supernatants were collected at 18 h post infection. IFN-β levels in the supernatants were measured by ELISA.

FIG. 69 shows that MVAΔE5R infection induces IFN-β protein secretion from CD103 + DCs through a cGAS-dependent pathway. CD103 + DCs were sorted from Flt3L-cultured BMDCs (CD11c + CD103 + ) obtained from WT, cGAS −/− , or MyD88 −/− mice. 2+10 5 cells were infected with either MVA or MVAΔE5R. NT control was included. Supernatants were collected at 18 h post infection. IFN-β levels in the supernatants were measured by ELISA.

FIG. 70 shows that MVAΔE5R infection of BMDCs results in lower levels of cell death compared with MVA. BMDCs were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were harvested at 16 h post infection and stained with LIVE/DEAD fixable viability dye and subjected for flow cytometry analysis.

FIGS. 71 A and 71 B show that MVAΔE5R infection promotes DC maturation in a cGAS-dependent manner. BMDCs from WT and cGAS −/− mice were infected with MVA-OVA or MVAΔE5R-OVA at MOI of 10. Cells were collected at 16 h post infection and stained for DC maturation markers: CD40 ( FIG. 71 A ) and CD86 ( FIG. 71 B ).

FIGS. 72 A- 72 G shows that MVAΔE5R infection of BMDCs promotes antigen cross-presentation as measured by T cell activation. BMDCs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R at MOI of 3 for 3 h and then incubated with OVA for 3 h. OVA was washed away and cells were then incubated with OT-I cells (which recognizes OVA 257-264 SIINFEKL peptide) for 3 days. OT-1 cells were stained with anti-CD69 and anti-CD8 antibodies and analyzed by flow cytometry. Supernatants were collected and IFN-γ levels were determined by ELISA. Dot plots demonstrate CD8 + cells expressing CD69.

FIG. 73 shows that MVAΔE5R infection of BMDCs promotes antigen cross-presentation as measured by IFN-γ production by activated T cells. BMDCs were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R at MOI of 3 for 3 h and then incubated with OVA for 3 h. OVA was washed away and cells were then incubated with OT-I cells (which recognizes OVA 257-264 SIINFEKL peptide) for 3 days. OT-1 cells were stained with anti-CD69 and anti-CD8 antibodies and analyzed by flow cytometry. Supernatants were collected and IFN-γ levels were determined by ELISA. FIG. 73 shows IFN-γ levels in the supernatants of the BMDC: T cells co-culture.

FIG. 74 shows that VACVΔE5R infection of BMDCs promotes antigen cross-presentation as measured by IFN-γ production by activated T cells. BMDCs were infected with either MVA, MVAΔE5R, or VACVΔE5R at MOI of 3 for 3 h; or BMDCs were incubated with cGAMP or mock control for 3 h. BMDCs were subsequently incubated with OVA for 3 h and then the OVA was washed away. Cells were then incubated with OT-I cells (which recognizes OVA257-264 SIINFEKL peptide) for 3 days. Supernatants were collected and IFN-γ levels were determined by ELISA.

FIGS. 75 A- 75 C show that deletion of the E5R gene from MVA improves vaccination efficacy. FIG. 75 A is a scheme of vaccination strategy. On day 0, C57BL/6J mice were vaccinated with MVA-OVA or MVAΔE5R-OVA at 2×10 7 pfu either through skin scarification or intradermal injection. Spleens were harvested from euthanized mice one week later and co-cultured with OVA257-264 (SIINFEKL) peptide (10 μg/ml) pulsed BMDCs for 12 h. The intracellular IFN-γ levels in CD8 + T cells was then measured by flow cytometry. (* p<0.05; ** p<0.01). FIG. 75 B shows activated CD8 + T cells after vaccination through skin scarification with MVA-OVA or MVAΔE5R-OVA. FIG. 75 C shows activated CD8 + T cells after vaccination through intradermal injection of MVA-OVA or MVAΔE5R-OVA.

FIGS. 76 A and 76 B show that MVAΔE5R infection induces IFNB gene expression and IFN-β secretion from murine primary fibroblasts in a cGAS-dependent manner. Skin dermal fibroblasts were generated from female WT and cGAS −/− C57BL/6J mice. Cells were infected with either MVA, MVAΔE5R, Heat-iMVA, or Heat-iMVAΔE5R. Cells and supernatants were collected at 16 h post infection. FIG. 76 A shows RT-PCR results of IFNB gene expression in WT and cGAS −/− cells. FIG. 76 B shows IFN-β protein levels in the supernatants of infected WT and cGAS −/− cells as measured by ELISA.

FIGS. 77 A- 77 D show that MVAΔE5R gains its capacity to replicate its DNA in cGAS- or IFNAR1-deficient skin primary dermal fibroblasts. Skin primary dermal fibroblasts from WT, cGAS −/− or IFNAR1 −/− mice were infected with either MVA or MVAΔE5R at a MOI of 3. Cells were collected 1, 4, 10 and 24 h post infection. Viral DNA copy numbers were determined by quantitative PCR. FIG. 77 A shows DNA copy numbers in MVA-infected WT, cGAS −/− or IFNAR1 −/− skin dermal fibroblasts. FIG. 77 B shows the fold-change compared with the DNA copy numbers at 1 h post infection with MVA. FIG. 77 C shows DNA copy numbers in MVAΔE5R-infected WT, cGAS −/− or IFNAR1 −/− skin dermal fibroblasts. FIG. 77 D shows the fold-change compared with the DNA copy numbers at 1 h post infection with MVAΔE5R.

FIGS. 78 A- 78 D show that MVAΔE5R gains its capacity to generate infectious progeny viruses in cGAS-deficient skin primary dermal fibroblasts. Skin primary dermal fibroblasts from WT, cGAS −/− or IFNAR1 −/− -mice were infected with either MVA or MVAΔE5R at a MOI of 0.05. Cells were collected 1, 24, and 48 h post infection. Viral titers were determined by titrating on BHK21 cells. FIG. 78 A shows MVA titers over time after infection in WT, cGAS −/− or IFNAR1 −/− skin dermal fibroblasts. FIG. 78 shows the fold-change compared with the viral titers at 1 h post infection with MVA. FIG. 78 C shows MVAΔE5R titers over time after infection in WT, cGAS −/− or IFNAR1 −/− skin dermal fibroblasts. FIG. 78 D shows the fold-change compared with the viral titers at 1 h post infection with MVAΔE5R.

FIGS. 79 A and 79 B show that MVAΔE5R infection of murine melanoma cells induce IFNB gene expression and IFN-β protein secretion in a STING-dependent manner. FIG. 79 A shows RT-PCR results of IFNB induction by MVAΔE5R in WT B16-F10 murine melanoma cells and STING −/− B16-F10 cells. WT and STING −/− B16-F10 cells were infected with either MVA or MVAΔE5R at a MOI of 10. Cells were collected at 18 h post infection. RNAs were extracted and quantitative real-time PCR analysis was performed. FIG. 79 B shows ELISA results of IFN-β protein levels in the supernatants of WT and STING −/− B16-F10 cells infected with either MVA or MVAΔE5R collected at 18 h post infection.

FIG. 80 shows that MVAΔE5R infection of murine melanoma cells induces ATP release, which is a hallmark of immunogenic cell death. B16-F10 cells were infected with WT vaccinia, MVA, or MVAΔE5R at a MOI of 10. Supernatants were collected at 48 h post infection. ATP levels were determined by using ATPlite 1step Luminescence ATP Detection Assay System (PerkinElmer, Waltham, MA).

FIG. 81 shows a scheme of generating recombinant MVAΔE5R expressing hFlt3L and hOX40L through homologous recombination at the E4L and E6R loci of the MVA genome. pUC57 vector is used to insert a single expression cassette designed to express both hFlt3L and hOX40L using the vaccinia viral synthetic early and late promoter (PsE/L). The coding sequence of the hFl3L and hOX40L was separated by a cassette including a furin cleavage site followed by a Pep2A sequence. Homologous recombination that occurred at the E4L and E6R loci results in the insertion of expression cassette for hFlt3L and hOX40L.

FIGS. 82 A and 82 B show that the recombinant MVAΔE5R-hFlt3L-hOX40LhOX40L virus has the expected insertion as determined by PCR analysis. Lane 1 shows the Fermentas 1 kb plus DNA ladder. Lane 2 shows a band with expected size of 1120 bp using F0/R5 primer pairs. Lane 3 shows a band with expected size of 1166 bp using F2/R2 primer pairs. Lane 4shows a band with expected size of 1136 bp using F5/R0 primer pairs.

FIGS. 83 A- 83 C show that MVAΔE5R-hFlt3L-hOX40L virus expresses both hFlt3L and hOX40L on the surface of infected cells. FIG. 83 A are dot plots of FACS analysis of hFlt3L expression on the Y axis and hOX40L expression on the X axis of BHK21cells infected with either MVA or MVAΔE5R-hFlt3L-hOX40LhOX40L for 24 h. Cells were infected at a MOI of 10. A mock infection, no virus control was included. FIG. 83 B are dot plots of FACS analysis of hFlt3L expression on the Y axis and hOX40L expression on the X axis of murine B16-F10 melanoma infected with either VA or MVAΔE5R-hFlt3L-hOX40L for 24 h. Cells were infected at a MOI of 10. A mock infection, no virus control was included. FIG. 83 C are dot plots of FACS analysis of hFl3L expression on the Y axis and hOX40L expression on the X axis of human melanoma cells SK-MEL28 infected with either MVA or MVAΔE5R-hFlt3L-hOX40LhOX40L for 24 h. Cells were infected at a MOI of 10. A mock infection, no virus control was included.

FIG. 84 shows Western blot results of the expression of hFl3L and hOX40L in MVAΔE5R-hFlt3L-hOX40LhOX40L-infected BHK21 cells. BHK21 cells were either mock infected, or infected with MVA or MVAΔE5R-hFlt3L-hOX40LhOX40L. Cells lysates were collected at 24 h post infection. Western blot analysis was performed using anti-hFl3L and anti-hOX40L antibodies.

FIGS. 85 A and 85 B show a scheme to generate VACV-TK − -anti-CTLA-4-ΔE5R-hFl3L-mOX40L. FIG. 85 A shows a schematic diagram of pCB vector with a single expression cassette designed to express the heavy chain and light of the antibody using the vaccinia viral synthetic early and late promoter (PsE/L). The coding sequence of the heavy chain (mulgG2a) and the light chain of 9D9 was separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence to enables ribosome skipping. The pCB plasmid containing the anti-mu-CTLA-4 gene under the control of the vaccinia PsE/L as well as the E. coli xanthine-guanine phosphoribosyl transferase gene (gpt) under the control of vaccinia P7.5 promoter flanked by the thymidine kinase (TK) gene on either side. Recombinant virus expressing anti-mu-CTLA-4 from TK locus was generated through homologous recombination at the TK locus between pCB plasmid DNA and viral genomic DNA. FIG. 85 B is the schematic diagram of using the vaccinia viral synthetic early and late promoter (PsE/L) to express both human Flt3L and murine OX40L as a fusion protein in a single expression cassette. The coding sequence of human Flt3L and murine OX40L was separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence. A pUC57 plasmid containing human Flt3L and murine OX40L fusion gene flanked by the E4L and E6R genes on either side was constructed. Recombinant virus expressing human Flt3L and murine OX40L fusion protein from E5R locus was generated through homologous recombination at E4L and E6R loci between pUC57 plasmid and viral genomic DNA.

FIGS. 86 A- 86 C show the scheme and the results of PCR analysis to verify the recombinant vaccinia virus VACV-TK − -anti-muCTLA-4-E5R − -hFl3L-mOX40L, as well as the Western blot results on the expression of anti-CTLA-4 antibodies by the cells infected with the recombinant virus. FIG. 86 A shows the schematic diagram of the primers used to amplify the different gene fragments from the inserted expression cassette of pUC57 expression plasmid. Primer pair f1/r1 was used to generate a 2795 bp PCR fragment, which contains the whole expression cassette. This primer pair was also used to check the purity of the recombinant virus. Primer pair f0/r5 was used to confirm the expression cassette was inserted into the right position in virus genome. Human Flt3L gene was amplified using primer pair f1/r3 or fo/r2 from VACV-TK − -anti-muCTLA-4-E5R − -hFl3L-mOX40L. Murine OX40L gene was generated using primer pair f4/r1. And finally, pCB-R4 and TK-F5 primer pair was used to generate the anti-mu-CTLA-4 gene inserted in TK locus from VACV-TK − -anti-muCTLA-4-E5R − -hFl3L-mOX40L or MVA-TK − -anti-muCTLA-4-E5R − -hFl3L-mOX40L recombinant virus. FIG. 86 B shows the gel image of the PCR results using the primer pairs described in FIG. 86 A and a table that displays the predicated sizes of the amplified DNA fragments with the primer pairs. FIG. 86 C shows Western blot results of human SK-MEL-28 melanoma cells mock infected or infected with E3LΔ83N-TK − -vector, E3LΔ83N-TK − -hFl3L-anti-muCTLA-4, E3LΔ83N-TK − -hFl3L-anti-muCTLA-4-C7L − -mOX40L, VACV, VACV-TK − -anti-muCTLA-4-C7L − -mOX40L, or VACV-TK − -anti-muCTLA-4-E5R − -hFl3L-mOX40L at a MOI of 10. Cell lysates were collected at 24 hours post-infection, and polypeptides were separated using 10% SDS-PAGE. HRP-linked anti-mouse IgG (heavy and light chain) antibody was used to detect full-length (FL), heavy chain (HC), and light chain (LC) of anti-muCTLA-4 antibodies.

FIG. 87 shows the protein sequence alignments of E5 orthologs from multiple members of the poxvirus family. Figure discloses SEQ ID NOs: 42-54, 20 and 55-60, respectively, in order of appearance.

FIG. 88 A shows the protein sequence alignments of E5 from vaccinia virus and Modified vaccinia virus Ankara (MVA). Figure discloses SEQ ID NOs: 20 and 58, respectively, in order of appearance. FIG. 88 B shows the protein sequence alignments of E5 from vaccinia virus and myxoma virus. Figure discloses SEQ ID NOs: 20-21, respectively, in order of appearance.

FIGS. 89 A and 89 B show that myxoma virus M31R inhibits cGAS and STING induced IFN-β pathway. FIG. 89 A shows that HEK293T cells were transfected with plasmids expressing murine cGAS, human STING together with either E5R, M31R or pcDNA vector control expressing plasmid. After 24 h, Luciferase signals were determined. FIG. 89 B shows that HEK293T cells were transfected with plasmids expressing murine STING together with either E5R, M31R or pcDNA vector control expressing plasmid. After 24 h, Luciferase signal were determined.

FIGS. 90 A and 90 B show that vaccinia E5 promotes cGAS ubiquitination. FIG. 90 A shows that HEK293T cells were transfected with Flag-cGAS and HA-ubiquitin. After 24 h, cells were infected with either WT VACV or VACVΔE5R. Cell lysis were collected after 6 h. cGAS was immunoprecipitated with anti-Flag antibody and ubiquitination was detected by anti-HA antibody. FIG. 90 B shows Western blot analysis of cGAS and β-actin in whole cell lysates (WCL).

FIGS. 91 A- 91 C are graphical representations of data showing IT MVAΔE5R delays tumor growth and prolongs survival in murine B16-F10 melanoma unilateral tumor implantation model. FIG. 91 A is a scheme of tumor implantation and treatment for a B16-F10 unilateral tumor implantation model. Briefly, 5×10 5 B16-F10 melanoma cells were implanted intradermally to the right flank of C57B/6J mice. Eight days post tumor implantation, PBS, 4×10 7 pfu of MVA, MVAΔE5R or Heat-iMVA were intratumorally injected twice weekly. The tumor sizes were measured and the survival of mice was monitored. FIG. 91 B is a graph of the Kaplan-Meier survival curve of tumor-bearing mice treated with either PBS, MVA, MVAΔE5R, or Heat-iMVA, treatments. (n=5, *P<0.05; ** P<0.01; Mantel-Cox test). FIG. 91 C is a table showing median survival of mice treated with either PBS, MVA, MVAΔE5R, or Heat-iMVA.

FIGS. 92 A- 92 E show that MVAΔC7L-hFl3L-TK(−) mOX40LΔE5R infection of BMDCs results in higher levels of IFNB gene expression and IFN-β protein secretion compared with MVAΔE5R. FIGS. 92 A- 92 C show schematic diagrams of generating MVAΔC7L-hFl3L-TK(−)-mOX40LAE5R virus. The first step involves the generation of MVAΔC7L-hFl3L through homologous recombination at the C8L and C6R loci, replacing C7L C7Lgene with hFl3L under the control of PsE/L promoter ( FIG. 92 A ). The second step involves the generation of MVAΔC7L-hFlt3L-TK(−)-mOX40LC7LhFl3LmOX40Lthrough homologous recombination at the TK loci, replacing TK gene with mOX40L under the control of PsE/L promoter ( FIG. 92 B ). The resulting virus was described in FIGS. 5 A and 5 B . The third step is to generate MVAΔC7L-hFl3L-TK(−)-mOX40LΔE5R through homologous recombination at the E4L and E6R loci, replacing E5R gene with mCherry under the control of P7.5 promoter ( FIG. 92 C ). FIG. 92 D shows RT-PCR results of IFNB gene expression in BMDCs infected with either MVAΔE5R, MVAΔC7L-hFl3L-TK(−)-mOX40L, or MVAΔC7L-hFl3L-TK(−)mOX40LΔE5R. WT and IFNAR −/− BMDCs were mock-infected or infected with MVAΔE5R, MVAΔC7L-hFl3L-TK(−)-mOX40L, or MVAΔC7L-hFl3L-TK(−)mOX40LΔE5R at a MOI of 10. Cells were collected at 16 h post infection and RT-PCR was performed. FIG. 92 E shows ELISA results of IFN-β protein levels in the supernatants of BMDCs infected with either MVAΔE5R, MVAΔC7L-hFl3L-TK(−)-mOX40L, or MVAΔC7L-hFl3L-TK(−)mOX40LΔE5R. WT and IFNAR −/− BMDCs were mock-infected or infected with MVAΔE5R, MVAΔC7L-hFl3L-TK(−)-mOX40L, or MVAΔC7L-hFl3L-TK(−)mOX40LΔE5R at a MOI of 10. Supernatants were collected at 16 h post infection and ELISA was performed to measure IFN-β protein levels.

FIGS. 93 A and 93 B show the scheme of generating MVAΔC7LΔE5R-hFl3L-mOX40L and MVAΔC7L-OVA-ΔE5R-hFl3L-mOX40L. FIG. 93 A shows the scheme of generating MVAΔC7LΔE5R-hFl3L-mOX40L. pUC57 plasmid is constructed to use the vaccinia viral synthetic early and late promoter (PsE/L) to express both human Flt3L and murine OX40L as a fusion protein in a single expression cassette. The coding sequence of human Flt3L and murine OX40L was separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence. Recombinant virus expressing human Flt3L and murine OX40L fusion protein from E5R locus was generated through homologous recombination at E4L and E5R loci between pUC57 plasmid and MVAΔC7L C7Lviral genome. FIG. 93 B shows the scheme of generating MVAΔC7L-OVA-ΔE5R-hFl3L-mOX40L. Recombinant virus expressing human Flt3L and murine OX40L fusion protein from E5R locus was generated through homologous recombination at E4L and E5R loci between pUC57 plasmid and MVAΔC7L-OVA viral genome.

FIGS. 94 A and 94 B . FIG. 94 A : Dual-luciferase assay of HEK293T cells transfected with ISRE-firefly luciferase reporter, a control plasmid pRL-TK that expresses Renilla luciferase, together with either myxoma M62R, Myxoma M62R-HA, Myxoma M64R, Myxoma M64R-HA, vaccinia C7L-expressing or control plasmid. 24 h post transfection, cells were treated with IFN-β for another 24 h before harvesting. FIG. 94 B : Dual-luciferase assay of HEK293T cells transfected with IFNB-firefly luciferase reporter, a control plasmid pRL-TK that expresses Renilla luciferase, and STING-expressing plasmid, together with either myxoma M62R, Myxoma M62R-HA, Myxoma M64R, Myxoma M64R-HA, vaccinia C7L-expressing, or control plasmid. Cells were harvested at 24 h post transfection.

FIG. 95 shows a scheme of generating recombinant MVAΔE5R expressing hFl3L and mOX40L through homologous recombination at the E4L and E6R loci of the MVA genome. pUC57 vector was used to insert a single expression cassette designed to express both hFl3L and mOX40L using the vaccinia viral synthetic early and late promoter (PsE/L). The coding sequence of the hFl3L and mOX40L was separated by a cassette including a furin cleavage site followed by a Pep2A sequence. Homologous recombination that occurred at the E4L and E6R loci results in the insertion of expression cassette for hFl3L and mOX40L.

FIGS. 96 A and 96 B show that MVAΔE5R-hFl3L-mOX40L virus expresses both hFl3L and mOX40L on the surface of infected cells. FIG. 96 A shows the dot plots of FACS analysis of hFl3L expression on the Y axis and mOX40L expression on the X axis of BHK21 cells, murine melanoma cells B16-F10, or human melanoma cells SK-MEL28. Cells were infected with either MVA or MVAΔE5R-hFl3L-mOX40L at a MOI of 10 for 24 h. No virus mock infection control was included. FIG. 96 B shows the graphs of medium fluorescence intensity (MFI) of human Flt3L and murine OX40L on infected BHK21, B16-F10, and SK-MEL28 cells infected with either MVA, MVAΔE5R-hFl3L-mOX40L, or PBS.

FIGS. 97 - 102 are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L generated more activated tumor-infiltrating effector T cells in injected and non-injected distant tumors as well as in the spleens compared with MVA, MVAΔE5R, or Heat-iMVA in a B16-F10 bilateral tumor model. FIG. 97 shows the experimental scheme. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, 2×10 7 pfu of either MVAΔE5R-hFl3L-mOX40L, MVA, MVAΔE5R, an equivalent amount of Heat-iMVA, or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice, three days apart. Spleens were harvested at 2 days post second injection, ELISPOT analyses were performed to evaluate tumor-specific T cells in the spleens. Both injected and non-injected tumors were also isolated and tumor infiltrating lymphocytes were analyzed by FACS.

FIGS. 98 A- 98 B . ELISPOT assay was performed by co-culturing irradiated B16-F10 cells (150,000) and splenocytes (1,000,000) in a 96-well plate. FIG. 98 A shows the image of ELISPOT of triplicate samples from left to right. FIG. 98 B shows the graph of IFN-γ + spots per 1,000,000 purified CD8 + T cells. Each bar represents spleen sample from individual mouse (n=3-8) (*P<0.05; **P<0.01,/test).

FIGS. 99 A- 99 C . FIG. 99 A shows the representative dot plots of Granzyme B + CD8 + T cells in non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 99 B shows the graph of percentages of CD8 + T cells out of CD3 + cells. Data are means ±SEM (n=5-9). (**P<0.01; ***P<0.001,/test). FIG. 99 C shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells). Data are means ±SEM (n=5-9) (**P<0.01; ***P<0.001,/test).

FIGS. 100 A- 100 C . FIG. 100 A shows the representative dot plots of Granzyme B + CD4 + T cells in non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 100 B shows the graph of percentages of CD4 + T cells out of CD3* cells. Data are means ±SEM (n=5-9). (*P<0.05;/test). FIG. 100 C shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells). Data are means ±SEM (n=5-9) (**P<0.01; ***P<0.001,/test).

FIGS. 101 A- 101 C . FIG. 101 A shows the representative dot plots of Granzyme B* CD8 + T cells in the injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 101 B shows the graph of percentages of CD8 + T cells out of CD3 + cells. Data are means ±SEM (n=5-9). ****P<0.0001,/test). FIG. 101 C shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells). Data are means ±SEM (n=5-9) (*P<0.05; ****P<0.0001,/test).

FIGS. 102 A- 102 C . FIG. 102 A shows the representative dot plots of Granzyme B + CD4 + T cells in the injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 102 B shows the graph of percentages of CD4 + T cells out of CD3 + cells. Data are means ±SEM (n=5-9). (**P<0.01;/test). FIG. 102 C shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells). Data are means ±SEM (n=5-9) (*P<0.05; **P<0.01; ****P<0.0001,/test).

FIGS. 103 A- 104 C are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L reduced FoxP3 + CD4 + regulatory T cells in the injected tumors, but not in the non-injected tumors. FIG. 103 A shows the representative dot plots of FoxP3 + CD4 + cells in the injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 103 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-9). (**P<0.01; ***P<0.001,/test). FIG. 103 C shows the graph of absolute numbers of FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9). (*P<0.05,/test). FIG. 104 A shows the representative dot plots of FoxP3 + CD4 + cells in the non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 104 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-9). FIG. 104 C shows the graph of absolute numbers of FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9).

FIGS. 105 A to 109 C are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L preferentially reduces OX40 + FoxP3 + CD4 + regulatory T cells in the injected tumors. FIG. 105 A shows the representative dot plots of OX40 + FoxP3 + CD4 + cells in the non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 105 B shows the graph of percentages of OX40 + FoxP3 − CD4 + T cells out of CD4 + cells in the injected tumors. Data are means ±SEM (n=5-9). (**P<0.01; ***P<0.001,/test). FIG. 105 C shows the graph of absolute numbers of OX40 + FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9). (*P<0.05,/test).

FIG. 106 A shows the representative dot plots of OX40 + FoxP3 + CD4 + cells in the non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 106 B shows the graph of percentages of OX40 + FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-9). FIG. 106 C shows the graph of absolute numbers of OX40 + FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9).

FIG. 107 A shows the representative dot plots of OX40 + FoxP3 + CD4 + cells in the non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS. FIG. 107 B shows the graph of percentages of OX40 + FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-9). FIG. 107 C shows the graph of absolute numbers of OX40 + FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9).

FIG. 108 shows the representative dot plots of OX40 + CD8 + cells in the non-injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, Heat-iMVA, or PBS.

FIGS. 109 A- 109 C are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L results in more reduction of FoxP3 + CD4 + regulatory T cells in the injected tumors compared with MVAΔE5R. FIG. 109 A shows the representative dot plots of FoxP3 + CD4 + cells in the injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, MVAΔE5R, or PBS. FIG. 109 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells in the injected tumors. Data are means ±SEM (n=5-9). (*P<0.05; **P<0.01, 1 test). FIG. 109 C shows the graph of absolute numbers of FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-9). (**P<0.01,/test).

FIGS. 110 - 115 C are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L generated more activated tumor-infiltrating effector CD8 + and CD4 + T cells in the injected and non-injected distant tumors in OX40 −/− mice compared with WT mice in a B16-F10 bilateral murine melanoma model. FIG. 110 shows the experimental scheme. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Ten days post tumor implantation, intratumoral injections (4×10 7 pfu) of either MVAΔE5R-hFl3L-mOX40L or PBS were performed to the larger tumors on the right flank twice, three days apart. Both the injected and non-injected distant tumors were harvested at 2 days post second injection and tumor-infiltrating lymphocytes were analyzed by FACS.

FIG. 111 A shows the representative dot plots of Granzyme B + CD8 + T cells in the injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 111 B shows the graph of percentages of CD8 + T cells out of CD3 + cells. Data are means ±SEM (n=4-10). FIG. 111 C shows the graph of absolute numbers of CD8 + T cells per gram of tumor. Data are means ±SEM (n=4-10). FIG. 111 D shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=4-10). FIG. 111 E shows the graph of absolute numbers of Granzyme B + CD8 + T cells per gram of tumor. Data are means ±SEM (n=4-10).

FIG. 112 A shows the representative dot plots of Granzyme B + CD4 + T cells in the injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 112 B shows the graph of percentages of CD4 + T cells out of CD3 + cells. Data are means ±SEM (n=4-10). FIG. 112 C shows the graph of absolute numbers of CD4 + T cells per gram of tumor. Data are means ±SEM (n=4-10). FIG. 112 D shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=4-10). FIG. 112 E shows the graph of absolute numbers of Granzyme B + CD4 + T cells per gram of tumor. Data are means ±SEM (n=4-10).

FIG. 113 A shows the IT injection of MVAΔE5R-hFl3L-mOX40L fails to reduce FoxP3 + CD4 + T cells in the injected tumors from OX40 −/− mice. Representative dot plots of FoxP3 + CD4 + T cells in the injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 113 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells Data are means ±SEM (n=4-10).

FIGS. 114 A- 115 C demonstrate that IT injection of MVAΔE5R-hFl3L-mOX40L reduces OX40 + FoxP3 + CD4 + and OX40 + FoxP3 + CD4 + T cells in the injected tumors from the WT mice. FIG. 114 A shows the representative dot plots of OX40 + FoxP3 + CD4 + T cells in the injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 114 B shows the graph of percentages of OX40 + FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=4-10). FIG. 114 C shows the graph of absolute numbers of OX40 + FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=4-10).

FIG. 115 A shows the representative dot plots of OX40 + FoxP3 + CD4 + T cells in the injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 115 B shows the graph of percentages of OX40 + FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=4-10). FIG. 115 C shows the graph of absolute numbers of OX40 + FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=4-10).

FIGS. 116 A- 119 C are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L results in more proliferation and activation of tumor-infiltrating effector CD8 + and CD4 + T cells in distant non-injected tumors from OX40 −/− mice compared with WT mice. FIG. 116 A shows the representative dot plots of Granzyme B + CD8 + T cells in non-injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 116 B shows the graph of percentages of CD8 + T cells out of CD45 + cells. Data are means ±SEM (n=5-10). FIG. 116 C shows the graph of absolute numbers of CD8 + T cells per gram of tumor. Data are means ±SEM (n=5-10). FIG. 116 D shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=5-10). FIG. 116 E shows the graph of absolute numbers of Granzyme B + CD8 + T cells per gram of tumor. Data are means ±SEM (n=5-10).

FIG. 117 A shows the representative dot plots of Ki67 + CD8 + T cells in non-injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 117 B shows the graph of percentages of Ki67 + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=5-10). FIG. 117 C shows the graph of absolute numbers of Ki67 + CD8 + T cells per gram of tumor. Data are means ±SEM (n=5-10).

FIG. 118 A shows the representative dot plots of Granzyme B + CD4 + T cells in none-injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 118 B shows the graph of percentages of CD4 + T cells out of CD45 + cells. Data are means ±SEM (n=5-10). FIG. 118 C shows the graph of absolute numbers of CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-10). FIG. 118 D shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-10). FIG. 118 E shows the graph of absolute numbers of Granzyme B + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-10).

FIG. 119 A shows the representative dot plots of Ki67 + CD4 + T cells in non-injected tumors from WT and OX40 −/− mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS. FIG. 119 B shows the graph of percentages of Ki67 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5-10). FIG. 119 C shows the graph of absolute numbers of Ki67 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5-10).

FIGS. 120 - 124 B are a series of graphical representations of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L was capable of inducing antitumor effects without recruiting T cells from the lymphoid organs. FIG. 120 shows the experimental scheme. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Nine days post tumor implantation, intratumoral injections (4×10 7 pfu) of either MVAAE5R-hFl3L-mOX40L, or PBS were performed to the larger tumors on the right flank twice on day 9 and 12. The injected tumors were harvested at 2 days post second injection and tumor-infiltrating lymphocytes were analyzed by FACS. FTY720 (25 μg), which blocks egress of lymphocytes from the lymphoid organs, was given to the mice intrapertoneally on day 7, 9, 11, and 13.

FIG. 121 (upper panel) shows the graphs of tumor volumes of both injected and non-injected tumors over time. Data are means +SEM (n=7-8). FIG. 121 (lower panel) shows the graphs of tumor volumes of both injected and non-injected tumors at day 6 post first injection. Data are means +SEM (n=7-8).

FIGS. 122 A- 122 D shows the representative dot plots of Granzyme B + CD8 + T cells in injected tumors from mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS in combination with intraperitoneal delivery of FTY720 or DMSO. FIG. 122 B shows the graph of percentages of CD8 + T cells out of CD45 + cells. Data are means ±SEM (n=7-8). FIG. 122 C shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=7-8). FIG. 122 D shows the graph of percentages of Ki67 + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=7-8).

FIG. 123 A shows the representative dot plots of Granzyme B + CD8 + T cells in TDLNs of the injected tumors from mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS in combination with intraperitoneal delivery of FTY720 or DMSO. FIG. 123 B shows the graph of percentages of CD8 + T cells out of CD3 + cells. Data are means ±SEM (n=7-8). FIG. 123 C shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=7-8).

FIG. 124 A shows the representative dot plots of Ki67 + CD8 + T cells in TDLNs of the injected tumors from mice after treatment with either MVAΔE5R-hFl3L-mOX40L or PBS in combination with intraperitoneal delivery of FTY720 or DMSO. FIG. 124 B shows the graph of percentages of Ki67 + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=7-8).

FIGS. 125 - 129 C are graphical representations of data showing intratumoral delivery of MVAΔE5R-hFl3L-mOX40L delays tumor growth, activates CD8 + T cells and reduces FoxP3 + CD4 + T cells in the injected tumors in a murine AT3 breast cancer fat pad implantation model. FIG. 125 is a scheme of tumor implantation and treatment for murine breast cancer AT3 fat pad implantation model. Briefly, AT3 cells (1×10 6 ) were implanted into the 4th fat pad of the C57B/6J mice. 14 days post tumor implantation, intratumoral injections (6×10 7 pfu) of MVAΔE5R-hFl3L-mOX40L, or Heat-iMVA, or PBS, were performed twice, three days apart. The injected tumors were measured and harvested for FACS analysis.

FIG. 126 A shows the graph of tumor volumes of injected AT3 tumors after treatment with MVAΔE5R-hFl3L-mOX40L, or Heat-iMVA, or PBS over time. Data are means ±SEM (n=5). Graph of tumor weight of injected tumors at day 6 post first injection. Data are means ±SEM (n=5). FIG. 126 B shows the tumor weighs on day 6. *=p<0.05.

FIG. 127 A shows the representative dot plots of Granzyme B + CD8 + T cells in injected AT3 tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, or Heat-iMVA, or PBS. FIG. 127 B shows the graph of percentages of CD8 + T cells out of CD3 + cells. Data are means ±SEM (n=5). FIG. 127 C shows the graph of absolute numbers of CD8 + T cells per gram of tumor. Data are means ±SEM (n=5). FIG. 127 D shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=5). FIG. 127 E shows the graph of absolute numbers of Granzyme B + CD8 + T cells per gram of tumor. Data are means ±SEM (n=5).

FIG. 128 A shows the representative dot plots of Granzyme B + CD4 + T cells in injected AT3 tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, or Heat-iMVA, or PBS. FIG. 128 B shows the graph of percentages of CD4 + T cells out of CD3 + cells. Data are means ±SEM (n=5). FIG. 128 C shows the graph of absolute numbers of CD4 + T cells per gram of tumor. Data are means ±SEM (n=5). FIG. 128 D shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5). FIG. 128 E shows the graph of absolute numbers of Granzyme B + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5).

FIG. 129 A shows the representative dot plots of FoxP3 + CD4 + T cells in injected AT3 tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, or Heat-iMVA, or PBS. FIG. 129 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=5). FIG. 129 C shows the graph of absolute numbers of FoxP3 + CD4 + T cells per gram of tumor. Data are means ±SEM (n=5).

FIGS. 130 - 131 B are graphical representations of data showing that combination of IT delivery of MVAΔE5R-hFl3L-mOX40L and intraperitoneal delivery of anti-PD-L1 results in enhanced therapeutic efficacy in a bilateral B16-F10 melanoma implantation model.

FIG. 130 shows the experimental scheme. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 1×10 5 to the left flank). Seven days post tumor implantation, 4×10 7 pfu of either MVAΔE5R-hFl3L-mOX40L or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice per week. One group of the mice also received anti-PD-L1 antibody (250 μg) twice a week in conjunction with IT MVAΔE5R-hFl3L-mOX40L. Tumor volumes and mice survival were monitored.

FIG. 131 A shows tumor volumes of both injected and non-injected tumors in mice treated with either PBS or MVAΔE5R-hFl3L-mOX40L intratumorally, or with the combination of IT MVAΔE5R-hFl3L-mOX40L plus IP anti-PD-L1. FIG. 131 B shows the Kaplan Meier survival curve of the three groups.

FIGS. 132 A- 134 B are graphical representations of data showing MVAΔE5R-hFlt3L-hOX40LhOX40L infection of BMDCs induces IFNB gene expression and IFN-β protein secretion; infection of human tumors (extramammary Paget's disease) with MVAΔE5R-hFlt3L-hOX40LhOX40L ex vivo results in the increase of Granzyme B + CD8 + T cells and the reduction of FoxP3 + CD4 + T cells.

FIG. 132 A shows the RT-PCR results of BMDCs that were infected with either MVA or MVAΔE5R-hFlt3L-hOX40LhOX40L at a MOI of 10. Cells were collected at 6 h post infection. RNAs were extracted and RT-PCRs were performed. FIG. 132 B shows the IFN-β protein levels in BMDCs infected with either MVA or MVAΔE5R-hFlt3L-hOX40LhOX40L at a MOI of 10. Supernatants were collected at 19 h post infection. IFN-β protein levels were determined by ELISA.

FIG. 133 A shows the representative dot plots of Granzyme B + CD8 + T cells in human tumors after infection with MVAΔE5R-hFl3L-mOX40L or PBS for two days. FIG. 133 B shows the representative dot plots of FoxP3 + CD4 + T cells in human tumors after infection with MVAΔE5R-hFl3L-mOX40L or PBS for two days.

FIG. 134 A shows the graph of percentages of Granzyme + CD8 + T cells out of CD8 + cells after infection with MVAΔE5R-hFl3L-mOX40L or PBS control for two days. Data are means ±SEM (n=3). FIG. 134 B shows the graph of percentages of FoxP3 + CD4 + T cells T cells out of CD4 + cells after infection with MVAΔE5R-hFl3L-mOX40L or PBS control for two days. Data are means ±SEM (n=3).

FIGS. 135 A- 137 B are graphical representations of data showing MVAΔE3LΔE5R induces higher levels of type I IFN in BMDCs and B16-F10 melanoma cells compared with MVAΔE5R or MVAΔE3L.

FIGS. 135 A- 135 C show a scheme of generating recombinant MVAΔE3LΔE5R and MVAΔE3LΔE5R-hFl3L-mOX40L through homologous recombination at the E2L and E4L loci of the MVAΔE5R or MVAΔE5R-hFl3L-mOX40L genome. Homologous recombination that occurred at the E2L and E4L loci results in the deletion of E3L gene from the MVAΔE5R or MVAΔE5R-hFl3L-mOX40L genome.

FIG. 136 shows that MVAΔE3LΔE5R infection of BMDCs induced higher levels of IFNB gene expression compared with MVAΔE5R, MVA, or Heat-iMVA. BMDCs from WT or cGAS −/− mice were infected with MVA, Heat-iMVA, MVAΔE5R, or MVAΔE3LΔE5R at a MOI of 10. Cells were collected at 6 h post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene.

FIGS. 137 A- 137 B show that MVAΔE3LΔE5R infection of murine B16-F10 melanoma cells induces higher levels of IFNB gene expression and IFN-β protein secretion compared with MVAΔE3L or MVAΔE5R. FIG. 137 A shows the quantitative RT-PCR analyses with WT or MDA5 −/− , or STING −/− MDA5 −/− B16-F10 cells infected with MVAΔE3L, or MVAΔE5R or MVAΔE3LΔE5R at a MOI of 10. Cells were collected at 16 h post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. FIG. 137 B shows the IFN-β protein levels in WT or MDA5 −/− , or STING −/− MDA5 −/− B16-F10 cells infected with MVAΔE3L, or MVAΔE5R, or MVAΔE3LΔE5R at a MOI of 10. Supernatants were collected at 24 h post infection and IFN-β protein levels in the supernatants were determined by ELISA.

FIGS. 138 - 139 B are graphical representations of data showing MVAΔE3LΔE5R-hFl3L-mOX40L expressed human Flt3L and murine OX40L transgenes in B16-F10 melanoma cells and intratumoral delivery of MVAΔE3LΔE5R-hFl3L-mOX40L induced stronger systemic antitumor T cell responses in a B16-F10 murine melanoma model.

FIG. 138 shows the FACS data demonstrating the mOX40L and hFl3L expression on B16-F10 cells infected with either MVAΔE5R-hFl3L-mOX40L or with MVAΔE3LΔE5R-hFl3L-mOX40L. B16-F10 cells were infected with either MVAΔE5R-hFl3L-mOX40L, or with MVAΔE3LΔE5R-hFl3L-mOX40L, or with MVAΔE3LΔE5R at a MOI of 10. Cells were washed 1 h later and harvested at 24 h post infection. Cells were strained with anti-mOX40L or anti-hFl3L antibody for FACS.

FIGS. 139 A- 139 B shows that intratumoral injection of MVAΔE3LΔE5R-hFl3L-mOX40L generated stronger antitumor-specific T cells in the spleens compared with MVAΔE5R-hFl3L-mOX40L. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, 2×10 7 pfu of either MVAΔE5R-hFl3L-mOX40L, MVAΔE3LΔE5R-hFl3L-mOX40L, an equivalent amount of Heat-iMVA, or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice, three days apart. Spleens were harvested at 2 days post second injection, ELISPOT analyses were performed to evaluate tumor-specific T cells in the spleens. ELISPOT assay was performed by co-culturing irradiated B16-F10 cells (150,000) and splenocytes (1,000,000) in a 96-well plate. FIG. 139 A shows the image of ELISPOT of triplicate samples of combined splenocytes from mice in the same treatment group. FIG. 139 B shows the graph of IFN-β + spots per 1,000,000 splenocytes. Each dot represents spleenocyte samples from an individual mouse (n=5-6) (*P<0.05; **P<0.01,/test).

FIGS. 140 - 141 B are graphical representations of data showing intratumoral delivery of MVAΔE3LΔE5R-hFl3L-mOX40L delayed the growth of both WT and β2M −/− B16-F10 tumor cells.

FIG. 140 shows the experimental scheme. Briefly, WT or b2M −/− B16-F10 tumor cells (2×10 5 ) (which were generated by CRISPR-cas9 technology in the inventors' lab) were implanted intradermally to the right flanks of C57BL/6J mice. 10 days after tumor implantation, tumors were injected with MVAΔE3LΔE5R-hFl3L-mOX40L twice a week. Tumor volumes were measured and mice survival were monitored.

FIG. 141 A shows tumor volumes of the injected WT and b2M −/− B16-F10 tumors in mice treated with either PBS or MVAΔE5R-hFl3L-mOX40L intratumorally. FIG. 141 B shows the Kaplan Meier survival curve of the four groups.

FIGS. 142 - 148 are a series of graphical representations of data showing that intratumoral injection of MVAΔE3LΔE5R-hFl3L-mOX40L generated more activated tumor-infiltrating effector T cells and reduced percentage of macrophage and DCs in injected tumors compared with MVAΔE5R-hFl3L-mOX40L in a AT3 bilateral tumor implantation model.

FIG. 142 shows the experimental scheme. Briefly, 10 5 AT3 breast cancer cells were implanted into the 4 th fat pad of the C57B/6J mice. Twelve days post tumor implantation, 6×10 7 pfu of either MVAΔE5R-hFl3L-mOX40L, MVAΔE3LΔE5R-hFl3L-mOX40L, or PBS was intratumorally (IT) injected into the tumors on both flanks twice, three days apart. Injected tumors were isolated. Tumor infiltrating lymphocytes and myeloid cells were analyzed by FACS.

FIG. 143 A shows the representative dot plots of Granzyme B + CD8 + T cells in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L, or MVAΔE3LΔE5R-hFl3L-mOX40L. FIG. 143 B shows the graph of percentages of CD8 + T cells out of CD45 + cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001,/test). FIG. 143 C shows the graph of absolute number of CD8 + T cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001,/test). FIG. 143 D shows the graph of percentages of Granzyme B + CD8 + T cells out of CD8 + cells. Data are means ±SEM (n=4-6) (**P<0.01; ***P<0.001,/test). FIG. 143 E shows the graph of absolute number of Granzyme B + CD8 + T cells. Data are means ±SEM (n=4-6) (**P<0.01; ***P<0.001,/test).

FIG. 144 A shows the representative dot plots of Ki67 + CD8 + T cells in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L. FIG. 144 B shows the graph of percentages of Ki67 + CD8 + T cells out of CD8 + cells Data are means ±SEM (n=4-6) (**P<0.01; ***P<0.001,/test). FIG. 144 C shows the graph of absolute number of Ki67 + CD8 + T cells. Data are means ±SEM (n=4-6) (**P<0.01; ***P<0.001,/test).

FIG. 145 A shows the representative dot plots of Granzyme B + CD4 + T cells in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L. FIG. 145 B shows the graph of percentages of CD4 + T cells out of CD3 + cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 145 C shows the graph of absolute number of CD4 + T cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 145 D shows the graph of percentages of Granzyme B + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=4-6) (**P<0.01; ***P<0.001, t test). FIG. 145 E shows the graph of absolute number of Granzyme B + CD4 + T cells. Data are means ¤T cells. Data aP<0.01; ***P<0.001, t test).

FIG. 146 A shows the representative dot plots of FoxP3 + CD4 + T cells in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L. FIG. 146 B shows the graph of percentages of FoxP3 + CD4 + T cells out of CD4 + cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 146 C shows the graph of absolute number of FoxP3 + CD4 + T cells. Data are means ±SEM. Data are P<0.01; ***P<0.001, t test).

FIG. 147 A shows the representative dot plots of OX40 + FoxP3 + CD4 + T cells in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L. FIG. 147 B shows the graph of percentages of OX40 + FoxP3 + CD4 + T cells out of FoxP3 + CD4 + cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 147 C shows the graph of absolute number of OX40 + FoxP3 + CD4 + T cells. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test).

FIGS. 148 A- 148 H are series of data showing that intratumoral injection of MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L reduces the percentage of macrophages and DCs in injected tumors. FIG. 148 A shows the graph of percentages of macrophages in injected tumors after treatment with either MVAΔE5R-hFl3L-mOX40L or MVAΔE3LΔE5R-hFl3L-mOX40L. Data are means ±SEM (n=4-6). FIG. 148 B shows the graph of absolute number of macrophages. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 C shows the graph of percentages of DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 D shows the graph of absolute number of DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 E shows the graph of percentages of CD11b + DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 F shows the graph of absolute number of CD11b + DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 G shows the graph of percentages of CD103 + DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test). FIG. 148 H shows the graph of absolute number of CD103 + DCs. Data are means ±SEM (n=4-6). (**P<0.01; ***P<0.001, t test).

FIGS. 149 - 150 are a series of graphical representations of data showing that the combination of intratumoral injection of MVAΔE3LΔE5R-hFl3L-mOX40L with anti-PD-L1 and anti-CTLA-4 antibody had superior anti-tumor efficacy in MMTV-PyMT breast cancer model. FIG. 149 shows the experimental scheme. After the first tumor became palpable, 4×10 7 pfu of MVAΔE3LΔE5R-hFl3L-mOX40L or PBS was intratumorally (IT) injected into the tumors twice a week. 250 μg Anti-PD-L1 and 100 μg anti-CTLA-4 antibody or isotype control antibody were injected intraperitoneally to each mouse twice a week. Tumor volumes were measured twice a week. FIG. 150 shows the graph of tumor growth curve after treatment with IT MVAΔE3LΔE5R-hFl3L-mOX40L and IP anti-PD-L1 and anti-CTLA-4 antibody. Data are means ±SEM (n=3-4).

FIGS. 151 - 152 B are a series of graphical representations of data showing that deletion of C11R gene from MVAΔE5R-hFl3L-mOX40L increases IFN production in BMDCs.

FIG. 151 is a schematic diagram of homologous recombination to generate MVAΔE5R-hFl3L-mOX40ΔC11R.

FIGS. 152 A- 152 B show that MVAΔE5R-hFl3L-mOX40ΔC11R infection of BMDCs induces higher levels of IFNB gene expression ( FIG. 152 A ) and protein secretion ( FIG. 152 B ) compared with MVAΔE5R or MVA. BMDCs from WT mice were infected with MVA, MVAΔE5R, or MVAΔE5R-hFl3L-mOX40ΔC11R at a MOI of 10. For assessing IFNB gene expression, cells were collected at 6 h post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. For testing IFN-b protein secretion from BMDCs, supernatants were collected at 19 h post infection. IFN-b protein levels were determined by ELISA.

FIGS. 153 - 154 C are a series of graphical representations of data showing that deletion of WR199 gene from MVA or MVAΔE5R-hFl3L-mOX40L increases IFN production in BMDCs.

FIG. 153 is a schematic diagram of homologous recombination to generate MVAΔE5R-hFl3L-mOX40ΔWR199. Homologous recombination that occurred at the B17L and B 19 R loci results in the deletion of WR199 and the insertion of expression cassette for mcherry flanked by two FRT sites.

FIG. 154 A shows the IFNB gene expression in PBS, MVA, MVAΔWR199, or Heat-iMVA infected BMDCs from WT or cGAS −/− mice at a MOI of 10. FIGS. 154 B- 154 C shows that MVAΔE5R-hFl3L-mOX40ΔC11R infection of BMDCs induces higher levels of IFNB gene expression ( FIG. 154 B ) and protein secretion ( FIG. 154 C ) compared with MVAΔE5R or MVA. BMDCs from WT mice were infected with MVA, MVAΔE5R, or MVAΔE5R-hFl3L-mOX40ΔC11R at a MOI of 10. For assessing IFNB gene expression, cells were collected at 6 h post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. For testing IFN-β protein secretion from BMDCs, supernatants were collected at 19 h post infection. IFN-β protein levels were determined by ELISA.

FIG. 155 shows a scheme of generating recombinant VACVΔB2R virus through homologous recombination at the BIR and B3R loci of the vaccinia virus (WR) genome. Homologous recombination that occurred at the BIR and B3R loci results in the deletion of B2R gene from the vaccinia virus (WR) genome.

FIGS. 156 A- 156 B show that VACVΔB2R was highly attenuated in an intranasal infection model. FIG. 156 A shows the weight loss after intranasal infection with either high dose of VACVΔB2R (H, 2×10 7 pfu), or low dose of VACVΔB2R (L, 2×10 6 pfu). FIG. 156 B shows the Kaplan-Meier survival curves of intranasal infected mice with high dose of VACVΔB2R (H, 2×10 7 pfu) or low dose of VACVΔB2R (L, 2×10 6 pfu).

FIGS. 157 A- 157 B shows a schematic diagrams of generating recombinant VACVΔE3L83NΔB2R, VACVΔE5RΔB2R, and VACVΔE3L83NΔE5RΔB2R viruses. FIG. 157 A shows that VACVΔE3L83NΔB2R was generated through homologous recombination at the B1R and B3R loci of the vaccinia VACVΔE3L83N genome, resulting in the deletion of B2R gene from the VACVΔE3L83N genome. FIG. 157 B shows that VACVΔE5RΔB2R and VACVΔE3L83NΔE5RΔB2R are generated through homologous recombination at the B1R and B3R loci of the vaccinia VACVΔE5R or VACVΔE3L83NΔE5R genome, respectively. Homologous recombination that occurred at the B1R and B3R loci results in the deletion of B2R gene from the VACVΔE5R or VACVΔE3L83NΔE5R genome, respectively.

FIG. 158 shows that VACVΔE3L83NΔE5R, VACVΔE5RΔB2R, and VACVΔE3L83NΔE5RΔB2R are highly attenuated in an intranasal infection model. WT mice were intranasally infected with 2×10 7 pfu of VACVΔB2R, VACVΔE5R, VACVΔE3L83NΔE5R, VACVΔE5RΔB2R, or VACVΔE3L83NΔE5RΔB2R, and survival and weight loss were monitored daily. This figure shows weight loss after intranasal infection with these five different viruses.

FIGS. 159 A- 159 B shows that VACVΔE5RΔB2R and VACVΔE3L83NΔE5RΔB2R infection of murine BMDC induce higher levels of IFNB gene expression and IFN-γ protein secretion compared with. VACVΔB2R or VACVΔE5R. FIG. 159 A shows the IFNB gene expression in WT and cGAS knockout BMDC cells infected with VACV, VACVΔ83N, VACVΔB2R, VACVΔE5R, VACVΔE5RΔB2R, VACVΔE3L83NΔE5R, or VACVΔE3L83NΔE5RΔB2R at a MOI of 10. Cells were collected at 6 hours post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. FIG. 159 B shows the IFN-γ protein secretion by WT and cGAS knockout BMDC cells infected with VACV, VACVΔ83N, VACVΔB2R, VACVΔE5R, VACVΔE5RΔB2R, VACVΔE3L83NΔE5R, or VACVΔE3L83NΔE5RΔB2R at a MOI of 10. Supernatants were collected at 24 h post infection and IFN-γ protein levels in the supernatants were determined by ELISA.

FIG. 160 shows that VACVΔE5RΔB2R and VACVΔE3L83NΔE5RΔB2R infection of murine BMDC induce higher levels of phosphorylation of STING, IRF3 and TBK1 compared with VACVΔB2R or VACVΔE5R. WT BMDC cells were infected with VACV, VACVΔB2R, VACVΔE5R, VACVΔE5RΔB2R, VACVΔE3L83NΔE5R, or VACVΔE3L83NΔE5RΔB2R at a MOI of 10. Cell lysis were collected at different time points. The phosphorylation of STING, IRF3,and TBK1 were detected by antibodies against phosphorylated STING, IRF3,and TBK1,respectively.

FIG. 161 shows a scheme of stepwise strategy to generate recombinant VACVΔE3L83NΔTKΔE5R virus expressing anti-muCTLA-4 antibody, and hFl3L, mOX40L and mIL12 proteins through homologous recombination first at the TK and then at the E5R loci of the VACVΔE3L83N genome. pCB vector was used to insert a single expression cassette to express the anti-muCTLA-4 antibody heavy and light chains under the control of the vaccinia virus synthetic early and late promoter (PsE/L). Homologous recombination that occurred at the TK-L and TK-R sites results in the insertion of expression cassette of anti-muCTLA-4 antibody into TK locus on VACVΔE3L83N genome. pUC57 vector was used to insert two expression cassettes designed to express both hFl3L-mOX40L fusion protein and mIL-12 using the vaccinia viral synthetic early and late promoter (PsE/L). The coding sequence of the hFl3L-mOX40L was separated by a furin cleavage site followed by a Pep2A sequence. The coding sequence of p40 and p30 subunits of mIL12 was separated by a furin cleavage site followed by a Pep2A sequence. The C-terminus of p30 subunit was tagged with a matrix binding sequence. Homologous recombination at the E4L and E6R loci results in the insertion of expression cassette for hFl3L-mOX40L and mIL12 into the E5L locus of VACVΔE3L83N-ΔTK-anti-muCTLA-4 genome.

FIG. 162 shows a scheme of generating recombinant VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 virus with deletion of B2R gene through homologous recombination at the B1R and B3R loci of the VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) genome. Homologous recombination that occurred at the B1R and B3R loci results in the deletion of B2R gene from the virus genome to generate the VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12-ΔB2R virus (OV-VACVΔE5RΔB2R).

FIG. 163 shows a multistep growth curve of the recombinant viruses VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) and VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12-ΔB2R (OV-VACVΔE5RΔB2R) in BSC40 cells compared with WT VACV. BSC40 cells were infected with VACV, VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12, and VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12-ΔB2R at a MOI of 0.01. Virus samples were collected at different time points and virus titers were determined using BSC40 cells.

FIGS. 164 A- 164 B shows that VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12-ΔB2R (OV-VACVΔE5RΔB2R) infection of murine BMDC induce higher levels of IFNB gene expression and IFN-γ protein secretion compared with VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R). FIG. 164 A shows the IFNB gene expression in WT BMDC cells infected with VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12, VACVΔE3L83N-ΔTK-anti-muCTLA-4ΔE5R-hFl3L-mOX40L-mIL-12-ΔB2R, or Heat-iMVA at a MOI of 10. Cells were collected at 6 hours post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. FIG. 164 B shows the IFN-γ protein secretion in same infection as in FIG. 164 A . Supernatants were collected at 24 h post infection and IFN-γ protein levels in the supernatants were determined by ELISA.

FIGS. 165 A- 165 C show the expression of the transgenes anti-muCTLA-4, mOX40L, or human Flt3L in VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) infected B16-F10 cells and in tumors injected with virus. FIG. 165 A shows a western blot demonstrating that murine anti-CTLA-4 and human Flt3L are expressed in VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R)-infected B16-F10 cells. FL: full length of anti-muCTLA-4; HC: heavy chain of anti-muCTLA-4; LC: light chain of anti-muCTLA-4. FIG. 165 B shows a dot plot of FACS analysis of mOX40 expression on murine melanoma cells B16-F10. Cells were infected with VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (expressing GFP) at a MOI of 10 for 24 h. No virus mock infection control was included. Cells were stained with anti-mOX40L antibody. FIG. 165 C shows the mIL-12 expression in B16-F10 melanoma tumors after intratumoral injection of VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) virus. Intradermally implanted B16-F10 melanoma tumors were injected with 4×10 7 pfu of VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) in 100 μl of PBS, and tumors were collected at 48 hours after treatment. Tumor samples were lysed and the expression of murine IL-12 were examined by western blot using anti-p40 antibody.

FIGS. 166 A- 166 D . FIGS. 166 A- 166 C show the expression and secretion of murine IL-12 after VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) virus infection of three different murine cancer cell lines. Tumor cells were infected with VACV, VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12, or mock infected. Supernatant were collected at 24 and 48 hours after virus infection and the concentration of IL-12 in cell culture supernatant were determined by ELISA. FIG. 166 A shows the mIL-12 levels in OV-VACVΔE5R-infected B16-F10 melanoma cells. FIG. 166 B shows the mIL-12 levels in OV-VACVΔE5R-infected 4T1 breast cancer cells. FIG. 166 C shows the mIL-12 level in OV-VACVΔE5R-infected MC38 colon cancer cells. FIG. 166 D shows serum mIL-12 levels in mice treated with OV-VACVΔE5R. At 48 h and 72 h post intratumoral injection of the virus, mice were euthanized and blood/serum was collected for cytokine measurement by ELISA.

FIGS. 167 A- 167 B shows antitumor efficacy of intratumoral delivery of VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) either alone or in combination with anti-PD-L1 antibody in a bilateral B16-F10 tumor implantation model. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 1×10 5 to the left flank). Seven days post tumor implantation, 4×10 7 pfu of either VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R), Heat-iMVA, or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice per week. One group of the mice also received anti-PD-L1 antibody (250 μg) twice a week in conjunction with IT VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFlt3L-mOX40L-mIL-12 (OV-VACVΔE5R). Tumor volumes and mice survival were monitored. FIG. 167 A shows tumor volumes of both injected and non-injected tumors in mice treated with either PBS or VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFlt3L-mOX40L-mIL-12 (OV-VACVΔE5R), Heat-iMVA intratumorally, or with the combination of IT VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFlt3L-mOX40L-mIL-12 (OV-VACVΔE5R) plus IP anti-PD-L1. FIG. 167 B shows the Kaplan Meier survival curve of the four groups.

FIGS. 168 A- 168 B show that intratumoral injection of VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12ΔB2R (OV-VACVΔE5RΔB2R) generated stronger antitumor-specific T cells in the spleens compared with VACVΔE3L83N-ΔTK-anti-muCTLA-4-ΔE5R-hFl3L-mOX40L-mIL-12 (OV-VACVΔE5R) or Heat-iMVA. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, 4×10 7 pfu of either OV-VACVΔE5RΔB2R, OV-VACVΔE5R, an equivalent amount of Heat-iMVA, or PBS was intratumorally (IT) injected into the larger tumors on the right flank twice, three days apart. Spleens were harvested at 2 days post second injection, ELISPOT analyses were performed to evaluate tumor-specific T cells in the spleens. ELISPOT assay was performed by co-culturing irradiated B16-F10 cells (150,000) and splenocytes (1,000,000) in a 96-well plate. FIG. 168 A : Graph of IFN-γ + spots per 1,000,000 splenocytes. Each dot represents splenocytes from an individual mouse (n=4) (*P<0.05; **P<0.01, ****P<0.0001, t test). FIG. 168 B : Image of ELISPOT of triplicate samples of combined splenocytes from mice in the same treatment group.

FIG. 169 shows a scheme of stepwise strategy to generate recombinant VACVΔE3L83NΔTKΔE5R virus expressing anti-huCTLA-4 antibody, and hFl3L, hOX40L and hIL12 proteins through homologous recombination first at the TK and then at the E5R loci of the VACVΔE3L83N genome. pCB vector was used to insert a single expression cassette to express the anti-huCTLA-4 antibody heavy and light chains under the control of the vaccinia virus synthetic early and late promoter (PsE/L). Homologous recombination that occurred at the TK-L and TK-R sites results in the insertion of expression cassette of anti-huCTLA-4 antibody into TK locus on VACVΔE3L83N genome, which was generated by deleting the DNA fragment of E3L gene encoding N-terminal 83 amino acid. pUC57 vector was used to insert two expression cassettes designed to express both hFlt3L-hOX40LhOX40L fusion protein and mIL-12 using the vaccinia viral synthetic early and late promoter (PsE/L). The coding sequence of the hFl3L-mOX40L was separated by a furin cleavage site followed by a Pep2A sequence. The coding sequence of p40 and p30 subunits of hIL12 was separated by a furin cleavage site followed by a Pep2A sequence. The C-terminus of p30 subunit was tagged with a matrix binding sequence. Homologous recombination at the E4L and E6R loci results in the insertion of expression cassette for hFlt3L-hOX40LhOX40L and hIL12 into the E5L locus of VACVΔE3L83N-ΔTK-anti-muCTLA-4 genome.

FIG. 170 shows a scheme of generating recombinant myxoma virus (Lausanne strain) with deletion of M063R gene through homologous recombination at the M062R and M064R loci of the myxomaΔM127-mcherry genome, as well as generating recombinant myxoma virus (Lausanne strain) with deletion of M064R gene through homologous recombination at the M063R and M065R loci of the myxomaΔM127-mcherry genome. Homologous recombination that occurred at the M062R and M064R loci results in the deletion of M063R gene from the virus genome to generate MyxomaΔM063R virus. Homologous recombination that occurred at the M063R and M065R loci results in the deletion of M064R gene from the virus genome to generate MyxomaΔM064R virus.

FIGS. 171 A- 171 B show that MyxomaΔM064R and MyxomaΔM063R infection of murine BMDC induce higher levels of IFNB gene expression and IFN-B protein secretion compared with the parental myxoma virus expressing mcherry (Myxoma-mcherry) which also contains a deletion of the M0127 gene. BMDC cells were infected with Myxoma-mcherry, MyxomaΔM063R, MyxomaΔM064R, or MVA at a MOI of 10. Cells were collected at 6 hours post infection and RNAs were extracted. Quantitative RT-PCR analyses were performed to examine the expression of IFNB gene. FIG. 171 A shows the RT-PCR result of IFNB gene expression in infected BMDCs. Supernatants were collected at 24 h post infection and IFN-B protein levels in the supernatants were determined by ELISA. FIG. 171 B shows the ELISA results of IFN-B protein levels in the supernatants of infected BMDCs.

FIGS. 172 - 174 B are a series of graphical representations of data showing that intratumoral injection of myxomaΔM064R or myxoma-mcherry leads to activation of effector CD4 + and CD8 + T cells in a B16-F10 melanoma model. FIG. 172 shows the experimental scheme. Briefly, B16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6J mice (5×10 5 to the right flank and 2.5×10 5 to the left flank). Seven days post tumor implantation, 2×10 7 pfu of either Myxoma-mCherry, MyxomaΔM064R, MVAΔE5R or PBS was intratumorally (IT) injected into the larger tumors on the right flank. Two days post injection, the injected tumors were isolated and tumor infiltrating lymphocytes were analyzed by FACS. FIG. 172 shows that intratumoral injection of myxomaAOM64R or myxoma-mcherry leads to activation of effector CD4 + and CD8 + T cells in a B16-F10 melanoma model.

FIG. 173 A shows the representative dot plots of Granzyme B + CD8 + T cells in the injected tumors with either Myxoma-mCherry, MyxomaΔM064R, MVAΔE5R or PBS treatment. FIG. 173 B shows the graph of absolute number of CD45 + cells in the injected tumors. Data are means ±SEM (n=5-8). FIG. 173 C shows the graph of percentage of Granzyme B + CD8 + T cells out of CD8 + T cells. Data are means ±SEM (n=5-8) (*P<0.05, t test).

FIG. 174 A shows the representative dot plots of Granzyme B + CD4 + T cells in the injected tumors with either Myxoma-mCherry, MyxomaΔM064R, MVAΔE5R or PBS treatment. FIG. 174 B shows the graph of percentage of Granzyme B + CD4 + T cells out of CD4 + T cells. Data are means ±SEM (n=5-8) (**P<0.01; ***P<0.001; ****P<0.0001, t test).

It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology.

I. Definitions

The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.

As used herein, the term “about” encompasses the range of experimental error that may occur in a measurement and will be clear to the skilled artisan.

As used herein, the term “adjuvant” refers to a substance that enhances, augments, or potentiates the host's immune response to antigens, including tumor antigens.

As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intradermally, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and the administration by another.

As used herein, the term “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the antigen is contained within a whole cell, such as in a tumor antigen-containing whole cell vaccine. In some embodiments, the target antigen encompasses cancer-related antigens or neoantigens and includes proteins or other molecules expressed by tumor or non-tumor cancers, such as molecules that are present in cancer cells but absent in non-cancer cells, and molecules that are up-regulated in cancer cells as compared to non-cancer cells.

As used herein, “attenuated,” as used in conjunction with a virus, refers to a virus having reduced virulence or pathogenicity as compared to a non-attenuated counterpart, yet is still viable or live. Typically, attenuation renders an infectious agent, such as a virus, less harmful or virulent to an infected subject compared to a non-attenuated virus. This is in contrast to a killed or completely inactivated virus.

As used herein, “conjoint administration” refers to administration of a second therapeutic modality in combination with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-AWR199). For example, an immune checkpoint blocking agent, immunomodulatory agent, and/or anti-cancer drug administered in close temporal proximity with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199). For example, a PD-1/PD-L1 inhibitor and/or a CTLA-4 inhibitor (in more specific embodiments, an antibody) can be administered simultaneously (i.e., concurrently) with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L- IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) (by intravenous or intratumoral injection when the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199 is administered intratumorally or systemically as stated above) or before or after the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199 administration. In some embodiments, if the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199 administration and the immune checkpoint blocking agent, immunomodulatory agent, and/or anti-cancer drug are administered about 1 to about 7 days apart or even up to three weeks apart, this would still be within “close temporal proximity” as stated herein, therefore such administration will qualify as “conjoint.”

The term “corresponding wild-type strain” or “corresponding wild-type virus” is used herein to refer to the wild-type MVA, vaccinia virus (VACV), or myxoma virus (MYXV) strain from which the engineered MVA, vaccinia, or myxoma strain or virus was derived. As used herein, a wild-type MVA, vaccinia, or myxoma strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) a particular gene of interest and/or to express a heterologous nucleic acid. For example, in some embodiments, a wild-type MVA, vaccinia, or myxoma strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) the C7 gene and express OX40L. In other embodiments, a wild-type MVA, vaccinia, or myxoma strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) the E5R (or M31R) gene. The engineered MVA, vaccinia, or myxoma strain or virus may have been modified to disrupt or delete (knock out) the C7 gene and express OX40L alone or in combination with further modifications (e.g., engineered to express additional immunomodulatory proteins and/or comprise additional gene deletions) as described herein. Additionally or alternatively, the engineered MVA, vaccinia, or myxoma strain or virus may have been modified to disrupt or delete (knock out) the E5R (or M31R) gene alone or in combination with further modifications (e.g., engineered to express additional immunomodulatory proteins and/or comprise additional gene deletions) as described herein. The term “corresponding MVAΔE3L strain” or “corresponding MVAΔE3L virus” is used herein to refer to the MVA strain or virus having an E3L deletion alone (i.e., an MVAΔE3L strain or virus comprising no other genetic deletions or additions). The term “corresponding MVAΔC7L strain” or “corresponding MVAΔC7L virus” is used herein to refer to the MVA strain or virus having a C7L deletion alone (i.e., an MVAΔC7L strain or virus comprising no other genetic deletions or additions). The term “corresponding MVAΔE5R strain” or “corresponding MVAΔE5R virus” is used herein to refer to the MVA strain or virus having an E5R deletion alone (i.e., an MVAΔE5R strain or virus comprising no other genetic deletions or additions). The term “corresponding VACVΔC7L strain” or “corresponding VACVΔC7L virus” is used herein to refer to the vaccinia strain or virus having a C7L deletion alone (i.e., a VACVΔC7L strain or virus comprising no other genetic deletions or additions). The term “corresponding VACVΔE5R strain” or “corresponding VACVΔE5R virus” is used herein to refer to the vaccinia strain or virus having an E5R deletion alone (i.e., a VACVΔE5R strain or virus comprising no other genetic deletions or additions).

As used herein, the terms “delivering” and “contacting” refer to depositing the one or more engineered poxviruses (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) of the present disclosure in the tumor microenvironment whether this is done by local administration to the tumor (intratumoral) or by, for example, intravenous route. The term focuses on engineered virus (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) that reaches the tumor itself. In some embodiments, “delivering” is synonymous with administering, but it is used with a particular administration locale in mind, e.g., intratumoral.

The terms “disruption” and “mutation” are used interchangeably herein to refer to a detectable and heritable change in the genetic material. Mutations may include insertions, deletions, substitutions (e.g., transitions, transversion), transpositions, inversions, knockouts, and combinations thereof. Mutations may involve only a single nucleotide (e.g., a point mutation or a single nucleotide polymorphism) or multiple nucleotides. In some embodiments, mutations are silent, that is, no phenotypic effect of the mutation is detected. In other embodiments, the mutation causes a phenotypic change, for example, the expression level of the encoded product is altered, or the encoded product itself is altered. In some embodiments, a disruption or mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain. In other embodiments, a disruption or mutation may result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wild-type strain.

As used herein, an “effective amount” or “therapeutically effective amount” refers to a sufficient amount of an agent, which, when administered at one or more dosages and for a period of time, is sufficient to provide a desired biological result in alleviating, curing, or palliating a disease. In the present disclosure, an effective amount of one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) comprises an amount that (when administered for a suitable period of time and at a suitable frequency) reduces the number of cancer cells; or reduces the tumor size or eradicates the tumor; or inhibits (i.e., slows down or stops) cancer cell infiltration into peripheral organs; inhibits (i.e., slows down or stops) metastatic growth; inhibits (stabilizes or arrests) tumor growth; allows for treatment of the tumor; and/or induces and promotes an immune response against the tumor. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation in light of the present disclosure. Such determination may begin with amounts found effective in vitro and amounts found effective in animals. The therapeutically effective amount will be initially determined based on the concentration or concentrations found to confer a benefit to cells in culture. Effective amounts can be extrapolated from data within the cell culture and can be adjusted up or down based on factors such as detailed herein. Effective amounts of the viral constructs are generally within the range of about 10 5 to about 10 10 plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage is about 10 6 -10 9 pfu. In some embodiments, a unit dosage is administered in a volume within the range from 1 to 10 mL. The equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, pfu is equal to about 5 to 100 virus particles. A therapeutically effective amount the hFlt3L transgene bearing viruses can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration. For example, a therapeutically effective amount of hFlt3L bearing viruses in accordance with the present disclosure may vary according to factors such as the disease state, age, sex, weight, and general condition of the subject, and the potency of the viral constructs to elicit a desired immunological response in the particular subject for the particular cancer.

With particular reference to the viral-based immunostimulatory agents disclosed herein, an “effective amount” or “therapeutically effective amount” refers to an amount of a composition comprising one or more one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYMVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) sufficient to reduce, inhibit, or abrogate tumor cell growth, thereby reducing or eradicating the tumor, or sufficient to inhibit, reduce or abrogate metastatic spread either in vitro, ex vivo, or in a subject or to elicit and promote an immune response against the tumor that will eventually result in one or more of metastatic spread reduction, inhibition, and/or abrogation as the case may be. The reduction, inhibition, or eradication of tumor cell growth may be the result of necrosis, apoptosis, or an immune response, or a combination of two or more of the foregoing (however, the precipitation of apoptosis, for example, may not be due to the same factors as observed with oncolytic viruses). The amount that is therapeutically effective may vary depending on such factors as the particular virus used in the composition, the age and condition of the subject being treated, the extent of tumor formation, the presence or absence of other therapeutic modalities, and the like. Similarly, the dosage of the composition to be administered and the frequency of its administration will depend on a variety of factors, such as the potency of the active ingredient, the duration of its activity once administered, the route of administration, the size, age, sex, and physical condition of the subject, the risk of adverse reactions and the judgment of the medical practitioner. The compositions are administered in a variety of dosage forms, such as injectable solutions.

With particular reference to combination therapy with an immune checkpoint inhibitor, an “effective amount” or “therapeutically effective amount” for an immune checkpoint blocking agent means an amount of an immune checkpoint blocking agent sufficient to reverse or reduce immune suppression in the tumor microenvironment and to activate or enhance host immunity in the subject being treated. Immune checkpoint blocking agents include, but are not limited to, inhibitory antibodies against CD28 inhibitor such as CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti-PD-1 (programmed Death 1) inhibitory antibodies (e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-L1 (Programmed death ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736, MSB 00107180), as well as inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, BTLA, or PDR001, and combinations thereof. Dosage ranges of the foregoing are known or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible.

In some embodiments, the tumor expresses the particular checkpoint, but in the context of the present technology, this is not strictly necessary as immune checkpoint blocking agents block more generally immune suppressive mechanisms within the tumors, elicited by tumor cells, stromal cells, and tumor-infiltrating immune cells.

For example, the CTLA-4 inhibitor ipilimumab, when administered as adjuvant therapy after surgery in melanoma, is administered at 1-2 mg/mL over 90 minutes for a total infusion amount of 3 mg/kg every three weeks for a total of 4 doses. This therapy is often accompanied by severe, even life-threatening, immune-mediated adverse reactions, which limits the tolerated dose as well as the cumulative amount that can be administered. It is anticipated that it will be possible to reduce the dose and/or cumulative amount of ipilimumab when it is administered conjointly with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199). In particular, in light of the experimental results set forth below, it is anticipated that it will be further possible to reduce the CTLA-4 inhibitor's dose if it is administered directly to the tumor conjointly with one or both the foregoing MVA viruses. Accordingly, the amounts provided above for ipilimumab may be a starting point for determining the particular dosage and cumulative amount to be given to a patient in conjoint administration.

As another example, pembrolizumab is prescribed for administration as adjuvant therapy in melanoma diluted to 25 mg/mL. It is administered at a dosage of 2 mg/kg over 30 minutes every three weeks. This may be a starting point for determining dosage and administration in the conjoint administration of one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199).

Nivolumab could also serve as a starting point in determining the dosage and administration regimen of checkpoint inhibitors administered in combination with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199). Nivolumab is prescribed for administration at 3 mg/kg as an intravenous infusion over 60 minutes every two weeks.

Immune stimulating agents such as agonist antibodies have also been explored as immunotherapy for cancers. For example, anti-ICOS antibody binds to the extracellular domain of ICOS leading to the activation of ICOS signaling and T-cell activation. Anti-OX40 antibody can bind to OX40 and potentiate T-cell receptor signaling leading to T-cell activation, proliferation and survival. Other examples include agonist antibodies against 4-1BB (CD137), GITR.

The immune stimulating agonist antibodies can be used systemically in combination with intratumoral injection of one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199). Alternatively, the immune stimulating agonist antibodies can be used conjointly with one or more engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199) via intratumoral delivery either simultaneously (i.e., concurrently) or sequentially.

The term “immunomodulatory drug” is used herein to refer to Fingolimod (FTY720).

The terms “engineered” or “genetically engineered” are used herein to refer to an organism that has been manipulated to be genetically altered, modified, or changed, e.g., by disruption of the genome. For example, an “engineered vaccinia virus strain,” “engineered modified vaccinia Ankara virus,” or “engineered myxoma virus” refers to a vaccinia, modified vaccinia Ankara, or myxoma strain that has been manipulated to be genetically altered, modified, or changed. In the present context, “engineered” or “genetically engineered” includes recombinant vaccinia viruses, recombinant modified vaccinia Ankara viruses, and recombinant myxoma viruses.

The term “gene cassette” is used herein to refer to a DNA sequence encoding and capable of expressing one or more genes of interest (e.g., OX40L, hFlt3L, a selectable marker, or a combination thereof) that can be inserted between one or more selected restriction sites of a DNA sequence. In some embodiments, insertion of a gene cassette results in a disrupted gene. In some embodiments, disruption of the gene involves replacement of at least a portion of the gene with a gene cassette, which includes a nucleotide sequence encoding a gene of interest (e.g., OX40L, hFlt3L, a selectable marker, or a combination thereof).

As used herein, “heterologous nucleic acid,” refers to a nucleic acid, DNA, or RNA, which has been introduced into a virus, and which is not a copy of a sequence naturally found in the virus into which it is introduced. Such heterologous nucleic acid may comprise segments that are a copy of a sequence that is naturally found in the virus into which it has been introduced.

As used herein, wherever a gene is described, the gene may be either human or murine such that the designation of human (h or hu) or murine (m or mu) may be used interchangeably and is not intended to be limiting. For example, where mIL-12 is described, hIL-12 may be substituted for mIL-12 in the described constructs, and vice versa.

As used herein, “IL-15/IL-15Rα” encompasses membrane bound hIL-15/IL-15Rα transpresentation constructs and fusion proteins as described in Van den Bergh et al. ( Pharmacology & Therapeutics 170:73-79 (2017); Kowalsky et al. ( Molecular Therapy 26(10):2476-2486 (2018); Stoklasek et al. ( J. Immunol. 177:6072-6080); Duboi et al. ( J. Immunol. 180:2099-2106 (2008); Epardaud et al, ( Cancer Res. 68:2972-2983 (2008); and Dubois et al. ( Immunity 17:537-547 (2002), each of which is herein incorporated by reference.

As used herein, “immune checkpoint inhibitor” or “immune checkpoint blocking agent” or “immune checkpoint blockade inhibitor” refers to molecules that completely or partially reduce, inhibit, interfere with, or modulate the activity of one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Checkpoint proteins include, but are not limited to, CD28 receptor family members, CTLA-4 and its ligands CD80 and CD86; PD-1 and its ligands PD-L1 and PD-L2; LAG3, B7-H3, B7-H4, TIM3, ICOS, II DLBCL, BTLA or any combination of two or more of the foregoing. Non-limiting examples of immune checkpoint blocking agents contemplated for use herein include, but are not limited to, inhibitory antibodies against CD28 inhibitor such as CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti-PD-1 (programmed Death 1) inhibitory antibodies (e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-L1 (Programmed death ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736, MSB 00107180), as well as inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS, DLBCL inhibitors, or BTLA, PDR001, and combinations thereof.

As used herein, “immune response” refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, etc. An immune response may include a cellular response, such as a T-cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, i.e., T-cell function. A T-cell response may include generation, proliferation or expansion, or stimulation of a particular type of T-cell, or subset of T-cells, for example, effector CD4 + , CD4 + helper, effector CD8 + , CD8 + cytotoxic, or natural killer (NK) cells. Such T-cell subsets may be identified by detecting one or more cell receptors or cell surface molecules (e.g., CD or cluster of differentiation molecules). A T-cell response may also include altered expression (statistically significant increase or decrease) of a cellular factor, such as a soluble mediator (e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin) that influences the differentiation or proliferation of other cells. For example, Type I interferon (IFN-α/β) is a critical regulator of the innate immunity (Huber et al., Immunology 132(4):466-474 (2011)). Animal and human studies have shown a role for IFN-α/β in directly influencing the fate of both CD4 + and CD8 + T-cells during the initial phases of antigen recognition and anti-tumor immune response. IFN Type I is induced in response to activation of dendritic cells, in turn a sentinel of the innate immune system. An immune response may also include humoral (antibody) response.

The term “immunogenic composition” is used herein to refer to a composition that will elicit an immune response in a mammal that has been exposed to the composition. In some embodiments, an immunogenic composition comprises MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, and/or MVAΔE5R-hFlt3L-OX40L-ΔWR199, an antigen, an adjuvant comprising any one or more of the foregoing engineered viruses, and/or an adjuvant comprising MVAΔC7L-hFlt3L-TK(−)-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, and/or Heat-iMVAΔE5R alone or in combination with immune checkpoint blockade inhibitors. As used herein, an immunogenic composition encompasses vaccines. In some embodiments, the immunogenic composition comprises a tumor antigen-containing whole cell vaccine (e.g., an irradiated whole cell vaccine).

As used herein, the term “inactivated MVA” refers to heat-inactivated MVA (Heat-iMVA) and/or UV-inactivated MVA which are infective, nonreplicative, and do not suppress IFN Type I production in infected DC cells. As used herein, the term “inactivated vaccinia virus” includes heat-inactivated vaccinia virus and/or UV-inactivated vaccinia virus. MVA or vaccinia virus inactivated by a combination of heat and UV radiation is also within the scope of the present disclosure.

As used herein, “Heat-inactivated MVA” (Heat-iMVA) and “Heat-inactivated vaccinia virus” refer to MVA and vaccinia virus, respectively, which have been exposed to heat treatment under conditions that do not destroy its immunogenicity or its ability to enter target cells (tumor cells) but remove residual replication ability of the virus as well as factors that inhibit the host's immune response. An example of such conditions is exposure to a temperature within the range of about 50 to about 60° C. for a period of time of about an hour. Other times and temperatures can be determined by one of skill in the art.

As used herein, “UV-inactivated MVA” and “UV-inactivated vaccinia virus” refer to MVA and vaccinia virus, respectively, that have been inactivated by exposure to UV under conditions that do not destroy its immunogenicity or its ability to enter target cells (tumor cells) but remove residual replication ability of the virus. An example of such conditions, which can be useful in the present methods, is exposure to UV using, for example, a 365 nm UV bulb for a period of about 30 min to about 1 hour. Other limits of these conditions of UV wavelength and exposure can be determined by one of skill in the art.

A “knock out,” “knocked out gene,” or a “gene deletion” refers to a gene including a null mutation (e.g., the wild-type product encoded by the gene is not expressed, expressed at levels so low as to have no effect, or is non-functional). In some embodiments, the knocked out gene includes heterologous sequences (e.g., one or more gene cassettes comprising a heterologous nucleic acid sequence) or genetically engineered non-functional sequences of the gene itself, which renders the gene non-functional. In other embodiments, the knocked out gene is lacking a portion of the wild-type gene. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 60% of the wild-type gene sequence is deleted. In other embodiments, the knocked out gene is lacking at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or at least about 100% of the wild-type gene sequence. In other embodiments, the knocked out gene may include up to 100% of the wild-type gene sequence (e.g., some portion of the wild-type gene sequence may be deleted) but also include one or more heterologous and/or non-functional nucleic acid sequences inserted therein.

As used herein, “metastasis” refers to the spread of cancer from its primary site to neighboring tissues or distal locations in the body. Cancer cells (including cancer stem cells) can break away from a primary tumor, penetrate lymphatic and blood vessels, circulate through the bloodstream, and grow in normal tissues elsewhere in the body. Metastasis is a sequential process, contingent on tumor cells (or cancer stem cells) breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. Once at another site, cancer cells re-penetrate through the blood vessels or lymphatic walls, continue to multiply, and eventually form a new tumor (metastatic tumor). In some embodiments, this new tumor is referred to as a metastatic (or secondary) tumor.

As used herein, “MVA” means “modified vaccinia Ankara” and refers to a highly attenuated strain of vaccinia derived from the Ankara strain and developed for use as a vaccine and vaccine adjuvant. The original MVA was isolated from the wild-type Ankara strain by successive passage through chicken embryonic cells. Treated thus, it lost about 15% of the genome of wild-type vaccinia including its ability to replicate efficiently in primate (including human) cells. (Mayr et al., Zentralbl Bakteriol B 167:375-390 (1978)). MVA is considered an appropriate candidate for development as a recombinant vector for gene or vaccination delivery against infectious diseases or tumors. (Verheust et al., Vaccine 30(16):2623-2632 (2012)). MVA has a genome of 178 kb in length and a sequence first disclosed in Antoine et al., Virol. 244(2):365-396 (1998). Sequences are also disclosed in GenBank Accession No. U94848.1 (SEQ ID NO: 1). Clinical grade MVA is commercially and publicly available from Bavarian Nordic A/S Kvistgaard, Denmark. Additionally, MVA is available from ATCC, Rockville, MD, and from CMCN (Institut Pasteur Collection Nationale des Microorganismes) Paris, France.

The term “MVAΔC7L,” is used herein to refer to a modified vaccinia Ankara (MVA) mutant virus or a vaccine comprising the virus, in which the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MVAΔC7L” includes a deletion mutant of MVA which lacks a functional C7L gene and is infective but non-replicative and it is further impaired in its ability to evade the host's immune system. As used herein, “MVAΔC7L” encompasses a recombinant MVA virus that does not express a functional C7 protein. In some embodiments, the ΔC7L mutant includes a heterologous nucleic acid sequence in place of all or a majority of the C7L gene sequence. For example, as used herein, “MVAΔC7L” encompasses a recombinant MVA nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of C7 in the MVA genome (e.g., position 18,407 to 18,859 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MVAΔC7L-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) (“MVAΔC7L-hFlt3L”). In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MVAΔC7L virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus of the MVA genome (e.g., position 75,560 to 76,093 of SEQ ID NO: 1), splitting the TK gene and obliterating it (“MVAΔC7L-OX40L-TK(−)”; “MVAΔC7L-hFlt3L-TK(−)”). In some embodiments, MVAΔC7L encompasses a recombinant MVA virus in which all or a majority of the C7L gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MVAΔC7L-hFlt3L-TK(−)-OX40L”). In some embodiments, the recombinant MVAΔC7L-OX40L viruses of the present technology are further modified to express at least one further heterologous gene, such as any one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L (where “h” or “hu” designates the human protein), and/or include at least one further viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. For example, in some embodiments, MVAΔC7L-hFlt3L-TK(−)-OX40 is further modified to comprise a deletion of E5R, in which the E5R gene is replaced by a selectable marker (e.g., mCherry) through homologous recombination at the E4L and E6R loci. In some embodiments, MVAΔC7L is modified to express one or more heterologous genes from within other loci, such as the E5R locus. For example, in some embodiments, MVAΔC7L encompasses a recombinant MVA virus in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MVAΔC7LΔE5R-hFlt3L-OX40L.” In other embodiments, the recombinant MVAΔC7L-OX40L viruses of the present technology contain no further heterologous genes and/or viral gene mutations other than those specifically referred to in the name of the virus.

The term “MVAΔE3L” means a deletion mutant of MVA which lacks a functional E3L gene and is infective but non-replicative and it is further impaired in its ability to evade the host's immune system. It has been used as a vaccine vector to transfer tumor or viral antigens. The mutant MVA E3L knockout and its preparation have been described in U.S. Pat. No. 7,049,145, for example. As used herein, “MVAΔE3L” encompasses a recombinant MVA modified to express a specific gene of interest (SG), such as OX40L (“MVAΔE3L-OX40L”). In some embodiments, the MVAΔE3L virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus, splitting the TK gene and obliterating it (“MVAΔE3L-OX40L-TK(−)”; “MVAΔE3L-hFlt3L-TK(−)”). In some embodiments, the recombinant MVAΔE3L-OX40L viruses of the present technology are further modified to express at least one further heterologous gene, such as any one or more hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following deletions: B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, the recombinant MVAΔE3L-OX40L viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “MVAΔE5R,” is used herein to refer to a modified vaccinia Ankara (MVA) mutant virus or a vaccine comprising the virus, in which the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MVAΔE5R” includes a deletion mutant of MVA which lacks a functional E5R gene and is infective but non-replicative and it is further impaired in its ability to evade the host's immune system. As used herein, “MVAΔE5R” encompasses a recombinant MVA virus that does not express a functional E5 protein. In some embodiments, the ΔE5R mutant includes a heterologous nucleic acid sequence in place of all or a majority of the E5R gene sequence. For example, as used herein, “MVAΔE5R” encompasses a recombinant MVA nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of E5R in the MVA genome (e.g., position 38,432 to 39,385 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MVAΔE5R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) (“MVAΔE5R-hFlt3L”). In some embodiments, MVAΔE5R encompasses a recombinant MVA wherein the E5R locus is modified to express one or more heterologous genes. For example, in some embodiments, MVAΔE5R encompasses a recombinant MVA in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MVAΔE5R-hFlt3L-OX40L.” In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MVAΔE5R virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus of the MVA genome (e.g., position 75,560 to 76,093 of SEQ ID NO: 1), splitting the TK gene and obliterating it (“MVAΔE5R-OX40L-TK(−)”; “MVAΔE5R-hFlt3L-TK(−)”). In some embodiments, MVAΔE5R encompasses a recombinant MVA virus in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MVAΔE5R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered MVAΔE5R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L (where “h” or “hu” designates the human protein), and/or include at least one further viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, the MVAΔE5R viruses of the present technology contain no further heterologous genes and/or viral gene mutations other than those specifically referred to in the name of the virus.

The term “MVAΔWR199,” is used herein to refer to a modified vaccinia Ankara (MVA) mutant virus or a vaccine comprising the virus, in which the WR199 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MVAΔWR199” includes a deletion mutant of MVA which lacks a functional WR199 gene and is infective but non-replicative and it is further impaired in its ability to evade the host's immune system. As used herein, “MVAΔWR199” encompasses a recombinant MVA virus that does not express a functional E5 protein. In some embodiments, the ΔWR199 mutant includes a heterologous nucleic acid sequence in place of all or a majority of the WR199 gene sequence. For example, as used herein, “MVAΔWR199” encompasses a recombinant MVA nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of WR199 in the MVA genome (e.g., position 158,399 to 160,143 of the sequence set forth in GenBank Accession No. AY603355) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MVAΔWR199-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) (“MVAΔWR199-hFlt3L”). In some embodiments, MVAΔWR199 encompasses a recombinant MVA wherein the WR199 locus is modified to express one or more heterologous genes. For example, in some embodiments, MVAΔWR199 encompasses a recombinant MVA in which all or a majority of the WR199 gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MVAΔWR199-hFlt3L-OX40L.” In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MVAΔWR199 virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus of the MVA genome (e.g., position 75,560 to 76,093 of SEQ ID NO: 1), splitting the TK gene and obliterating it (“MVAΔWR199-OX40L-TK(−)”; “MVAΔWR199-hFlt3L-TK(−)”). In some embodiments, MVAΔWR199 encompasses a recombinant MVA virus in which all or a majority of the WR199 gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MVAΔWR199-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered MVAΔWR199 viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L (where “h” or “hu” designates the human protein), and/or include at least one further viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; and/or N2L. In other embodiments, the MVAΔWR199 viruses of the present technology contain no further heterologous genes and/or viral gene mutations other than those specifically referred to in the name of the virus.

The term “VACVΔC7L,” is used herein to refer to a vaccinia mutant virus or vaccine comprising the virus in which the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “VACVΔC7L” encompasses a recombinant vaccinia virus (VACV) that does not express a functional C7 protein. In some embodiments, the vaccinia virus is derived from the Western Reserve (WR) strain. In some embodiments, the ΔC7L mutant includes a heterologous sequence in place of all or a majority of the C7L gene sequence. For example, as used herein, “VACVΔC7L” encompasses a recombinant vaccinia virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of C7 in the VACV genome (e.g., position 15,716 to 16,168 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“VACVΔC7L-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“VACVΔC7L-hFlt3L”). In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the VACVΔC7L virus encompasses a recombinant vaccinia virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), splitting the TK gene and obliterating it (“VACVΔC7L-OX40L-TK(−)”; “VACVΔC7L-hFlt3L-TK(−)”). In some embodiments, VACVΔC7L encompasses a recombinant vaccinia virus in which all or a majority of the C7L gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“VACVΔC7L-hFlt3L-TK(−)-OX40L”). In some embodiments, the recombinant VACVΔC7L-OX40L viruses of the present technology are further modified to express at least one additional heterologous gene, such as any one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. For example, in some embodiments, the disclosure of the present technology provides a recombinant VACVΔ E3L83N-hFlt3L-anti-CTLA-4-ΔC7L-OX40L virus. In other embodiments, the recombinant VACVΔC7L-OX40L viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “VACVΔE5R,” is used herein to refer to a vaccinia mutant virus or vaccine comprising the virus in which the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “VACVΔE5R” encompasses a recombinant vaccinia virus (VACV) that does not express a functional E5 protein. In some embodiments, the vaccinia virus is derived from the Western Reserve (WR) strain. In some embodiments, the ΔE5R mutant includes a heterologous sequence in place of all or a majority of the E5R gene sequence. For example, as used herein, “VACVΔE5R” encompasses a recombinant vaccinia virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of E5R in the VACV genome (e.g., position 49,236 to 50,261 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“VACVΔE5R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“VACVΔE5R-hFlt3L”). In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the VACVΔE5R virus encompasses a recombinant vaccinia virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), splitting the TK gene and obliterating it (“VACVΔE5R-OX40L-TK(−)”; “VACVΔE5R-hFlt3L-TK(−)”). In some embodiments, VACVΔE5R encompasses a recombinant vaccinia virus in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“VACVΔE5R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered VACVΔE5R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. For example, in some embodiments, the disclosure of the present technology provides a recombinant VACVΔE3L-hFlt3L-anti-CTLA-4-OX40L-ΔE5R virus. As another example, in some embodiments, the TK locus of the vaccinia genome is modified through homologous recombination to express both the heavy and light chain of an antibody, such as anti-CTLA-4, wherein the coding sequences of the heavy chain and light chain are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence to produce VACV-TK(−)-anti-CTLA-4. In some embodiments, the VACV-TK(−)-anti-CTLA-4 genome is further modified to comprise a deletion of E5R, in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as VACV-TK − -anti-CTLA-4-E5R − -hFlt3L-OX40L (or VACVΔE5R-TK(−)-anti-CTLA-4-hFlt3L-OX40L). In other embodiments, the VACVΔE5R viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “VACVΔB2R,” is used herein to refer to a vaccinia mutant virus or vaccine comprising the virus in which the B2R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “VACVΔB2R” encompasses a recombinant vaccinia virus (VACV) that does not express a functional B2 protein. In some embodiments, the vaccinia virus is derived from the Western Reserve (WR) strain. In some embodiments, the ΔB2R mutant includes a heterologous sequence in place of all or a majority of the B2R gene sequence. For example, as used herein, “VACVΔB2R” encompasses a recombinant vaccinia virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of B2R in the VACV genome (e.g., position 164,856 to 165,530 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“VACVΔB2R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“VACVΔB2R-hFlt3L”). In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the VACVΔB2R virus encompasses a recombinant vaccinia virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), splitting the TK gene and obliterating it (“VACVΔB2R-OX40L-TK(−)”; “VACVΔB2R-hFlt3L-TK(−)”). In some embodiments, VACVΔB2R encompasses a recombinant vaccinia virus in which all or a majority of the B2R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“VACVΔB2R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered VACVΔB2R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. As another example, in some embodiments, the TK locus of the vaccinia genome is modified through homologous recombination to express both the heavy and light chain of an antibody, such as anti-CTLA-4, wherein the coding sequences of the heavy chain and light chain are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence to produce VACV-TK(−)-anti-CTLA-4. In some embodiments, the VACV-TK(−)-anti-CTLA-4 genome is further modified to comprise a deletion of B2R, in which all or a majority of the B2R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence. In other embodiments, the VACVΔB2R viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “MYXVΔM31R,” is used herein to refer to a myxoma mutant virus or vaccine comprising the virus in which the M31R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). Myxoma virus M31R is the ortholog of the vaccinia virus E5R. As used herein, “MYXVΔM31R” encompasses a recombinant myxoma virus (MYXV) that does not express a functional M31R protein. In some embodiments, the ΔM31R mutant includes a heterologous sequence in place of all or a majority of the M31R gene sequence. For example, as used herein, “MYXVΔM31R” encompasses a recombinant myxoma virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of M31R in the MYXV genome (e.g., position 30,138 to 31,319 of the MYXV genome) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MYXVΔM31R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“MYXVΔM31R-hFlt3L”). In some embodiments, MYXVΔM31R encompasses a recombinant MYXV wherein the M31R locus is modified to express one or more heterologous genes. For example, in some embodiments, MYXVΔM31R encompasses a recombinant MYXV in which all or a majority of the M31R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MYXVΔM31R-hFlt3L-OX40L.” In some embodiments, the heterologous nucleic acid sequence further comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MYXVΔM31R virus encompasses a recombinant myxoma virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 57,797 to 58,333 of the myxoma genome), splitting the TK gene and obliterating it (“MYXVΔM31R-OX40L-TK(−)”; “MYXVΔM31R-hFlt3L-TK(−)”). In some embodiments, MYXVΔM31R encompasses a recombinant myxoma virus in which all or a majority of the M31R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MYXVΔM31R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered MYXVΔM31R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following myxoma orthologs of vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, the MYXVΔM31R viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “MYXVΔM63R,” is used herein to refer to a myxoma mutant virus or vaccine comprising the virus in which the M63R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MYXVΔM63R” encompasses a recombinant myxoma virus (MYXV) that does not express a functional M63R protein. In some embodiments, the ΔM63R mutant includes a heterologous sequence in place of all or a majority of the M63R gene sequence. For example, as used herein, “MYXVΔM63R” encompasses a recombinant myxoma virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of M63R in the MYXV genome is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MYXVΔM63R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“MYXVΔM63R-hFlt3L”). In some embodiments, MYXVΔM63R encompasses a recombinant MYXV wherein the M63R locus is modified to express one or more heterologous genes. For example, in some embodiments, MYXVΔM63R encompasses a recombinant MYXV in which all or a majority of the M63R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MYXVΔM63R-hFlt3L-OX40L.” Additinally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MYXVΔM63R virus encompasses a recombinant myxoma virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 57,797 to 58,333 of the myxoma genome), splitting the TK gene and obliterating it (“MYXVΔM63R-OX40L-TK(−)”; “MYXVΔM63R-hFlt3L-TK(−)”). In some embodiments, MYXVΔM63R encompasses a recombinant myxoma virus in which all or a majority of the M63R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MYXVΔM63R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered MYXVΔM63R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following myxoma orthologs of vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In some embodiments, MYXVΔM63R is further engineered to comprise additional myxoma gene deletions (e.g., ΔM31R, ΔM62R, and/or ΔM64R). In other embodiments, the MYXVΔM63R viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

The term “MYXVΔM64R,” is used herein to refer to a myxoma mutant virus or vaccine comprising the virus in which the M64R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). As used herein, “MYXVΔM64R” encompasses a recombinant myxoma virus (MYXV) that does not express a functional M64R protein. In some embodiments, the ΔM64R mutant includes a heterologous sequence in place of all or a majority of the M64R gene sequence. For example, as used herein, “MYXVΔM64R” encompasses a recombinant myxoma virus nucleic acid sequence, wherein the nucleic acid sequence corresponding to the position of M64R in the MYXV genome is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L (“MYXVΔM64R-OX40L”) or human Fms-like tyrosine kinase 3 ligand (hFlt3L) gene (“MYXVΔM64R-hFlt3L”). In some embodiments, MYXVΔM64R encompasses a recombinant MYXV wherein the M64R locus is modified to express one or more heterologous genes. For example, in some embodiments, MYXVΔM64R encompasses a recombinant MYXV in which all or a majority of the M64R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as “MYXVΔM64R-hFlt3L-OX40L.” Additinally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MYXVΔM64R virus encompasses a recombinant myxoma virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the TK locus (e.g., position 57,797 to 58,333 of the myxoma genome), splitting the TK gene and obliterating it (“MYXVΔM64R-OX40L-TK(−)”; “MYXVΔM64R-hFlt3L-TK(−)”). In some embodiments, MYXVΔM64R encompasses a recombinant myxoma virus in which all or a majority of the M64R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second gene of interest (e.g., OX40L) is inserted into the TK locus (“MYXVΔM64R-hFlt3L-TK(−)-OX40L”). In some embodiments, the engineered MYXVΔM64R viruses of the present technology are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15RΔ, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one viral gene mutation or deletion, such as any one or more of the following myxoma orthologs of vaccinia viral deletions: E3L (ΔE3L); E3LΔ83N; C7L (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In some embodiments, MYXVΔM64R is further engineered to comprise additional myxoma gene deletions (e.g., ΔM31R, ΔM62R, and/or ΔM63R). In other embodiments, the MYXVΔM64R viruses of the present technology do not express any further heterologous genes and/or do not include any additional viral gene mutations or deletions other than those specifically indicated in the name of the virus.

As used herein, “oncolytic virus” refers to a virus that preferentially infects cancer cells, replicates in such cells, and induces lysis of the cancer cells through its replication process. Nonlimiting examples of naturally occurring oncolytic viruses include vesicular stomatitis virus, reovirus, as well as viruses engineered to be oncoselective such as adenovirus, Newcastle disease virus and herpes simplex virus (See, e.g., Nemunaitis, J. Invest. New Drugs 17(4):375-86 (1999); Kirn, DH et al., Nat. Rev. Cancer 9(1):64-71(2009); Kirn et al., Nat. Med. 7:781 (2001); Coffey et al., Science 282:1332 (1998)). Vaccinia virus infects many types of cells but replicates preferentially in tumor cells due to the fact that tumor cells have a metabolism that favors replication, exhibit activation of certain pathways that also favor replication and create an environment that evades the innate immune system, which also favors viral replication.

As used herein, “parenteral,” when used in the context of administration of a therapeutic substance or composition, includes any route of administration other than administration through the alimentary tract. Particularly relevant for the methods disclosed herein are intravenous (including, for example, through the hepatic portal vein for hepatic delivery), intratumoral, or intrathecal administration.

The terms “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” refer to an excipient, diluent, carrier, and/or adjuvant useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient, diluent, carrier, and adjuvant that is acceptable for pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used in the specification and claims includes one and more such excipients, diluents, carriers, and adjuvants.

As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

As used herein, the term “recombinant” when used with reference, e.g., to a virus, or cell, or nucleic acid, or protein, or vector, indicates that the virus, cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a virus or cell so modified. Thus, for example, recombinant viruses or cells express genes that are not found within the native (non-recombinant) form of the virus or cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

As used herein, “solid tumor” refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, except for hematologic cancers such as lymphomas, leukemias, and multiple myeloma. Examples of solid tumors include, but are not limited to: soft tissue sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain/CNS tumors (e.g., astrocytoma, glioma, glioblastoma, childhood tumors, such as atypical teratoid/rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Some of the most common solid tumors for which the compositions and methods of the present disclosure would be useful include: head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) ovarian cancer, cervical cancer prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, bladder cancer, ductal carcinoma in situ, renal cell carcinoma, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder, gastrointestinal, Wilms' tumor, heart, head and neck, laryngeal and hypopharyngeal, oral (e.g., lip, mouth, salivary gland), nasopharyngeal, neuroblastoma, peritoneal, pituitary, Kaposi's sarcoma, small intestine, stomach, testicular, thymus, thyroid, parathyroid, vaginal tumor, and the metastases of any of the foregoing.

As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably herein, and can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, “subject” means any animal (mammalian, human, or other) patient that can be afflicted with cancer and when thus afflicted is in need of treatment. In some embodiments, “subject” means human.

As used herein, a “synergistic therapeutic effect” in some embodiments reflects a greater-than-additive therapeutic effect that is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. In some embodiments, a “synergistic therapeutic effect” reflects an enhanced therapeutic effect that is produced by a combination of at least two agents relative to the individual administration of the agents. For example, lower doses of one or more agents may be used in treating a disease or disorder, resulting in increased therapeutic efficacy and decreased side-effects.

“Treating,” “treat,” “treated,” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission. In some embodiments, “inhibiting,” means reducing or slowing the growth of a tumor. In some embodiments, the inhibition of tumor growth may be, for example, by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In some embodiments, the inhibition may be complete.

It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

As used herein, “tumor immunity” refers to one or more processes by which tumors evade recognition and clearance by the immune system. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated or eliminated, and the tumors are recognized and attacked by the immune system (the latter being termed herein “anti-tumor immunity”). An example of tumor recognition is tumor binding, and examples of tumor attack are tumor reduction (in number, size, or both) and tumor clearance.

As used herein, “T-cell” refers to a thymus derived lymphocyte that participates in a variety of cell-mediated adaptive immune reactions. As used herein, “effector T-cell” includes helper, killer, and regulatory T-cells.

As used herein, “helper T-cell” refers to a CD4 + T-cell; helper T-cells recognize antigen bound to MHC Class II molecules. There are at least two types of helper T-cells, Th1 and Th2, which produce different cytokines.

As used herein, “cytotoxic T-cell” refers to a T-cell that usually bears CD8 molecular markers on its surface (CD8 + ) and that functions in cell-mediated immunity by destroying a target T-cell having a specific antigenic molecule on its surface. Cytotoxic T-cells also release Granzyme, a serine protease that can enter target T-cells via the perforin-formed pore and induce apoptosis (cell death). Granzyme serves as a marker of cytotoxic phenotype. Other names for cytotoxic T-cell include CTL, cytolytic T-cell, cytolytic T lymphocyte, killer T-cell, or killer T lymphocyte. Targets of cytotoxic T-cells may include virus-infected cells, cells infected with bacterial or protozoal parasites, or cancer cells. Most cytotoxic T-cells have the protein CD8 present on their cell surfaces. CD8 is attracted to portions of the Class I MHC molecule. Typically, a cytotoxic T-cell is a CD8 + cell.

As used herein, “tumor-infiltrating leukocytes” refers to white blood cells of a subject afflicted with a cancer (such as melanoma), that are resident in or otherwise have left the circulation (blood or lymphatic fluid) and have migrated into a tumor.

As used herein, “vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned,” “operatively linked,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from a transcribed gene. A non-limiting example of a pCB-OX40L-gpt vector according to the present technology is set forth in SEQ ID NO: 3. A non-limiting example of a pUC57-hFlt3L-GFP vector according to the present technology is set forth in SEQ ID NO: 4. A non-limiting example of a pUC57-delC7-hOX40L-mCherry vector is set forth in SEQ ID NO: 5.

The term “virulence” as used herein to refer to the relative ability of a pathogen to cause disease. The term “attenuated virulence” or “reduced virulence” is used herein to refer to a reduced relative ability of a pathogen to cause disease.

II. Immune System and Cancer

Malignant tumors are inherently resistant to conventional therapies and present significant therapeutic challenges. Immunotherapy has become an evolving area of research and an additional option for the treatment of certain types of cancers. The immunotherapy approach rests on the rationale that the immune system may be stimulated to identify tumor cells and target them for destruction.

Numerous studies support the importance of the differential presence of immune system components in cancer progression (Jochems et al., Exp. Biol. Med. 236(5):567-579 (2011)). Clinical data suggest that high densities of tumor-infiltrating lymphocytes are linked to improved clinical outcome (Mlecnik et al., Cancer Metastasis Rev. 30:5-12, (2011)). The correlation between a robust lymphocyte infiltration and patient survival has been reported in various types of cancer, including melanoma, ovarian, head and neck, breast, bladder, urothelial, colorectal, lung, hepatocellular, gallbladder, and esophageal cancer (Angell et al., Current Opinion in Immunology 25:1-7, (2013)). Tumor immune infiltrates include macrophages, dendritic cells (DC), monocytes, neutrophils, natural killer (NK) cells, naïve and memory lymphocytes, B cells and effector T-cells (T lymphocytes), primarily responsible for the recognition of antigens expressed by tumor cells and subsequent destruction of the tumor cells by cytotoxic T-cells.

Despite presentation of antigens by cancer cells and the presence of immune cells that could potentially react against tumor cells, in many cases the immune system does not get activated or is affirmatively suppressed. Key to this phenomenon is the ability of tumors to protect themselves from immune response by coercing cells of the immune system to inhibit other cells of the immune system. Tumors develop a number of immunomodulatory mechanisms to evade antitumor immune responses. For example, tumor cells secrete immune inhibitory cytokines (such as TGF-β) or induce immune cells, such as CD4 + T regulatory cells and macrophages, in tumor lesions to secrete these cytokines. Tumors also have the ability to bias CD4 + T-cells to express the regulatory phenotype. The overall result is impaired T-cell responses and impaired induction of apoptosis or reduced anti-tumor immune capacity of CD8 + cytotoxic T-cells. Additionally, tumor-associated altered expression of MHC class I on the surface of tumor cells makes them “invisible” to the immune response (Garrido et al. Cancer Immunol. Immunother. 59(10):1601-1606 (2010)). Inhibition of antigen-presenting functions and dendritic cell (DC) additionally contributes to the evasion of anti-tumor immunity (Gerlini et al. Am. J. Pathol. 165(6):1853-1863 (2004)).

Moreover, the local immunosuppressive nature of the tumor microenvironment, along with immune editing, can lead to the escape of cancer cell subpopulations that do not express the target antigens. Thus, finding an approach that would promote the preservation and/or restoration of anti-tumor activities of the immune system would be of considerable therapeutic benefit.

Immune checkpoints have been implicated in the tumor-mediated downregulation of anti-tumor immunity and used as therapeutic targets. It has been demonstrated that T-cell dysfunction occurs concurrently with an induced expression of the inhibitory receptors, CTLA-4 and programmed death 1 polypeptide (PD-1), members of the CD28 family of receptors. PD-1 is an inhibitory member of the CD28 family of receptors that in addition to PD-1 includes CD28, CTLA-4, ICOS, and BTLA. However, while promise regarding the use of immunotherapy in the treatment of melanoma has been underscored by the clinical use and even regulatory approval of anti-CTLA-4 (ipilimumab) and anti-PD-1 drugs (e.g., pembrolizumab and nivolumab), the response of patients to these immunotherapies has been limited. Clinical trials, focused on blocking these inhibitory signals in T-cells (e.g., CTLA-4, PD-1, and the ligand of PD-1, PD-L1), have shown that reversing T-cell suppression is critical for successful immunotherapy (Sharma et al., Science 348(6230):56-61 (2015); Topalian et al., Curr. Opin. Immunol. 24(2):202-217 (2012)). These observations highlight the need for development of novel therapeutic approaches for harnessing the immune system against cancer.

III. Poxviruses: Vaccinia Virus (VACV), Modified Vaccinia Ankara (MVA) Virus, and Myxoma Virus (MYXV)

Poxviruses, such as engineered vaccinia viruses, are in the forefront as oncolytic therapy for metastatic cancers (Kirn et al., Nature Review Cancer 9:64-71 (2009)). Vaccinia viruse (VACV), a member of the Poxvirus family, is a large DNA virus, which has a rapid life cycle and efficient hematogenous spread to distant tissues. Poxviruses are well-suited as vectors to express multiple transgenes in cancer cells and thus to enhance therapeutic efficacy (Breitbach et al., Current pharmaceutical biotechnology 13:1768-1772 (2012)). Preclinical studies and clinical trials have demonstrated efficacy of using oncolytic vaccinia viruses and other poxviruses for treatment of advanced cancers refractory to conventional therapy (Park et al., Lacent Oncol. 9:533-542 (2008); Kirn et al., PLOS Med 4: e 353 (2007); Thorne et al., J. Clin. Invest. 117:3350-3358 (2007)). Poxvirus-based oncolytic therapy has the advantage of killing cancer cells through a combination of cell lysis, apoptosis, and necrosis. It also triggers innate immune sensing pathway that facilitates the recruitment of immune cells to the tumors and the development of anti-tumor adaptive immune responses. The current oncolytic vaccinia strains in clinical trials (JX-594, for example) are replicative strains. They use wild-type vaccinia with deletion of thymidine kinase to enhance tumor selectivity, and with expression of transgenes such as granulocyte macrophage colony stimulating factor (GM-CSF) to stimulate immune responses (Breitbach et al., Curr. Pharm. Biotechnol. 13:1768-1772 (2012)). Many studies have shown, however, that wild-type vaccinia has immune suppressive effects on antigen presenting cells (APCs) (Engelmayer et al., J. Immunol. 163:6762-6768 (1999); Jenne et al., Gene Therapy 7:1575-1583 (2000); P. Li et al., J. Immunol. 175:6481-6488 (2005); Deng et al., J. Virol. 80:9977-9987 (2006)), and thus adds to the immunosuppressive and immunoevasive effects of tumors themselves.

The vaccinia virus (Western Reserve strain; WR) genome sequence is set forth in SEQ ID NO: 2, and is given by GenBank Accession No. AY243312.1.

Modified Vaccinia Ankara (MVA) virus is also a member of the Poxvirus family. MVA was generated by approximately 570 serial passages on chicken embryo fibroblasts (CEF) of the Ankara strain of vaccinia virus (CVA) (Mayr et al., Infection 3:6-14 (1975)). As a consequence of these long-term passages, the resulting MVA virus contains extensive genome deletions and is highly host cell restricted to avian cells (Meyer et al., J. Gen. Virol. 72:1031-1038 (1991)). It was shown in a variety of animal models that the resulting MVA is significantly avirulent (Mayr et al., Dev. Biol. Stand. 41:225-34 (1978)).

The safety and immunogenicity of MVA has been extensively tested and documented in clinical trials, particularly against the human smallpox disease. These studies included over 120,000 individuals and have demonstrated excellent efficacy and safety in humans. Moreover, compared to other vaccinia based vaccines, MVA has weakened virulence (infectiousness) while it triggers a good specific immune response. Thus, MVA has been established as a safe vaccine vector, with the ability to induce a specific immune response.

Due to the above mentioned characteristics, MVA became an attractive candidate for the development of engineered MVA vectors, used for recombinant gene expression and vaccines. As a vaccine vector, MVA has been investigated against numerous pathological conditions, including HIV, tuberculosis and malaria, as well as cancer (Sutter et al., Curr. Drug Targets Infect. Disord. 3:263-271(2003); Gomez et al., Curr. Gene Ther. 8:97-120 (2008)).

It has been demonstrated that MVA infection of human monocyte-derived dendritic cells (DC) causes DC activation, characterized by the upregulation of co-stimulatory molecules and secretion of proinflammatory cytokines (Drillien et al., J. Gen. Virol. 85:2167-2175 (2004)). In this respect, MVA differs from standard wild type vaccinia virus (WT-VAC), which fails to activate DCs. Dendritic cells can be classified into two main subtypes: conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). The former, especially the CD103 + /CD8α + subtype, are particularly adapted to cross-presenting antigens to T-cells; the latter are strong producers of Type I IFN.

Viral infection of human cells results in activation of an innate immune response (the first line of defense) mediated by type I interferons, notably interferon-alpha (α). This normally leads to activation of an immunological “cascade,” with recruitment and proliferation of activated T-cells (both CTL and helper) and eventually with antibody production. However, viruses express factors that dampen immune responses of the host. MVA is a better immunogen than WT-VAC and replicates poorly in mammalian cells. (See, e.g., Brandler et al., J. Virol. 84:5314-5328 (2010)).

However, MVA is not entirely non-replicative and contains some residual immunosuppressive activity. Nevertheless, MVA has been shown to prolong survival of treated subjects.

The MVA genome sequence is set forth in SEQ ID NO: 1 and is given by GenBank Accession No. U94848.1.

Myxoma virus (MYXV) is the prototypic member of the Leporipoxvirus genus within the Poxviridae family. The MYXV Lausanne strain genome (given by, e.g., GenBank Accession No. AF170726.2) is 161.8 kbp in size, encoding about 171 genes. The central region of the genome encodes less than 100 genes that are highly conserved in all poxviruses while the terminal genomic regions are enriched for more unique genes that encode immunomodulatory and host-interactive factors that are involved in subverting the host immune system and other anti-viral responses. Myxoma virus exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Attractive features of MYXV as an oncolytic agent include its ability to productively infect various human cancer cells and its consistent safety in all non-rabbit hosts tested, including mice and humans. In some embodiments, the myxoma virus is derived from strain Lausanne.

V. Vaccinia virus C7 protein and MVA or Vaccinia Virus comprising deletion of C7 (MVAΔC7L, VACVΔC7L), or Myxoma Virus comprising deletion of myxoma C7 orthologs

Vaccinia virus C7 protein is an important host range factor for vaccinia virus life cycle in mammalian cells. C7L homologs are present in almost all of the poxviruses that infect mammalian hosts. Deletion of both host range gene C7L and K1L renders the virus incapable of replication in human cells (Perkus et al., Virology, 1990). The mutant virus deficient of both K1L and C7L gains its ability to replicate in human Hela cells when SAMD9 is knocked-out (Sivan et al., MBio, 2015). Both K1 and C7 have been found to interact with SAMD9 (Sivan et al., MBio, 2015). Overexpression of IRF1 leads to host restriction of C7L and K1L double deleted vaccinia virus (Meng et al., Journal of Virology, 2012). Both C7 and K1 interact with SAMD9 in vitro (Sivan et al., MBio. 2015). Whether C7 directly modulates IFN production or signaling is unknown. Type I IFN plays an important role in host defense of viral infection, and yet, the role of C7 in immune modulation of the IFN pathway is unclear.

Without wishing to be bound by theory, it is thought that vaccinia C7 is an inhibitor of type I IFN induction and IFN signaling. TANK Binding Kinase 1 (TBK1) is a serine/threonine kinase that plays a critical role in the induction of innate immune responses to various pathogen-associated molecular patterns (PAMPs), including nucleic acids. On the one hand, RIG-I-like receptors such as RIG-I and MDA5, which detect 5′ triphosphate RNA and dsRNA, respectively, interact with a mitochondrial protein IPS-1 or MAVS, leading to the activation and phosphorylation of TBK1. Endosomal dsRNA binds to Toll-like receptor 3 (TLR3), which results in the recruitment of TRIF and TRAF3 and activation of TBK1. On the other hand, cytosolic DNA can be detected by the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), which leads to the production of cyclic GMP-AMP (cGAMP). cGAMP, in turn, binds to the endoplasmic reticulum (ER)-localized adaptor STING, leading to the recruitment and activation of TBK1. TBK1 phosphorylates transcription factor IRF3, which translocates to the nucleus to activate IFNB gene expression. Without wishing to be bound by theory, it is believed that C7 inhibits IFNB induction by various stimuli, including RNA virus, DNA virus, poly (I:C), immunostimulatory DNA (ISD). C7 may exert its inhibitory effect at the level of TBK1/IRF3 complex. Once secreted, type I IFN binds to IFNAR, which leads to the activation of the JAK/STAT signaling pathway. Phosphorylated STAT1 and STAT2 translocate to the nucleus, where together with IRF9, they activate the expression of IFN-stimulated genes (ISGs). Without wishing to be bound by theory, it is believed that in addition to its ability to inhibit IFNB induction, C7 can also block IFNAR signaling through its interaction of STAT2, thereby preventing IFN-β-induced STAT2 phosphorylation. Without wishing to be bound by theory, it is believed that vaccinia C7 has dual inhibitory role of type I IFN production and signaling. Previous studies have shown that the deletion of C7L from WT vaccinia (VACVΔC7L) results in the attenuation of the virus and deletion of C7L from MVA (MVAΔC7L) leads to enhanced immunostimulatory functions compared with MVA.

Ectopic C7 expression has been shown to block STING, TBK1, or IRF3-induced IFNB and ISRE (interferon stimulated response element) promoter activation. Murine or human macrophage cell lines that overexpress C7 have been shown to have blunted innate immune responses to DNA or RNA stimuli, or the infection of DNA or RNA viruses. It has also been shown that overexpression of C7 attenuates ISG gene expression induced by IFN-β treatment. MVA with deletion of C7L (MVAΔC7L) infection of cDCs has been shown to induce higher levels of type I IFN than MVA. C7 has been shown to block IFN-β-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway via preventing Stat2 phosphorylation. C7 has been shown to directly interact with Stat2 as demonstrated by co-immunoprecipitation studies.

An illustrative full-length vaccinia virus C7 host range protein, given by GenBank Accession No. AAB96405.1 (SEQ ID NO: 6) is provided below.

1 mgiqhefdii ingdialrnl qlhkgdnygc klkiisndyk klkfrfiirp dwseidevkg

61 ltvfannyav kvnkvddtfy yviyeavihl ynkkteiliy sddenelfkh yypyislnmi

121 skkykvkeen ysspyiehpl ipyrdyesmd

Myxoma virus has three C7 orthologs, M62, M63, M64. Myxoma M64 shares similar structure features with vaccinia C7, despite having only 23% sequence identity. Myxoma M62 can rescue replication defects of VACVΔK1LΔC7L in human cells. Myxoma M63 deletion results in a recombinant virus that is non-replicative in rabbit cells. In some embodiments, the technology of the present disclosure provides an engineered myxoma virus such as MYXVΔM64R or MYXVΔM64R-hFlt3L-mOX40L.

VI. OX40 ligand (OX40L)

The OX40 ligand (OX40L) and its binding partner, tumor necrosis factor receptor OX40, are members of the TNFR/TNF superfamily and are expressed on activated CD4 and CD8 T-cells as well as a number of other lymphoid and non-lymphoid cells. The OX40L-OX40 interaction provides survival and activation signals for T-cells expressing OX40. OX40 additionally suppresses the differentiation and activity of Treg, further amplifying this process. OX40 and OX40L also regulate cytokine production from T-cells, antigen-presenting cells, NK cells, and NKT cells, and modulate cytokine receptor signaling. The OX40L of the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔE5R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, and MYXVΔM31R recombinant viruses of the present technology can be either huOX40L or muOX40L.

Illustrative human OX40L (huOX40L) nucleic acid (SEQ ID NO: 7) and polypeptide sequences (SEQ ID NO: 8) are provided below.

Illustrative murine OX40L (muOX40L) nucleic acid (SEQ ID NO: 9) and polypeptide sequences (SEQ ID NO: 10) are provided below.

muOX40L-ORF (codon optimized)(SEQ ID NO: 9):

muOX40L polypeptide (SEQ ID NO: 10):

M E G E G V Q P L D E N L E N G S R P R F K W K K T L R L V V S

G I K G A G  M L L C F I Y V C L Q L S S S P A K D P P I Q R L R

G A V T R C E D G Q L F I S S Y K N E Y Q T  M E V Q N N S V V I

K C D G L Y I I Y L K G S F F Q E V K I D L H F R E D H N P I

S I P  M L N D G R R I V F T V V A S L A F K D K V Y L T V N A P

D T L C E H L Q I N D G E L I V V Q L T P G Y C A P E G S Y H

S T V N Q V P L  Stop

VII. Human Fms-like tyrosine kinase 3 ligand (hFlt3L)

Human Fms-like tyrosine kinase 3 ligand (hFlt3L), a type I transmembrane protein that stimulates the proliferation of bone marrow cells, was cloned in 1994 (Lyman et al., 1994). The use of hFlt3L has been explored in various preclinical and clinical settings including stem cell mobilization in preparation for bone marrow transplantation, cancer immunotherapy such as expansion of dendritic cells, as well as a vaccine adjuvant. Recombinant human Flt3L (rhuFlt3L) has been tested in more than 500 human subjects and is bioactive, safe, and well-tolerated. Much progress has been made in understanding the critical role of the growth factor Flt3L in the development of DC subsets, including CD8α + /CD103 + DCs and pDCs.

CD103 + /CD8α + DCs are required for spontaneous cross-priming of tumor antigen-specific CD8 + T-cells. It has been reported that CD103 + DCs are sparsely present within the tumors and they compete for tumor antigens with abundant tumor-associated macrophages. CD103 + DCs are uniquely capable of stimulating naïve as well as activated CD8 + T-cells and are critical for the success of adoptive T-cell therapy (Broz, et al. Cancer Cell, 26(5):638-52 (2014)). Spranger et al. reported that the activation of oncogenic signaling pathway WNT/β-catenin leads to reduction of CD103 + DCs and anti-tumor T-cells within the tumors (Spranger et al., 2015). Intratumoral delivery of Flt3L-cultured bone marrow derived dendritic cells (BMDCs) leads to responsiveness to the combination of anti-CTLA-4 and anti-PD-L1 immunotherapy (Spranger et al., 2015). Systemic administration of Flt3L, a growth factor for CD103 + DCs, and intratumor injection of poly I:C (TLR3 agonist) expanded and activated the CD103 + DC populations within the tumors and overcame resistance or enhanced responsiveness to immunotherapy in a murine melanoma and MC38 colon cancer models.

The recent discovery of tumor neoantigens in various solid tumors indicates that solid tumors harbor unique neoantigens that usually differ from person to person (Castle et al., Cancer Res 72:1081-1091 (2012); Schumacher et al., Science 348:69-74 (2015)). The genetically engineered or recombinant viruses disclosed herein do not exert their activity by expressing tumor antigens. Intratumoral delivery of the present genetically engineered or recombinant MVA viruses allows efficient cross-presentation of tumor neoantigens and generation of anti-tumor adaptive immunity within the tumors (and also extending systemically), and therefore leads to “in situ cancer vaccination” utilizing tumor differentiation antigens and neoantigens expressed by the tumor cells in mounting an immune response against the tumor.

Despite the presence of neoantigens generated by somatic mutations within tumors, the functions of tumor antigen-specific T-cells are often held in check by multiple inhibitory mechanisms (Mellman et al., Nature 480, 480-489 (2011)). For example, the up-regulation of cytotoxic T lymphocyte antigen 4 (CTLA-4) on activated T-cells can compete with T-cell co-stimulator CD28 to interact with CD80 (B71)/CD86 (B7.2) on dendritic cells (DCs), and thereby inhibit T-cell activation and proliferation. CTLA-4 is also expressed on regulatory T (Treg) cells and plays an important role in mediating the inhibitory function of Tregs (Wing et al., Science 322:271-275 (2008); Peggs, et al., J. Exp. Med. 206:1717-1725 (2009)). In addition, the expression of PD-L/PD-L2 on tumor cells can lead to the activation of the inhibitory receptor of the CD28 family, PD-1, leading to T-cell exhaustion. Immunotherapy utilizing antibodies against inhibitory receptors, such as CTLA-4 and programmed death 1 polypeptide (PD-1), have shown remarkable preclinical activities in animal studies and clinical responses in patients with metastatic cancers, and have been approved by the FDA for the treatment of metastatic melanoma, non-small cell lung cancer, as well as renal cell carcinoma (Leach et al., Science 271:1734-1746 (1996); Hodi et al., NEJM 363:711-723 (2010); Robert et al., NEJM 364:2517-2526 (2011); Topalian et al., Cancer Cell 27:450-461 (2012); Sharma et al., Science 348(6230):56-61 (2015)).

VIII. ΔE3L and E3LΔ83N

Poxviruses are extraordinarily adept at evading and antagonizing multiple innate immune signaling pathways by encoding proteins that interdict the extracellular and intracellular components of those pathways (Seet et al. Annu. Rev. Immunol. 21:377-423 (2003)). Chief among the poxvirus antagonists of intracellular innate immune signaling is the vaccinia virus duel Z-DNA and dsRNA-binding protein E3, which can inhibit the PKR and NF-κB pathways (Cheng et al., Proc. Natl. Acad. Sci. USA 89:4825-4829 (1992); Deng et al., J. Virol. 80:9977-9987 (2006)) that would otherwise be activated by vaccinia virus infection. A mutant vaccinia virus lacking the E3L gene (ΔE3L) has a restricted host range, is highly sensitive to IFN, and has greatly reduced virulence in animal models of lethal poxvirus infection (Beattie et al., Virus Genes 1289-94 (1996); Brandt et al., Virology 333263-270 (2004)). Recent studies have shown that infection of cultured cell lines with ΔE3L virus elicits proinflammatory responses that are masked during infection with wild-type vaccinia virus (Deng et al., J. Virol. 80:9977-9987 (2006); Langland et al. J. Virol. 80:10083-10095). Infection of a mouse epidermal dendritic cell line with wild-type vaccinia virus attenuated proinflammatory responses to the TLR agonists lipopolysaccharide (LPS) and poly (I:C), an effect that was diminished by deletion of E3L. Moreover, infection of the dendritic cells with ΔE3L virus triggered NF-κB activation in the absence of exogenous agonists (Deng et al., J. Virol. 80:9977-9987 (2006)). Whereas wild-type vaccinia virus infection of murine keratinocytes does not induce the production of proinflammatory cytokines and chemokines, infection with ΔE3L virus does induce the production of IFN-β, IL-6, CCL4 and CCL5 from murine keratinocytes, which is dependent on the cytosolic dsRNA-sensing pathway mediated by the mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytosolic RNA sensors RIG-I and MDA5) and the transcription factor IRF3 (Deng et al., J. Virol. 82(21):10735-10746 (2008)).

E3LΔ83N virus with deletion of the Z-DNA-binding domain is 1,000-fold more attenuated than wild-type vaccinia virus in an intranasal infection model (Brandt et al., 2001). E3LΔ83N also has reduced neurovirulence compared with wild-type vaccinia in an intra-cranial inoculation model (Brandt et al., 2005). A mutation within the Z-DNA binding domain of E3 (Y48A) resulting in decreased Z-DNA-binding leads to decreased neurovirulence (Kim et al., 2003). Although the N-terminal Z-DNA binding domain of E3 is important in viral pathogenesis, how it affects host innate immune sensing of vaccinia virus is not well understood. Myxoma virus but not wild-type vaccinia infection of murine plasmacytoid dendritic cells induces type I IFN production via the TLR9/MyD88/IRF5/IRF7-dependent pathway (Dai et al., 2011). Myxoma virus E3 ortholog M029 retains the dsRNA-binding domain of E3 but lacks the Z-DNA binding domain of E3. It was found that the Z-DNA-binding domain of E3 (but probably not Z-DNA-binding activity per se) plays an important role in inhibiting poxviral sensing in murine and human pDCs (Dai et al., 2011; Cao et al., 2012).

Deletion of E3L sensitizes vaccinia virus replication to IFN inhibition in permissive RK13 cells and results in a host range phenotype, whereby ΔE3L cannot replicate in HeLa or BSC40 cells (Chang et al., 1995). The C-terminal dsRNA-binding domain of E3 is responsible for the host range effects, whereas E3LΔ83N virus with deletion of the N-terminal Z-DNA-binding domain is replication competent in HeLa and BSC40 cells (Brandt et al., 2001).

Vaccinia virus (Western Reserve strain; WR) with deletion of thymidine kinase is highly attenuated in non-dividing cells but is replicative in transformed cells (Buller et al., 1988). TK-deleted vaccinia virus selectively replicates in tumor cells in vivo (Puhlmann et al., 2000). Thorne et al. showed that compared with other vaccinia strains, WR strain has the highest burst ratio in tumor cell lines relative to normal cells (Thorne et al., 2007).

IX. Vaccinia virus E5 is a dominant inhibitor of the cytosolic DNA sensor cGAS

The cytosolic DNA sensor cGAS plays an important role in detecting viral nucleic acid, which leads to type I IFN production. It has been shown that infection of conventional dendritic cells with modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia strain, induces IFN production via a cGAS/STING-dependent mechanism. However, MVA infection triggers cGAS degradation. Vaccinia virus (VACV) is a cytoplasmic DNA virus, which encodes more than 200 genes. As described in the experimental examples section, seventy vaccinia viral early genes were screened for inhibition of cGAS/STING pathway in HEK293 T cells using a dual luciferase system. It was found that vaccinia E5R is a dominant inhibitor of the cGAS and is the key protein mediating cGAS degradation. MVAΔE5R induces much higher levels of type I IFN than MVA in multiple cell types, including bone marrow derived dendritic cells (BMDC), bone marrow-derived macrophages (BMDM), and skin primary fibroblasts ( FIGS. 57 C and 57 D ; 76 A and 76 B). MVAΔE5R-mediated type I IFN production is dependent on cGAS ( FIGS. 58 A- 58 C ). Furthermore, MVAΔE5R gains replication capability in cGAS −/− skin fibroblasts ( FIGS. 77 C and 77 D ). As a vaccine vector, skin scarification or intradermal vaccination with MVAΔE5R-OVA leads to much higher OVA-specific CD8 + T cell responses than MVA-OVA in vivo ( FIGS. 75 B and 75 C ). Intratumoral injection of MVAΔE5R leads to stronger anti-tumor immune responses and better survival compared with MVA ( FIGS. 91 A- 91 C ). Finally, in an intranasal infection model, VACVΔE5R is at least 1000-fold attenuated compared with WT VACV ( FIG. 55 B ). Taken together, these results provide strong evidence that E5 is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production. The inventors of the present technology are the first to describe the role of E5R in immune evasion.

An illustrative full-length vaccinia virus E5R host range protein, given by GenBank Accession No. AAB59825.1 (SEQ ID NO: 20) is provided below.

The myxoma ortholog of vaccinia virus E5R is M31R. An illustrative full-length myxoma virus M31R protein, given by GenBank Accession No. AAF14919.1 (SEQ ID NO: 21), is provided below.

X. Engineered Poxvirus Strains of the Present Technology

MVAΔC7L

The disclosure of the present technology relates to a C7L mutant modified vaccinia Ankara (MVA) virus (i.e., MVAΔC7L; MVA virus comprising a C7L deletion; MVA genetically engineered to comprise a mutant C7L gene), or immunogenic compositions comprising the virus, in which the virus is engineered to express one or more specific genes of interest (SG), such as OX40L (MVAΔC7L-OX40L), and their use as a cancer immunotherapeutic. In some embodiments, the C7 gene of the MVA virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a C7 knockout such that the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔC7L mutant includes a heterologous nucleic acid sequence in place of all or a majority of the C7L gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of C7 in the MVA genome (e.g., position 18,407 to 18,859 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in MVAΔC7L-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes hFl3L, resulting in MVAΔC7L-hFl3L. (See, e.g., FIG. 5 A ).

Additionally or alternatively, in some embodiments, MVAΔC7L is engineered to express both OX40L and hFlt3L. In some embodiments, the thymidine kinase (TK) gene of the MVA virus (e.g., position 75,560 to 76,093 of SEQ ID NO: 1), through homologous recombination, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which results in a TK gene knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting MVAΔCL-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side (see, e.g., FIG. 5 B ). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L or hFlt3L using the vaccinia viral synthetic early and late promoter (PsE/L), resulting in MVAΔC7L-TK(−)-OX40L. In some embodiments, the recombinant virus is further modified at the C7 locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a C7 knockout such that the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in MVAΔC7L-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the C7 locus while the expression cassette encoding hFlt3L is inserted into the TK locus.

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence. For example, in some embodiments, MVAΔC7L encompasses a recombinant MVA virus in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as MVAΔC7LΔE5R-hFl3L-OX40L (see, e.g., FIG. 93 A ). As another example, in some embodiments, the present technology provides a recombinant MVAΔC7L-hFl3L-TK(−)-OX40LΔE5R virus that is formed by making three separate modifications. The first modification involves replacing the C7L C7Lgene, through homologous recombination at the C8L and C6R loci, with a heterologous nucleic acid sequence comprising an expression cassette comprising an open reading frame encoding hFl3L, thereby forming MVAΔC7L-hFl3L. For the second modification, through homologous recombination at the TK locus, the TK gene is replaced with a heterologous nucleic acid sequence comprising an expression cassette comprising an open reading frame encoding OX40L, thereby forming MVAΔC7L-hFl3L-TK(−)-OX40L. For the third modification, through homologous recombination at the E4L and E6R loci, the E5R gene is replaced with a heterologous nucleic acid sequence comprising an expression cassette comprising an open reading frame encoding a selectable marker, such as mCherry, thereby forming MVAAC 7 L-hFl3L-TK(−)-OX40LΔE5R (see, e.g., FIG. 92 A ).

In some embodiments, the recombinant MVAΔC7L-OX40L viruses described above are modified to express at least one additional heterologous gene, such as any one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L, N2L, and/or WR199.

Although in certain embodiments described above, the transgene may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, MVA encodes several immune modulatory genes, including but not limited to C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus and allow insertion of transgenes. For example, in some embodiments, the present technology provides an MVAΔC7L-hFlt3L-TK(−)-OX40L virus expressing transgenes IL-2, IL-12, IL-18, IL-15, and/or IL-21 from within the E5R gene locus. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g. OX40L or OX40L and hFlt3L), and/or no further viral genes other than C7L or C7L and TK are disrupted or deleted.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker ( FIGS. 5 A- 5 B ). In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

Non-limiting examples of OX40L expression construct open reading frames according to the present technology are shown in SEQ ID NOs: 3 and 5 (Table 1). A non-limiting example of an hFlt3L expression construct according to the present technology is shown in SEQ ID NO: 4 (Table 1).

The MVA virus genome sequence (SEQ ID NO: 1) given by GenBank Accession No. U94848.1 is provided in FIG. 22 . In some embodiments, engineered MVAΔC7L virus expressing OX40L is generated by inserting an expression construct such as those illustrated by SEQ ID NOs: 3 and 5 into the MVA genomic region that corresponds to the position of the TK locus (e.g., position 75,560 to 76,093 of SEQ ID NO: 1). Additionally or alternatively, in some embodiments engineered MVAΔC7L-OX40L is further modified to express hFlt3L by inserting an expression construct such as that which is ilustrated by SEQ ID NO: 4 into the MVA genomic region that corresponds to the C7 locus (e.g., position 18,407 to 18,859 of SEQ ID NO: 1).

MVAΔE3L

The disclosure of the present technology relates to an E3L mutant modified vaccinia Ankara (MVA) virus (i.e., MVAΔE3L; MVA virus comprising an E3L deletion; MVA virus genetically engineered to comprise a mutant E3L gene), or immunogenic compositions comprising the virus, in which the virus is engineered to express one or more specific genes of interest (SG), such as OX40L (MVAΔE3L-OX40L), and their use as a cancer immunotherapeutic. In some embodiments, the thymidine kinase (TK) gene of the MVA virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a TK knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting MVAΔE3L-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. For example, in some embodiments, the nucleic acid sequence corresponding to the position of TK in the MVAΔE3L genome (e.g., position 75,798 to 75,868 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in MVAΔE3L-TK(−)-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L).

Although in certain embodiments described above, the transgene (e.g., OX40L) may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, MVA encodes several immune modulatory genes, including but not limited to C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus, and allow insertion of transgenes.

In some embodiments, the recombinant MVAΔE3L-OX40L viruses described above are modified to express at least one other heterologous gene, such as any one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: C7; E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L), and/or no further viral genes other than E3L or E3L and TK are disrupted or deleted.

In some embodiments, MVAΔE3L is engineered to express both OX40L and hFlt3L. In some embodiments, the recombinant virus is further modified at the E3 locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in an E3 knockout such that the E3 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in MVAΔE3L-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the E3 locus while the expression cassette encoding hFlt3L is inserted into the TK locus.

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker (see, e.g., FIG. 1 ). In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

Non-limiting examples of OX40L expression construct open reading frames according to the present technology are shown above in Table 1.

MVAΔE5R

The disclosure of the present technology relates to an E5R mutant modified vaccinia Ankara (MVA) virus (i.e., MVAΔE5R; MVA virus comprising an E5R deletion; MVA genetically engineered to comprise a mutant E5R gene), or immunogenic compositions comprising the virus, and their use as a cancer immunotherapeutic. In some embodiments, the E5R gene of the MVA virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in an E5R knockout such that the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔE5R mutant includes a heterologous nucleic acid sequence in place of all or a majority of the E5R gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of E5R in the MVA genome (e.g., position 38,432 to 39,385 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in MVAΔE5R-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes hFlt3L, resulting in MVAΔE5R-hFlt3L.

In some embodiments, the MVAΔE5R virus is engineered to express one or more specific genes of interest (SG), such as a heterologous gene selected from any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions or mutations: C7 (ΔC7); E3L (ΔE3L); E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R (ΔE5R); K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In some embodiments, the MVAΔE5R virus is selected from MVAΔE3LΔE5R, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15Rα, or MVAΔE5R-hFlt3L-OX40L-ΔWR199.

In some embodiments, the thymidine kinase (TK) gene of the MVA virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a TK knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting MVAΔE5R-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. For example, in some embodiments, the nucleic acid sequence corresponding to the position of TK in the MVAΔE5R genome (e.g., position 75,798 to 75,868 of SEQ ID NO: 1) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in MVAΔE5R-TK(−)-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L).

Although in certain embodiments described above, the transgene (e.g., OX40L) may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, MVA encodes several immune modulatory genes, including but not limited to C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus, and allow insertion of transgenes.

In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L, hFlt3L), and/or no further viral genes other than E5R or E5R and TK are disrupted or deleted.

In some embodiments, MVAΔE5R is engineered to express both OX40L and hFlt3L. In some embodiments, the recombinant virus is further modified at the E5R locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in an E5R knockout such that the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in MVAΔE5R-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the E5R locus while the expression cassette encoding hFlt3L is inserted into the TK locus.

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence. For example, in some embodiments, MVAΔE5R encompasses a recombinant MVA in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as MVAΔE5R-hFl3L-OX40L (see, e.g., FIG. 81 ).

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

Non-limiting examples of expression and deletion construct open reading frames according to the present technology are shown in (Table 2).

In some embodiments, engineered MVAΔE5R virus is generated by inserting an expression construct such as those illustrated by SEQ ID NOs: 22-24 and 32 (Table 2) into the MVA genomic region that corresponds to the position of the E5R locus (e.g., position 38,432 to 39,385 of SEQ ID NO: 1; or position 38,389 to 39,389 of the sequence set forth in GenBank Accession No. AY603355). In some embodiments, the MVAΔE5R virus is further engineered by inserting an expression construct such as that illustrated by SEQ ID NO: 31 into the MVA genomic region that corresponds to the E3L locus (e.g., position 36,931 to 37,497 of the sequence set forth in GenBank Accession No. AY603355). In some embodiments, the MVAΔE5R virus is further engineered by inserting an expression construct such as that illustrated by SEQ ID NO: 33 into the MVA genomic region that corresponds to the C11R locus (e.g., position 4,160-4,785 of the sequence set forth in GenBank Accession No. AY603355).

A non-limiting example of an MVAΔK7R construct open reading frame according to the present technology is shown in SEQ ID NO: 25 (Table 3).

VACVΔC7L

The disclosure of the present technology relates to a C7L mutant vaccinia virus (i.e., VACVΔC7L; VACV comprising a C7L deletion; VACV genetically engineered to comprise a mutant C7L gene), or immunogenic compositions comprising the virus, in which the virus is engineered to express one or more specific genes of interest (SG), such as OX40L, and its use as a cancer immunotherapeutic (VACVΔC7L-OX40L). In some embodiments, the C7 gene of the vaccinia virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a C7 knockout such that the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔC7L mutant includes a heterologous nucleic acid sequence in place of all or a majority of the C7L gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of C7 in the VACV genome (e.g., position 15,716 to 16,168 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in VACVΔC7L-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes hFlt3L, resulting in VACVΔC7L-hFlt3L.

Additionally or alternatively, in some embodiments, VACVΔC7L is engineered to express both OX40L and hFlt3L. In some embodiments, the thymidine kinase (TK) gene of the vaccinia virus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), through homologous recombination, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which results in a TK gene knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting VACVΔC7L-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L), resulting in VACVΔC7L-TK(−)-OX40L. In some embodiments, the recombinant virus is further modified at the C7 locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a C7 knockout such that the C7 gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in VACVΔC7L-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the C7 locus while the expression cassette encoding hFlt3L is inserted into the TK locus. In some embodiments, a VACVΔC7L-anti-CTLA-4-hFlt3L-TK(−) virus is further modified to encode OX40L, resulting in VACVΔC7L-anti-CTLA-4-TK(−)-hFlt3L-OX40L. In some embodiments, the VACVΔC7L-anti-CTLA-4-TK(−)-hFlt3L-OX40L virus is further modified to express hIL-12, resulting in VACVΔC7L-anti-CTLA-4-TK(−)-hFlt3L-OX40L-hIL-12.

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence.

In some embodiments, the disclosure of the present technology provides a VACVΔC7L-TK(−)-anti-CTLA-4-OX40L virus. In some embodiments, the disclosure of the present technology provides a VACVΔC7L-E3LΔ83N-TK(−)-hFlt3L-anti-CTLA-4-OX40L virus.

In some embodiments, the recombinant VACVΔC7L-OX40L viruses described above are modified to express at least one further heterologous gene, such as any one or more of hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L or OX40L and hFlt3L), and/or no further viral genes other than C7L or C7L and TK are disrupted or deleted.

Although in certain embodiments described above, the transgene may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, VACV encodes several immune modulatory genes, including but not limited to C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus and allow insertion of transgenes.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

VACVΔE5R

The disclosure of the present technology relates to a E5R mutant vaccinia virus (i.e., VACVΔE5R; VACV comprising an E5R deletion; VACV genetically engineered to comprise a mutant E5R gene), or immunogenic compositions comprising the virus, in which the virus is engineered to express one or more specific genes of interest (SG), such as OX40L, and its use as a cancer immunotherapeutic (VACVΔE5R-OX40L). In some embodiments, the E5R gene of the vaccinia virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in an E5R knockout such that the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔE5R mutant includes a heterologous nucleic acid sequence in place of all or a majority of the E5R gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of E5R in the VACV genome (e.g., position 49,236 to 50,261 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in VACVΔE5R-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes hFlt3L, resulting in VACVΔE5R-hFlt3L.

Additionally or alternatively, in some embodiments, VACVΔE5R is engineered to express both OX40L and hFlt3L. In some embodiments, the thymidine kinase (TK) gene of the vaccinia virus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), through homologous recombination, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which results in a TK gene knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting VACVΔE5R-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L), resulting in VACVΔE5R-TK(−)-OX40L. In some embodiments, the recombinant virus is further modified at the E5R locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in an E5R knockout such that the E5R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in VACVΔE5R-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the E5R locus while the expression cassette encoding hFlt3L is inserted into the TK locus. In some embodiments, a VACVΔE5R-anti-CTLA-4-hFlt3L-TK(−) virus is further modified to encode OX40L, resulting in VACVΔE5R-anti-CTLA-4-TK(−)-hFlt3L-OX40L. In some embodiments, the VACVΔE5R-anti-CTLA-4-TK(−)-hFlt3L-OX40L virus is further modified to express hIL-12, resulting in VACVΔE5R-anti-CTLA-4-TK(−)-hFlt3L-OX40L-hIL-12. In some embodiments, the VACVΔE5R is engineered to comprise a nucleic acid encoding IL-15/IL-15Rα (VACVΔE5R-IL-15/IL-15Rα) alone or in combination with one or more additional modifications as described herein. For example, in some embodiments, the VACVΔE5R-IL-15/IL-15Rα is further engineered to comprise a nucleic acid encoding OX40L (VACVΔE5R-IL-15/IL-15Rα-OX40L).

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence. For example, in some embodiments, the TK locus of the vaccinia genome is modified through homologous recombination to express both the heavy and light chain of an antibody, such as anti-CTLA-4, wherein the coding sequences of the heavy chain and light chain are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence to produce VACV-TK(−)-anti-CTLA-4. In some embodiments, the VACV-TK(−)-anti-CTLA-4 genome is further modified to comprise a deletion of E5R, in which all or a majority of the E5R gene sequence is replaced by a first specific gene of interest (e.g., hFlt3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as VACV-TK − -anti-CTLA-4-E5R − -hFlt3L-OX40L (or VACVΔE5R-TK(−)-anti-CTLA-4-hFlt3L-OX40L) (see, e.g., FIG. 85 A ).

In some embodiments, the genetically engineered or recombinant VACVΔE5R viruses described above are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7 (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L or OX40L and hFlt3L), and/or no further viral genes other than E5R or E5R and TK are disrupted or deleted.

Although in certain embodiments described above, the transgene may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, VACV encodes several immune modulatory genes, including but not limited to C7, C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus and allow insertion of transgenes.

In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L), and/or no further viral genes other than E5R or E5R and TK are disrupted or deleted.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

Non-limiting examples of VACVΔE5R construct open reading frames according to the present technology are shown in SEQ ID NOs: 26-28 (Table 4).

In some embodiments, engineered VACVΔE5R virus is generated by inserting an expression construct such as those illustrated by SEQ ID NOs: 26-28 (Table 4) into the VACV genomic region that corresponds to the position of the E5R locus (e.g., position 49,236 to 50,261 of SEQ ID NO: 2).

A non-limiting example of an anti-CTLA-4 antibody open reading for insertion into the TK locus of VACV, using, e.g., a pCB plasmid-based vector, is shown in SEQ ID NO: 29 (Table 5).

Non-limiting examples of IL-12 expression constructs for insertion into, for example, the E5R locus, using, for example, a pUC57 vector, according to the present technology by which, for example, the VACV-E3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFl3L-OX40L-hIL-12 constuct is engineered are shown in SEQ ID NOs: 35-38 (Table 5A) (see also FIG. 169 ).

In some embodiments, engineered VACV viruses of the present technology comprise an expression construct such as that illustrated by SEQ ID NO: 29 (Table 5) inserted into the VACV genomic region that corresponds to the position of the TK locus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2).

VACVΔB2R

The VACV B2R gene encodes poxin, a nuclease that plays a role in viral evasion of host cGAS-STING innate immunity. The disclosure of the present technology relates to a B2R mutant vaccinia virus (i.e., VACVΔB2R; VACV comprising a B2R deletion; VACV genetically engineered to comprise a mutant B2R gene), or immunogenic compositions comprising the virus, in which the virus is engineered to express one or more specific genes of interest (SG), such as OX40L (VACVΔB2R-OX40L), and its use as a cancer immunotherapeutic. In some embodiments, the B2R gene of the vaccinia virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a B2R knockout such that the B2R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔB2R mutant includes a heterologous nucleic acid sequence in place of all or a majority of the B2R gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of B2R in the VACV genome (e.g., position 164,856 to 165,530 of SEQ ID NO: 2) is replaced with a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence comprises an open reading frame that encodes a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the VACVΔB2R virus encompasses a recombinant VACV that does not express a functional B2R protein. In some embodiments, a specific gene of interest (e.g., OX40L, hFlt3L) is inserted into the B2R locus of the VACV genome, splitting the B2R gene and obliterating it.

Additionally or alternatively, in some embodiments, VACVΔB2R is engineered to express both OX40L and hFlt3L. In some embodiments, the thymidine kinase (TK) gene of the vaccinia virus (e.g., position 80,962 to 81,032 of SEQ ID NO: 2), through homologous recombination, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which results in a TK gene knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting VACVΔB2R-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L), resulting in VACVΔB2R-TK(−)-OX40L. In some embodiments, the recombinant virus is further modified at the B2R locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a B2R knockout such that the B2R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in VACVΔB2R-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the B2R locus while the expression cassette encoding hFlt3L is inserted into the TK locus. In some embodiments, a VACVΔB2R-anti-CTLA-4-hFlt3L-TK(−) virus is further modified to encode OX40L, resulting in VACVΔB2R-anti-CTLA-4-TK(−)-hFlt3L-OX40L. In some embodiments, the VACVΔB2R-anti-CTLA-4-TK(−)-hFlt3L-OX40L virus is further modified to express hIL-12, resulting in VACVΔB2R-anti-CTLA-4-TK(−)-hFlt3L-OX40L-hIL-12. In some embodiments, a VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFl3L-OX40L-hIL-12 genome is modified to comprise a B2R deletion (VACVΔE3L83N-ΔTK-anti-CTLA-4-ΔE5R-hFl3L-OX40L-IL-12-ΔB2R) (see, e.g., FIG. 168 ).

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence.

In some embodiments, the genetically engineered or recombinant VACVΔB2R viruses described above are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7 (ΔC7L); B19R (B18R; ΔWR200); E5R (ΔE5R); K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In some embodiments, the genetically engineered or recombinant VACVΔB2R viruses are selected from VACVΔB2R-ΔE5R, VACVΔB2R-ΔE5R-E3LΔ83N, and VACVΔB2R-E3LΔ83N. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein, and/or no further viral genes are disrupted or deleted other than those provided in the name of the virus herein.

Although in certain embodiments described above, the transgene may be inserted into the B2R locus, splitting the B2R gene and obliterating it or replacing it, other suitable integration loci can be selected. For example, VACV encodes several immune modulatory genes, including but not limited to C7, C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus and allow insertion of transgenes.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

A non-limiting example of a VACVΔB2R deletion construct comprising an open reading frame encoding a selectable marker according to the present technology is shown in SEQ ID NO: 30 (Table 7).

In some embodiments, engineered VACVΔB2R virus is generated by inserting an expression construct such as that illustrated by SEQ ID NO: 30 (Table 7) into the VACV genomic region that corresponds to the position of the B2R locus (e.g., position 164,856 to 165,530 of SEQ ID NO: 2).

MVAΔWR199

A non-limiting example of a MVAΔWR199 construct open reading frame according to the present technology is shown in SEQ ID NO: 34 (Table 8). In some embodiments, MVA comprising a ΔWR199 mutant is generated by inserting an expression construct, with, e.g., a WR199 knockout plasmid, such as that illustrated by SEQ ID NO: 34 into the MVA genomic region that corresponds to the position of the WR199 locus (e.g., position 158,399 to 160,143 of the sequence set forth in GenBank Accession No. AY603355) (see, e.g., FIG. 153 ).

MYXVΔM31R

Myxoma M31R is orthologous to VACV E5R (see FIG. 88 B ). The disclosure of the present technology relates to a M31R mutant myxoma virus (i.e., MYXVΔM31R; MYXV comprising an M31R deletion; MYXV genetically engineered to comprise a mutant M31R gene), or immunogenic compositions comprising the virus, and its use as a cancer immunotherapeutic. In some embodiments, the MYXVΔM31R virus is engineered to express one or more specific genes of interest (SG), such as OX40L, for use as a cancer immunotherapeutic (MYXVΔM31R-OX40L). In some embodiments, the myxoma virus is derived from strain Lausanne (given by, e.g., GenBank Accession No. AF170726.2). In some embodiments, the M31R gene of the myxoma virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a M31R knockout such that the M31R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔM31R mutant includes a heterologous nucleic acid sequence in place of all or a majority of the M31R gene sequence. For example, in some embodiments, the nucleic acid sequence corresponding to the position of M31R in the MYXV genome (e.g., position 30,138 to 31,319 of the myxoma genome) is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a specific gene of interest (SG), such as human OX40L, resulting in MYXVΔM31R-OX40L. In some embodiments, the expression cassette comprises a single open reading frame that encodes hFlt3L, resulting in MYXVΔM31R-hFlt3L.

Additionally or alternatively, in some embodiments, MYXVΔM31R is engineered to express both OX40L and hFlt3L. In some embodiments, the thymidine kinase (TK) gene of the myxoma virus (e.g., position 57,797 to 58,333 of the myxoma genome), through homologous recombination, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which results in a TK gene knockout such that the TK gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). The resulting MYXVΔM31R-TK(−) virus is further engineered to comprise one or more expression cassettes that are flanked by a partial sequence of the TK gene (TK-L and TK-R) on either side. In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as OX40L using the vaccinia viral synthetic early and late promoter (PsE/L), resulting in MYXVΔM31R-TK(−)-OX40L. In some embodiments, the recombinant virus is further modified at the M31R locus, through homologous recombination techniques, to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a M31R knockout such that the M31R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the expression cassette comprises a single open reading frame that encodes a specific gene of interest (SG), such as hFlt3L, resulting in MYXVΔM31R-hFlt3L-TK(−)-OX40L. In some embodiments, the expression cassette encoding OX40L is inserted into the M31R locus while the expression cassette encoding hFlt3L is inserted into the TK locus. In some embodiments, a MYXVΔM31R-anti-CTLA-4-hFlt3L-TK(−) virus is further modified to encode OX40L, resulting in MYXVΔM31R-anti-CTLA-4-TK(−)-hFlt3L-OX40L. In some embodiments, the MYXVΔM31R-anti-CTLA-4-TK(−)-hFlt3L-OX40L virus is further modified to express hIL-12, resulting in MYXVΔM31R-anti-CTLA-4-TK(−)-hFlt3L-OX40L-hIL-12.

In some embodiments, the genetically engineered or recombinant MYXVΔM31R viruses described above are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15RΔ, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7 (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein (e.g., OX40L or OX40L and hFlt3L), and/or no further viral genes other than M31R or M31R and TK are disrupted or deleted.

Although in certain embodiments described above, the transgene may be inserted into the TK locus, splitting the TK gene and obliterating it, other suitable integration loci can be selected. For example, MYXV encodes several immune modulatory genes, including but not limited to C7, C11, K3, F1, F2, F4, F6, F8, F9, F11, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, N1L, C11R, K1L, C16, M1L, N2L, and WR199. Accordingly, in some embodiments, these genes can be deleted to potentially enhance immune activating properties of the virus and allow insertion of transgenes.

Additionally or alternatively, in some embodiments, the heterologous nucleic acid sequence comprises an expression cassette comprising two or more open reading frames encoding two or more specific genes of interest, separated by a nucleotide sequence that encodes, in the 5′ to 3′ direction, a protease cleavage site (e.g., a furin cleavage site) and a 2A peptide (Pep2A) sequence. For example, in some embodiments, MYXVΔM31R encompasses a recombinant MYXV in which all or a majority of the M31R gene sequence is replaced by a first specific gene of interest (e.g., hFtl3L) and a second specific gene of interest (e.g., OX40L), wherein the coding sequences of the first and second specific genes of interest are separated by a cassette including a furin cleavage site followed by a 2A peptide (Pep2A) sequence, thereby forming a recombinant virus such as MYXVΔM31R-hFlt3L-OX40L.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

MYXVΔM63R and MYXVΔM64R

The disclosure of the present technology relates to an M63R mutant myxoma virus (i.e., MYXVΔM63R; MYXV comprising an M63R deletion; MYXV genetically engineered to comprise a mutant M63R gene), and an M64R mutant myxoma virus (i.e., MYXVΔM64R, MYXV comprising an M64R deletion; MYXV genetically engineered to comprise a mutant M64R gene), or immunogenic compositions comprising the viruses, and its use as a cancer immunotherapeutic. In some embodiments, the M63R or M64R mutants are inserted into a MYXVΔM127 mCherry genome (see, e.g., FIG. 170 ). In some embodiments, the MYXVΔM63R or MYXVΔM64R virus is engineered to express one or more specific genes of interest (SG), such as those disclosed herein, for use as a cancer immunotherapeutic. In some embodiments, the myxoma virus is derived from strain Lausanne (given by, e.g., GenBank Accession No. AF170726.2). In some embodiments, the M63R or M64R gene of the myxoma virus, through homologous recombination techniques, is engineered to contain a disruption comprising a heterologous nucleic acid sequence comprising one or more expression cassettes, which result in a M63R or M64R knockout such that the M63R or M64R gene is not expressed, expressed at levels so low as to have no effect, or the expressed protein is non-functional (e.g., is a null-mutation). In some embodiments, the ΔM63R or ΔM64R mutant includes a heterologous nucleic acid sequence (e.g., a heterologous nucleic acid sequence comprising an open reading frome that encodes a specific gene of interest (SG)).

In some embodiments, the genetically engineered or recombinant MYXVΔM63R or MYXVΔM64R viruses described above are modified to express at least one heterologous gene, such as any one or more of hOX40L, hFlt3L, hIL-2, hIL-12, hIL-15, hIL-15/IL-15Rα, hIL-18, hIL-21, anti-huCTLA-4, anti-huPD-1, anti-huPD-L1, GITRL, 4-1BBL, or CD40L, and/or include at least one other viral gene mutation or deletion, such as any one or more of the following deletions: E3L (ΔE3L); E3LΔ83N; C7 (ΔC7L); B2R (ΔB2R), B19R (B18R; ΔWR200); E5R; K7R; C12L (IL18BP); B8R; B14R; N1L; C11R; K1L; M1L; N2L; and/or WR199. In other embodiments, no further heterologous genes are added other than those provided in the name of the virus herein, and/or no further viral genes other than M63R or M64R are disrupted or deleted.

In some embodiments, the heterologous nucleotide sequence further comprises an additional expression cassette comprising an open reading frame that encodes a selectable marker operably linked to a promoter that is capable of directing expression of the selectable marker. In some embodiments, the selectable marker is a xanthine-guanine phosphoribosyl transferase (gpt) gene. In some embodiments, the selectable marker is a green fluorescent protein (GFP) gene. In some embodiments, the selectable marker is an mCherry gene encoding a red fluorescent protein.

Non-limiting examples of a MVAΔ63R and MVAΔ64R construct open reading frames according to the present technology are shown in SEQ ID NOs: 39 and 40 (Table 9).

XI. Melanoma

Melanoma, one of the deadliest cancers, is the fastest growing cancer in the U.S. and worldwide. In most cases, advanced melanoma is resistant to conventional therapies, including chemotherapy and radiation. As a result, people with metastatic melanoma have a very poor prognosis, with a life expectancy of only 6 to 10 months. The discovery that about 50% of melanomas have mutations in BRAF (a key tumor-promoting gene) opened the door for targeted therapy of this disease. Early clinical trials with BRAF inhibitors showed remarkable, but unfortunately not sustainable, responses in patients with melanomas with BRAF mutations. Therefore, alternative treatment strategies for these patients, as well as others with melanoma without BRAF mutations, are urgently needed.

Human pathological data indicate that the presence of T-cell infiltrates within melanoma lesions correlates positively with longer patient survival (Oble et al., Cancer Immun. 9:3 (2009)). The importance of the immune system in protection against melanoma is further supported by partial success of immunotherapies, such as the immune activators IFN-α2b and IL-2 (Lacy et al., Expert Rev. Dermatol. 7(1):51-68 (2012)) as well as the unprecedented clinical responses of patients with metastatic melanoma to immune checkpoint therapy, including anti-CTLA-4 and anti-PD-1/PD-L1 as an agent alone or in combination therapy (Sharma & Allison, Science 348(6230)” 56-61 (2015); Hodi et al., NEJM 363(8):711-723 (2010); Wolchok et al., Lancet Oncol. 11(6):155-164 (2010); Topalian et al., NEJM 366(26):2443-2454 (2012); Wolchok et al., NEJM 369(2):122-133 (2013); Hamid et al., NEJM 369( 2 ):134-144 (2013); Tumeh et al., Nature 515(7528):568-571 (2014)). However, many patients fail to respond to immune checkpoint blockade therapy alone.

XII. Type I IFN and the Cytosolic DNA-Sensing Pathway in Tumor Immunity

Type I IFN plays important roles in host antitumor immunity (Fuertes et al., Trends Immunol. 34:67-73 (2013)). IFNAR1-deficent mice are more susceptible to developing tumors after implantation of tumor cells; spontaneous tumor-specific T-cell priming is also defective in IFNAR1-deficient mice (Diamond et al., J. Exp. Med. 208:1989-2003 (2011); Fuertes et al., J. Exp. Med. 208:2005-2016 (2011)). More recent studies have shown that the cytosolic DNA-sensing pathway is important in the innate immune sensing of tumor-derived DNA, which leads to the development of antitumor CD8 + T-cell immunity (Woo et al., Immunity 41:830-842 (2014)). This pathway also plays a role in radiation-induced antitumor immunity (Deng et al., Immunity 4:843-852 (2014)). Although spontaneous anti-tumor T-cell responses can be detected in patients with cancers, cancers eventually overcome host antitumor immunity in most patients. Novel strategies to alter the tumor immune suppressive microenvironment would be beneficial for cancer therapy.

XIII. Immune Response

In addition to induction of the immune response by up-regulation of particular immune system activities (such as antibody and/or cytokine production, or activation of cell mediated immunity), immune responses may also include suppression, attenuation, or any other down-regulation of detectable immunity, so as to reestablish homeostasis and prevent excessive damage to the host's own organs and tissues. In some embodiments, an immune response that is induced according to the methods of the present disclosure generates effector T-cells (e.g., helper, killer, regulatory T-cells). In some embodiments, an immune response that is induced according to the methods of the present disclosure generates effector CD8 + (antitumor cytotoxic CD8 + ) T-cells or activated T helper (T H ) cells (e.g., effector CD4 + T-cells), or both that can bring about directly or indirectly the death, or loss of the ability to propagate, of a tumor cell.

Induction of an immune response by the compositions and methods of the present disclosure may be determined by detecting any of a variety of well-known immunological parameters (Takaoka et al., Cancer Sci. 94:405-11 (2003); Nagorsen et al., Crit. Rev. Immunol. 22:449-62 (2002)). Induction of an immune response may therefore be established by any of a number of well-known assays, including immunological assays. Such assays include, but need not be limited to, in vivo, ex vivo, or in vitro determination of soluble immunoglobulins or antibodies; soluble mediators such as cytokines, chemokines, hormones, growth factors and the like as well as other soluble small peptide, carbohydrate, nucleotide and/or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for example cell proliferation, altered motility, altered intracellular cation gradient or concentration (such as calcium); phosphorylation or dephosphorylation of cellular polypeptides; induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles, or the onset of apoptosis (programmed cell death); or any other criterion by which the presence of an immune response may be detected. For example, cell surface markers that distinguish immune cell types may be detected by specific antibodies that bind to CD4 + , CD8 + , or NK cells. Other markers and cellular components that can be detected include but are not limited to interferon γ (IFN-γ), tumor necrosis factor (TNF), IFN-α, IFN-β (IFNB), IL-6, and CCL5. Common methods for detecting the immune response include, but are not limited to, flow cytometry, ELISA, immunohistochemistry. Procedures for performing these and similar assays are widely known and may be found, for example in Letkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, Current Protocols in Immunology, 1998).

XIV. Pharmaceutical Compositions and Preparations of the Present Technology

Disclosed herein are pharmaceutical compositions comprising MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 that may contain a carrier or diluent, which can be a solvent or dispersion medium containing, for example, water, saline, Tris buffer, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be effected by various antibacterial and antifungal agents and preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars or sodium chloride, and buffering agents are included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin or carrier molecules. Other excipients may include wetting or emulsifying agents. In general, excipients suitable for injectable preparations can be included as apparent to those skilled in the art.

Pharmaceutical compositions and preparations comprising MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4- ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical viral compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate formulating virus preparations suitable for in vitro, in vivo, or ex vivo use. The compositions can be combined with one or more additional biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent or excipient to generate pharmaceutical (including biologic) or veterinary compositions of the instant disclosure suitable for parenteral or intratumoral administration.

Many types of formulation are possible as is appreciated by those skilled in the art. The particular type chosen is dependent upon the route of administration chosen, as is well-recognized in the art. For example, systemic formulations will generally be designed for administration by injection, e.g., intravenous, as well as those designed for intratumoral delivery. In some embodiments, the systemic or intratumoral formulation is sterile.

Sterile injectable solutions are prepared by incorporating MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the virus plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 compositions of the present disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks's solution, Ringer's solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 compositions may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, 1-dodecylhexahydro-2H-azepin-2-one (laurocapran), oleic acid, sodium citrate, Tris HCl, dextrose, propylene glycol, mannitol, polysorbate polyethylenesorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol sucrose, trehalose and other such generally known in the art may be used in any of the compositions of the instant disclosure. (Pramanick et al., Pharma Times 45(3):65-76 (2013)).

The biologic or pharmaceutical compositions of the present disclosure can be formulated to allow the virus contained therein to be available to infect tumor cells upon administration of the composition to a subject. The level of virus in serum, tumors, and if desired other tissues after administration can be monitored by various well-established techniques, such as antibody-based assays (e.g., ELISA, immunohistochemistry, etc.).

The engineered poxviruses of the present technology can be stored at −80° C. with a titer of 3.5×10 7 pfu/mL formulated in about 10 mM Tris, 140 mM NaCl pH 7.7. For the preparation of vaccine shots, e.g., 10 2 -10 8 or 10 2 -10 9 viral particles can be lyophilized in 100 mL of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, the injectable preparations can be produced by stepwise freeze-drying of the engineered poxvirus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose, or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 4° C. and room temperature for several months. In some embodiments, the ampoule is stored at temperatures below −20° C.

For therapy, the lyophilisate can be dissolved in an aqueous solution, such as physiological saline or Tris buffer, and administered either systemically or intratumorally. The mode of administration, the dose, and the number of administrations can be optimized by those skilled in the art.

The pharmaceutical composition according to the present disclosure may comprise an additional adjuvant. As used herein, an “adjuvant” refers to a substance that enhances, augments, or potentiates the host's immune response to tumor antigens. A typical adjuvant may be aluminum salts, such as aluminum hydroxide or aluminum phosphate, Quil A, bacterial cell wall peptidoglycans, virus-like particles, polysaccharides, toll-like receptors, nano-beads, etc. (Aguilar et al., Vaccine 25:3752-3762 (2007)).

XV. Therapeutic Methods of the Present Technology

In one aspect, the present disclosure provides a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of: a recombinant modified vaccinia Ankara (MVA) virus comprising a deletion of E3L (MVAΔE3L) genetically engineered to express OX40L (MVAΔE3L-OX40L); a recombinant MVA virus comprising a deletion of C7L (MVAΔC7L) genetically engineered to express OX40L (MVAΔC7L-OX40L); a recombinant MVAΔC7L engineered to express OX40L and hFlt3L (MVAΔC7L-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of C7L, a deletion of E5R, and to express hFlt3L and OX40L (MVAΔC7LΔE5R-hFlt3L-OX40L); a recombinant MVA genetically engineered to comprise a deletion of E5R (MVAΔE5R); a recombinant MVA genetically engineered to comprise a deletion of E5R and to express hFlt3L and OX40L (MVAΔE5R-hFlt3L-OX40L); a recombinant vaccinia virus comprising a deletion of C7L (VACVΔC7L) genetically engineered to express OX40L (VACVΔC7L-OX40L); a recombinant VACVΔC7L genetically engineered to express both OX40L and hFlt3L (VACVΔC7L-hFlt3L-OX40L); a VACV genetically engineered to comprise a deletion of E5R (VACVΔE5R); a recombinant VACV genetically engineered to comprise a deletion of E5R, a deletion of thymidine kinase (TK), and to express ani-CTLA-4, hFlt3L, and OX40L (VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L); a MYXV genetically engineered to comprise a deletion of M31R (MYXVΔM31R); a recombinant MYXV genetically engineered to comprise a deletion of M31R and to express hFl3L and OX40L (MYXVΔM31R-hFlt3L-OX40L); and/or additional engineered poxviruses selected from MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4, or combinations thereof. In some embodiments, the treatment comprises one or more of the following: inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject, reducing the size of the tumor, eradicating the tumor, inhibiting growth of the tumor, inhibiting metastatic growth of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, the induction, enhancement, or promotion of the immune response comprises one or more of the following: increased levels of effector T-cells in tumor cells as compared to tumor cells infected with the corresponding MVA, MVAΔE3L, MVAΔC7L, VACV, VACVΔC7L, or MYXV strain; and increased splenic production of effector T-cells as compared to the corresponding MVA, MVAΔE3L, MVAΔC7L, VACV, VACVΔC7L, or MYXV strain. In some embodiments, the subject is a human. In some embodiments, the composition of the present technology comprising MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 is administered to the subject by intratumoral or intravenous injection or a simultaneous (i.e., concurrent) or sequential combination of intratumoral and intravenous injection.

In some embodiments, the subject is diagnosed with a cancer such as melanoma, colon carcinoma, breast cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor and other bone tumors (e.g., osteosarcoma, malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, pancreatic cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, brain/CNS tumors (e.g., astrocytoma, glioma, glioblastoma, childhood tumors, such as atypical teratoid/rhabdoid tumor, germ cell tumor, embryonal tumor, ependymoma) medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, head-and-neck cancer, rectal adenocarcinoma, glioma, urothelial carcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer) non-small cell lung cancer (squamous and adenocarcinoma), ductal carcinoma in situ, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal cancer, eye cancer (e.g., melanoma, retinoblastoma), gallbladder cancer, gastrointestinal cancer, heart cancer, laryngeal and hypopharyngeal cancer, oral cancer (e.g., lip, mouth, salivary gland), nasopharyngeal cancer, neuroblastoma, peritoneal cancer, pituitary cancer, Kaposi's sarcoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, parathyroid cancer, vaginal tumor, and the metastases of any of the foregoing.

XVI. Combination Therapy with Other Active Agents

In some embodiments, the engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4) are combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with any combination of: (i) one or more immune checkpoint blocking agents and/or one or more immune system stimulators; (ii) one or more anti-cancer drugs; and (iii) an immunomodulatory drug (i.e., fingolimod (FTY720)). In some embodiments, the combined administration of the engineered poxviruses of the present technology (e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4) with any one or more of: (i) one or more immune checkpoint blocking agents and/or one or more immune system stimulators; (ii) one or more anti-cancer drugs; and (iii) an immunomodulatory drug (i.e., fingolimod (FTY720)) results in a synergistic effect with respect to the treatment of solid tumors.

A. Immune Checkpoint Blocking Agents and Immune System Stimulators

In some embodiments, MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 is combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with one or more immune checkpoint blocking agents and/or one or more immune system stimulators. The one or more immune checkpoint blocking agents may target any one or more of PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), or CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., anti-huPD-1, anti-huPD-L1, or anti-huCTLA-4 antibodies).

In some embodiments, the one or more immune checkpoint blocking agents are selected from the group consisting of ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, and durvalumab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, B7-H4, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, and any combination thereof. Dosage ranges of the foregoing are known in or readily within the skill in the art as several dosing clinical trials have been completed, making extrapolation to other agents possible.

By way of example, but not by way of limitation, in some embodiments, the one or more immune system stimulators are selected from among a natural killer cell (NK) stimulator, an antigen presenting cell (APC) stimulator, a granulocyte macrophage colony-stimulating factor (GM-CSF), and a toll-like receptor stimulator.

In some embodiments, the NK stimulator includes, but is not limited to, IL-2, IL-15, IL-15/IL-15RA complex, IL-18, and IL-12. In some embodiments, the NK stimulator includes an antibody that stimulates at least one of the following receptors NKG2, KIR2DL1/S1, KRI2DL5A, NKG2D, NKp46, NKp44, or NKp30.

In some embodiments, the APC stimulator includes, but is not limited to, CD28, ICOS, CD40, CD30, CD27, OX-40, and 4-1BB.

In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and one or more immune checkpoint inhibitors and/or one or more immune system stimulators results in a synergistic effect. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hil-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and one or more immune checkpoint inhibitors and/or one or more immune system stimulators results in an enhanced anti-tumor effect. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and anti-PD-L1 results in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-C11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15RΔ-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and anti-PD-1 results in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199μWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and anti-CTLA-4 results in a synergistic effect with respect to the treatment of solid tumors.

In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 with one or more immune checkpoint blocking agents and/or one or more immune system stimulators is further combined or separately, sequentially, or simultaneously (i.e., concurrently) administered with one or more anti-cancer drugs and/or immunomodulatory drugs described below in Sections XVI B and C. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RΔ, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R, MYXVΔM62R, MYXVΔM62RΔM63RΔM64R, MYXVΔM31R, MYXVΔM62RΔM63RΔM64RΔM31R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and/or MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 with one or more immune checkpoint blocking agents and/or one or more immune system stimulators and one or more anti-cancer drugs and/or immunomodulatory drugs described below in Sections XVI B and C results in a synergistic effect with respect to the treatment of solid tumors.

It has been reported that the sequential (i.e., serial) administration of anti-OX40 antibody followed by the immune checkpoint inhibitor, anti-PD-1 antibody, improves the therapeutic efficacy of the combination, resulting in delayed tumor progression and, in some cases, complete tumor regression. (See, e.g., Shrimali et al., Cancer Immunol. Res. 5(9): OF1-OF12 (2017); Messenheimer et al., Clin. Cancer Res. 23(20):6165-6177 (2017)). However, the same studies show that the simultaneous (i.e., concurrent) administration of anti-OX40 antibody and anti-PD-1 antibody negates the anti-tumor effects of OX40 antibody and results in poor treatment outcomes in mice. (See, Shrimali et al., (2017); Messenheimer et al., (2017)). By contrast, as shown in FIGS. 11 B- 11 G , the combined, simultaneous (i.e., concurrent) administration of the viruses expressing the OX40L transgene of the present technology (e.g., MVAΔC7L-hFl3L-OX40L) and an immune checkpoint inhibitor (e.g., MVAΔC7L-hFl3L-OX40L+anti-PD-L1 or MVAΔC7L-hFl3L-OX40L+anti-CTLA-4 or MVAΔC7L-hFl3L-OX40L+anti-PD-1 (not shown)) in a mouse melanoma model surprisingly and unexpectedly results in enhanced anti-tumor effects in both injected and non-injected tumors and increased survival as compared to controls. In some embodiments, the combination of the viruses expressing the OX40L transgene of the present technology, e.g., MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-1 5 /IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L- hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RΔ, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and/or and one or more immune checkpoint inhibitors (e.g., anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody) results in a surprising and unexpected enhanced anti-tumor effect as compared to the combination of an immune checkpoint inhibitor and anti-OX40 agonist antibody. In some embodiments, the combination of MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15RαΔC11R, MYXVΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα, MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-anti-CTLA-4, and MYXVΔM62RΔM63RΔM64RΔM31R-hFlt3L-OX40L-IL-12-IL-15/IL-15Rα-anti-CTLA-4 and one or more immune checkpoint inhibitors (e.g., anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody) results in a surprising and unexpected synergistic effect with respect to the treatment of solid tumors as compared to the combination of an immune checkpoint inhibitor and anti-OX40 agonist antibody.

B. Anti-Cancer Drugs

Receptor tyrosine kinases, such as EGFR and HER2, have been implicated in promoting tumor cell proliferation and survival through the Ras-Raf-Mek-Erk (Ras-MAPK) pathway. Raf and Mek are also targets for inhibiting oncogenic signals arising from upstream receptor tyrosine kinases or from gain-of-function mutations in RAS or RAF that drive Ras-MAPK signaling. The identification of key activating mutations in cancers including melanoma have led to the development of targeted therapies along the MAPK-pathway. For example, activating mutations in BRAF occur in over half of the melanoma cancers, a majority of which include the BRAF V600E mutation, which constitutively activates the MAPK signaling pathway. This in turn leads to increased metastatic behavior including invasiveness, while reducing apoptosis (i.e., increasing cancer cell survival). Several anti-cancer drugs have been developed to mitigate the pathogenic signaling from this pathway. Focused therapies targeting this pathway include inhibitors of EGFR, HER 2 , BRAF, RAF, and MEK.

Immunotherapies consisting of checkpoint inhibitors (PD-1/PD-L1, CTLA-4) and combinations of MAPK-pathway targeted therapies have shown promising results in, for example, BRAF-positive advanced melanoma. It has been reported that MAPK pathway activation contributes to immune escape, while MAPK pathway inhibition contributes to a more favorable immune environment via abrogation of immunosuppressive factors as well as dysregulation of certain other immunoregulatory proteins such as PD-L1. Furthermore, oncolytic herpes virus (T-Vec) in dual combination with MAPK pathway inhibition has been shown in preclinical models to increase cancer cell death as compared with single agent alone, while the triple combination of MAPK-inhibitor, T-Vec, and a checkpoint inhibitor (e.g., anti-PD-L1 antibody) showed synergistic immunostimulatory effects. Robust immune response with MAPK pathway inhibition has been strongly associated with increased activation of CD8 T-cell influx and increased levels of secreted IFN and TNF-α.

As demonstrated herein, the recombinant viral constructs of the present technology comprising deletions of E5R (or its orthologue), such as MVAΔE5R, VACVΔE5R, and MYXVΔM31R, significantly increase IFN gene expression levels greater than 1000-fold compared to a corresponding wild-type virus (see FIG. 60 A ).

In some embodiments, MVAΔE3L-OX40L, MVAΔC7L-OX40L, MVAΔC7L-hFlt3L-OX40L, MVAΔC7LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L, MVAΔE3LΔE5R-hFlt3L-OX40L, MVAΔE5R-hFlt3L-OX40L-ΔC11R, MVAΔE3LΔE5R-hFlt3L-OX40L-ΔC11R, VACVΔC7L-OX40L, VACVΔC7L-hFlt3L-OX40L, VACVΔE5R, VACV-TK − -anti-CTLA-4-ΔE5R-hFlt3L-OX40L, VACVΔB2R, VACVE3LΔ83NΔB2R, VACVΔE5RΔB2R, VACVE3LΔ83NΔE5RΔB2R, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12, VACVE3LΔ83N-ΔTK-anti-CTLA-4-ΔE5R-hFlt3L-OX40L-IL-12-ΔB2R, MYXVΔM31R, MYXVΔM31R-hFlt3L-OX40L, MYXVΔM63R, MYXVΔM64R, MVAΔWR199, MVAΔE5R-hFlt3L-OX40L-ΔWR199, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R, MVAΔE3LΔE5R-hFlt3L-mOX40LΔWR199-hIL-12ΔC11R-hIL-15/IL-15α, VACVΔE5R-IL-15/IL-15Rα, VACVΔE5R-IL-15/IL-15Rα-OX40L, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200-hIL-15/IL-15Rα, VACVΔE3L83N-ΔTK-anti-huCTLA-4-ΔE5R-hFlt3L-hOX40L-hIL-12ΔB2RΔWR199ΔWR200ΔC11R, VACVΔE3L83N-ΔTK-anti-huC