RECOMBINANT POXVIRUSES FOR CANCER IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/381,996, filed November 2, 2022, the contents of which are incorporated by reference in their entirety for any and all purposes.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under CA280657 and CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] 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 (MV A) virus comprising a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding OX40L or human OX40L (hOX40L), a heterologous nucleic acid molecule encoding Flt3L or human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding IL- 12 or human IL- 12 (hIL-12) (MVAAE3LAE5R-Flt3L-OX40LAWR199-IL-12 or MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12) alone or in combination with immune checkpoint blockade inhibitors. In particular, the present technology relates to the use of MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12, alone or in combination with immune checkpoint blockade inhibitors, as an immunotherapeutic composition, in methods for treating a solid tumor wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment, methods of preventing cancer recurrence for a period of time in a subject in need thereof, methods for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive immune system response, and methods for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. BACKGROUND

[0004] 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.

[0005] Malignant tumors 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.

SUMMARY

[0006] In one aspect, the present disclosure proides a method for treating a solid tumor in a subject in need thereof, the method comprising delivering to a tumor an immunogenic composition comprising an effective amount of a modified vaccinia Ankara (MV A) virus genetically engineered to comprise: a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding human OX40L (hOX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding human IL- 12 (hIL-12) (MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12); wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment and/or wherein the tumor comprises tumor cells comprising loss or mutation of the beta-2-microglobulin (B2M) gene or characterized by downregulation of B2M, and/or wherein the tumor is MHC-1 antigen presentation deficient. In some embodiments, the tumor is resistant to a CD8+ T-cell-mediated immune response. In some embodiments, the tumor is MHC-I antigen presentation deficient. In some embodiments, the tumor comprises tumor cells comprising loss or mutation of the B2M gene. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant. 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 one or more metastatic growths of the tumor, inducing apoptosis of tumor cells, or prolonging survival of the subject. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and/or expansion of one or more of stem-like T-cells, neutrophils, macrophages, and/or monocytes. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and/or expansion of stem-like T-cells. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and/or expansion of neutrophils. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and/or expansion of neutrophils and macrophages. In some embodiments, inducing an immune response in the subject against the tumor or enhancing or promoting an ongoing immune response against the tumor in the subject comprises increasing activation and/or expansion of neutrophils, macrophages, and monocytes. In some embodiments, the composition is administered to the subject by intratumoral injection. In some embodiments, the tumor is melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD).

[0007] In one aspect, the present disclosure provides, a method of preventing cancer recurrence for a period of time in a subject in need thereof comprising delivering to the subject an immunogenic composition comprising an effective amount of a modified vaccinia Ankara (MV A) virus genetically engineered to comprise: a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding human OX40L (hOX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding human IL-12 (hIL-12) (MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12); wherein the subject either currently has cancer or has previously had cancer; and wherein the method is effective for treating a current cancer and preventing recurrence thereof for a period of time, preventing recurrence of a previous cancer for a period of time, or preventing occurrence of a new cancer for a period of time. In some embodiments, the period of time for preventing recurrence or a current cancer, preventing recurrence of a previous cancer, and preventing occurrence of a new cancer is about 2 months. In one aspect, the present disclosure provides, a method for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive immune system response, the method comprising administering to the subject an effective amount of an immunogenic composition comprising an effective amount of a modified vaccinia Ankara (MV A) virus genetically engineered to comprise: a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding human OX40L (hOX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding human IL-12 (hIL-12) (MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12). In some embodiments, the solid tumor is resistant to immune checkpoint blockade inhibitor treatment and/or wherein the tumor comprises tumor cells comprising loss or mutation of the beta-2 - microglobulin (B2M) gene or characterized by downregulation of B2M, and/or wherein the tumor is MHC-1 antigen presentation deficient. In some embodiments, the tumor is resistant to a CD8+ T-cell-mediated immune response. In some embodiments, the tumor is MHC-I antigen presentation deficient. In some embodiments, the tumor comprises tumor cells comprising loss or mutation of the B2M gene. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant. In some embodiments, administration of the virus increases activation and/or expansion of one neutrophils. In some embodiments, the composition is administered to the subject by intratumoral injection.

[0008] In one aspect, the present disclosure provides, a method for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof, the method comprising in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising an effective amount of a modified vaccinia Ankara (MV A) virus genetically engineered to comprise: a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding human OX40L (hOX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding human IL-12 (hIL-12) (MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12). In some embodiments, the solid tumor is resistant to immune checkpoint blockade inhibitor treatment and/or wherein the tumor comprises tumor cells comprising loss or mutation of the beta-2 - microglobulin (B2M) gene or characterized by downregulation of B2M, and/or wherein the tumor is MHC-1 antigen presentation deficient. In some embodiments, the tumor is resistant to a CD8+ T-cell-mediated immune response. In some embodiments, the tumor is MHC-I antigen presentation deficient. In some embodiments, the tumor comprises tumor cells comprising loss or mutation of the B2M gene. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant. In some embodiments, administration of the virus increases the proportion of one or more of neutrophils or monocytes in the TIME and/or decreases the proportion of M2-like macrophages in the TIME. In some embodiments, administration of the virus increases the proportion of neutrophils in the TIME. In some embodiments, administration of the virus increases the proportion of monocytes in the TIME. In some embodiments, administration of the virus decreases the proportion of M2-like macrophages in the TIME. In some embodiments, administration of the virus increases the proportion of neutrophils and monocytes and decreases the proportion of M2-like macrophages in the TIME. In some embodiments, the composition is administered to the subject by intratumoral injection. In any of the preceding embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGs. 1A-1M show MQ833 infection of tumor and immune cells activates cGAS/STING mediated dsDNA sensing pathway and promotes IFN production. FIG. 1A is a diagram of MQ833 design. FIG. IB is a diagram of the IL12 cassette. For FIGs. 1C and ID, B16-F10 cells were infected by MVAAE5R or MQ833 at a MOI of 10. Cells and supernatant were collected at 16 hours post infection (hpi). FIG. 1C is a flow cytometric analysis of mOX40L and hFlt3L transgene expression. FIG. ID is a chart showing the results of an ELISA test for mIL12p40. For FIGs. IE and IF, B16-F10 cells were infected with indicated viruses at a MOI of 10, and cells and supernatant were collected 16 hpi. FIG. IE is a chart showing RT-PCR for Ifnb gene expression. FIG. IF is a chart showing the results of an ELISA test for IFN-p. FIG. 1G is a Western blot for indicated protein and phosphorylated proteins in the cGAS-STING pathway. B16-F10 cells were infected with indicated viruses at a MOI of 10, and cells were collected at 2, 4, 6, 8, 16 hours post infection (hpi). For FIGs. 1H and II, BMDCs from WT or Sting ^GllGl mice were infected with indicated viruses at a MOI of 10. Cells were lysed and collected for Western blot at indicated time points post infection. Supernatants were collected at 16 hours post infection. FIG. 1H is a bar chart showing the results of an ELISA for IFN-y. FIG. II is a Western blot for cGAS, p-IRF3, IRF3, and GAPDH. FIG. 1 J is a schematic diagram for DC and T cell coculture experiment. FIGs. 1K-1M are the results of ELISA for IFN-y level in supernatants from the coculture system. For FIG. IK, CD1 lc ⁺ DCs were isolated from the spleens of WT C57BL/6J or cGAS' ^/_ mice, pulsed with OVA, and infected with different viruses. OT-1 cells isolated from OT-1 mice were then added to the DCs and cocultured for 3 days. Supernatants were collected and IFN-y level was measured by ELISA. Data are means ± SD (n=3; *P < 0.05, **** < 0.000 J, t test). For FIG. IL, CD103 ⁺ DCs were sorted from Flt3L+ DCs and and subsequently pulsed with OVA and infected with different viruses. OT-1 cells isolated from OT-1 mice were then added to the DCs and co-cultured for 3 days. Supernatants were collected and IFN-y level was measured by ELISA. For FIG. IM, BMDCs were pulsed with OVA and infected with MQ833. OT-1 cells were either cocultured with pulsed and infected BMDCs or cultured in medium from the infected BMDC for 3 days.

[0010] FIGs. 2A-2H show intratumoral (IT) delivery of MQ833 generates an 80-100% cure in a B16-F10 melanoma model in a STING-MDA5-STAT2-dependent manner. FIG. 2A is a schematic for the experimental design showing 5 xlO ⁵ of B16-F10 cells intradermally implanted into the right flank of WT C57BL/6J mice (FIGs. 2B-2E), Mda5' ^/_ , Sting ^Gt/Gt , Mda5' ^/_ Sting ^Gt/Gt , or Stat2' ^/_ mice (FIGs. 2F-2G). Intratumoral virus treatments were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG. 2B is a chart showing the initial tumor volume for each group. FIG. 2C is a Kaplan-Meier survival curve of B16-F10 tumor bearing mice treated with MQ833 virus. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5- 10, **P<0.01, ****P < 0.0001). FIG. 2D are charts showing tumor volumes of mice injected with MQ832, MQ833, or PBS over days post treatment. FIG. 2E is a schematic diagram of flow cytometric analysis of tumor infiltrating lymphocytes. WT C57BL/6J mice were implanted with B16-F10 cells on the right (5 xlO ⁵ ) and left (2.5 xlO ⁵ ) flanks. Once the tumors were established, 2 doses of 4 xlO ⁷ PFU of MQ833 were intratumorally delivered to the right flanks twice, 3 days apart. Injected and non-injected tumors were harvested at day 2 post the second injection and then stained and analyzed by flow cytometry. FIG. 2F are charts showing percentages of CD8 ⁺ Granzyme B ⁺ , KLRG1 ⁺ CD8 ⁺ , and CD4 ⁺ Foxp3 ⁺ T cells in the injected and non-injected tumors in different treatment groups. Data are means ± SD (n=5; *P<0.05, **P<0.01, ***P<0.001, ****P < 0.0001, t test). FIG. 2G is a Kaplan- Meier survival curve of B16-F10 tumor bearing mice treated with MQ833 virus. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=8- 16, **p<0.01, ****P < 0.0001). FIG. 2H are charts showing tumor volume curves in different groups over days post treatment.

[0011] FIGs. 3A-3J show IT MQ833 injection reprograms the tumor-infiltrated myeloid cells. FIG. 3A is a schematic diagram of the experimental schedule. 5xl0 ⁵ of B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice, MdaS' ¹ ' Sting ^{G ,G} or Stat2' ^! ' mice. One IT MQ833 injection was given once the tumors were established. Tumors were collected and processed into single cell suspension, and CD45 ⁺ cells were sorted and scRNA-seq analyses were performed. FIG. 3B is a UMAP display of sorted CD45 ⁺ cells from 6 samples combined following 10X Genomics scRNA-seq workflow (n = 47020 cells). FIG. 3C are expression plots of 8 main cell types using their top marker genes. FIG. 3D are UMAP plots of cell clusters separated by sample. FIG. 3E is a stacked bar plot showing the percentage of each cluster across different samples. FIG. 3F is a dot plot for the top enriched and suppressed gene sets from the Molecular Signature Database (MSigDB) Hallmark immunologic signature gene sets in WT MQ833 versus WT PBS. FIG. 3G is a gene set enrichment analysis (GSEA) for individual pathways from the representative enriched and suppressed gene sets in WT MQ833 sample compared with WT PBS. FIG. 3H are heatmaps of the average expression of genes in the hallmark IFNa and fFNy response gene sets. Selected genes of interest are labeled in the right panel. FIG. 31 are volcano plots of differentially expressed genes in neutrophil clusters between indicated samples. Important genes are highlighted. FIG. 3J are heatmaps of the average expression of representative genes from the hallmark IFNa and fFNy response gene sets (left panel) and marker genes for anti-tumor or pro-tumor neutrophils (right panel).

[0012] FIGs. 4A-4F shows IT MQ833 injection reprograms the tumor-infiltrated T cells. 5 xlO ⁵ of B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice, MdaS' ¹ ' Sting ^{G ,G} or Stat2' ¹ ' mice. One dose of IT MQ833 injection was given once the tumors were established. Tumors were collected and processed into single cell suspension, and CD45 ⁺ cells were sorting and sent for scRNA-seq. FIG. 4A is a UMAP display of subclustered CD45 ⁺ CD3 ⁺ cells from 6 samples combined following 10X Genomics scRNA- seq workflow (n = 9,540 cells). FIG. 4B are UMAP plots of cell clusters separated by sample. FIG. 4C is a heat-map of selected gene expression in different clusters shown in FIG. 4 A. FIG. 4D is a stacked bar plot showing the percentage of each cluster across different samples. FIGs. 4E-4F are developmental pseudotimes and trajectory analyses of T cells in a UMAP display.

[0013] FIGs. 5A-5I show IT MQ833 reprograms TIME in both injected and non-injected tumors in a bilateral B16-F10 melanoma model. FIG. 5A is a schematic diagram of the experimental schedule. B16-F10 cells were intradermally implanted into the right (5 xlO ⁵ ) and left (2.5 xlO ⁵ ) flanks of WT C57BL/6J mice. Once the tumors were established, two doses of IT MQ833 injection were given 3 days apart. Tumors were collected 2 days after the second injection and processed into single cell suspension. CD45+ cells were sorted and sent for scRNA-seq. FIG. 5B is a UMAP display of sorted CD45+ cells from 3 samples combined following 10X Genomics scRNA-seq workflow (n = 21,675 cells). FIG. 5C are expression plots of 7 main cell types using their top marker genes. FIG. 5D are UMAP plots of cell clusters separated by sample. FIG. 5E is a stacked bar plot showing the percentage of each cluster across different samples. FIG. 5F is a UMAP display of subclustered CD45 ⁺ CD3 ⁺ cells from 6 samples combined following 10X Genomics scRNA-seq workflow (n = 4,901 cells). FIG. 5G are UMAP plots of cell clusters separated by sample. FIG. 5H is a stacked bar plot showing the percentage of each cluster across different samples. FIG. 51 are heatmaps of the average expression of representative marker genes for each cluster.

[0014] FIGs. 6A-6N show MQ833-induced antitumor immunity requires CD8 ⁺ T cells, neutrophils, and macrophages and nos2 expression in myeloid cells plays a crucial role. FIG. 6A is a schematic diagram of the experimental schedule. 5 xlO ⁵ of B16-F10 cells were intradermally implanted into the right flanks of WT C57BL/6J mice. IT MQ833 injections were given twice a week once the tumors were established. I.p. aCD8, aCD4, aNKl.l, and aLy6G antibodies were given one day prior to every virus injection. I.p. aCSFl were given every 5 days starting at one day prior to the first virus injection. Tumor size and mice survival were monitored. FIG. 6B is a Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log -rank (Mantel-Cox) test (n= 10-20; *P < 0.05, **P < 0.01, ****p < 0.0001). FIG. 6C are tumor volumes of mice separated by group over days post treatment. FIG. 6D is a schematic diagram of the flow cytometry analysis experiment. FIG. 6E are representative_contour plots and FIG. 6F are bar graphs showing average percentages of iNOS ⁺ cells among CD45 ⁺ CD3‘ CD1 lb ⁺ Ly6G ⁺ neutrophils (FIGs. 6E-6F top row), CD45 ⁺ CD3‘ CD68 ⁺ CD1 lb ⁺ macrophages (FIGs. 6E-6F middle row), and percentages of CD206 ⁺ cells among macrophages (FIGs. 6E-6F bottom row) in MQ833 or PBS treated tumors. FIGs. 6G and 6H show a survival study for B 16-F10 tumor-bearing mice treated with MQ833 in a NOS2' ¹ ' mouse model.

5xl0 ⁵ of B16-F10 cells were intradermally implanted into the right flank of NOS2' ¹ ' mice. IT MQ833 injections were given twice a week once the tumors were established. Tumor size and mice survival were monitored. FIG. 6G is a Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. (n=10) Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-10; *P < 0.05, *** < 0.001, **** < 0.0001). FIG. 6H are tumor volumes of mice separated by group over days post treatment. FIGs. 61 and 6J show scRNA-seq analysis of CD45 ⁺ cells isolated from rMVAAE3L or MQ833 treated tumors. FIG. 61 is a UMAP display showing nos2 expression (right panel) in highlighted clusters (left panel) FIG. 6J are Heat maps showing average expression of selected antitumor marker genes in all the CD45 ⁺ cells (first panel), neutrophil (second panel), Ml- like macrophage (third panel), and monocyte (fourth panel) clusters divided by treatment groups. FIG. 6K are flow cytometric analyses and FIG. 6L are dot plots showing the percentage of iNOS ⁺ cells among neutrophils in tumors from WT mice treated with PBS (left), WT mice treated with MQ833 (middle), and StatP^ mice treated with MQ833. FIG. 6M are graphs showing the kinetics of Ly6G ⁺ neutrophil and Ly6C ⁺ monocytes infiltration to the MQ833 -injected tumors and their iNOS expression. FIG. 6N are graphs showing that the wild-type neutrophils responded to the stimulation with either IFN-P or IFN-y, or both for tumor-killing function, whereas Nos2-deficient neutrophils had much weaker tumor-killing activities in response to IFN-P, or IFN-y, or both.

[0015] FIGs. 7A-7I show IT MQ833 treatment is effective in mice lacking an adaptive immune response and that IT MQ833 treatment can overcome ICB resistance. FIG. 7A is a schematic diagram of the survival and flow cytometry experimental design. 5 xlO ⁵ of Bl 6- F10 cells were intradermally implanted into the right flank of Rag ¹ ' mice. IT MQ833 injections were given twice weekly once the tumors were established. For survival study, tumor size and mice survival were monitored. For flow cytometric analysis, tumors were harvested at day 14 post implantation and tumor infiltrating immune cells were stained by fluorescent antibodies and analyzed by flow cytometry. FIG. 7B is a Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=5-10; ****P < 0.0001'). FIG. 7C are tumor volumes of mice separated by group over days post treatment. FIG. 7D are dot plots and FIG. 7E are bar graphs showing percentage of CD45 ⁺ CD3'CD1 lb ⁺ Ly6G ⁺ neutrophils among total CD45 ⁺ cells (top row) and iNOS ⁺ cells among neutrophils (bottom row) in MQ833 or PBS treated tumors. For FIGs. 7F and 7G, 1 xlO ⁶ of B2m' ^/_ B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice. IT MQ833 injections were given twice weekly once the tumors were established. Tumor sizes and mice survival were monitored. FIG. 7F is a Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=10; **P < 0.01, ****P < 0.0001). FIG. 7G are tumor volumes of mice separated by group over days post treatment. For FIG. 7H, Mice rejected primary B16-F10 or B2m" ^/_ B16-F10 tumors were rechallenged with a lethal dose of B16-F10 tumor cells. FIG. 7H is a Kaplan-Meier survival curve of the rechallenged mice. FIG. 71 is a working model illustrating the mechanisms of action MQ833.

[0016] FIGs. 8A-8C show MVAAE3LAE5R promotes type I IFN expression in multiple tumor cell lines. FIG. 8A show qRT-PCR analyses of Ijnb gene expression in B16-F10, 4T1, MC38, E0771, PyMT, and ID8 tumor lines with MVA, MVAAE3L, MVAAE5R, MVAAE3LAE5R virus infection. FIG. 8B is a Western blot testing the expression of MDA5, p-IRF3, IRF3, STING, and GAPDH in different Bl 6-F 10 cell lines infected with MVAAE3LAE5R. WT, MDAS' ⁷ ' (clone 27 and clone 13), and MDAS^STING’ ⁷ ’ B16-F10 cells were infected with MVAAE3LAE5R viruses at a MOI of 10, and cells were collected at 2, 4, 8 hours post infection (hpi). FIG. 8C is an ELISA test for IFN-P production. WT, MDA5' ^/_ , and MDAS^STING’ ⁷ ’ B16-F10 cells were infected by MVAAE5R or MQ833 at a MOI of 10. Supernatant were collected at 16 hours post infection (hpi).

[0017] FIGs. 9A-9F show antitumor activities generated by IT rMVAAE3L and rMVAAE3LAWR199 (MQ832). FIG. 9A is a diagram of the experimental design. FIG. 9B are dot plots showing the percentages of Gzmb ⁺ cells among CD8 T cells (left) and OX40 ⁺ cells among CD45 ⁺ CD3 ⁺ CD4 ⁺ Foxp3 ⁺ Tregs in B16-F10 tumors treated with Heat-iMVA, rMVA, or rMVAAE3L using a flow cytometric analysis. FIGs. 9C and 9D show the results of an ELISPOT assay analyzing CD8 ⁺ T cells isolated from splenocytes of mice treated with different viruses for anti-tumor IFN-y ⁺ T cells. FIG. 9C are representative images from the ELISPOT assay. FIG. 9D shows IFN-y ⁺ spots per 1,000,000 purified CD8 ⁺ T cells from the spleens of the mice treated with IT PBS, Heat-iMVA, rMVA, or rMVAAE3L (n=5, *P < 0.05; **P < 0.01). FIGs. 9E and 9F are a flow cytometric analysis of tumor infiltrating T cells in virus treated B16-F10 tumors. FIG. 9E are dot plots showing percentages of Gzmb ⁺ cells among CD8 ⁺ T cells (top) and OX40 ⁺ cells among CD45 ⁺ CD3 ⁺ CD4 ⁺ Foxp3 ⁺ Tregs in B16-F10 tumors treated with PBS, rMVAAE3L, or rMVAAE3LAWR199 (MQ832). FIG. 9F shows percentages of CD8 ⁺ T cells expressing Gzmb (left) and Tregs expressing 0X40 (right) within tumors from different treatment groups. (n=5, *P < 0.05; **P < 0.01; ***p < 0.001; ****P < 0.0001).

[0018] FIGs. 10A-10E show MQ833 promotes T cell activation by DCs in human PBMC. FIGs. 10A and 10B show human monocyte-derived dendritic cells (MoDCs) were infected with indicated viruses at a MOI of 10. Cells and supernatant were collected 16 hpi. FIG. 10A is a flow cytometric analysis of hOX40L and hFlt3L transgene expression. FIG. 10B is an ELISA assay for hIL12p70. FIG. 10C is a schematic diagram for human MoDC and T cell coculture experiment. MoDCs were isolated and cultured from the human PBMC, pulsed with OVA, and infected with different viruses. Allogenic or autologous CD8 ⁺ or CD4 ⁺ T cells were isolated from PBMC of a different or the same donor, respectively, and co-cultured with moDCs. Supernatants were collected and IFN-y level was measured by ELISA. FIG. 10D is an ELISA for IFN-y level in supernatants from the coculture system. FIG. 10E are qRT-PCR analyses of Ifnb, CxcllO, and 116 gene expression on MoDCs infected with various viruses.

[0019] FIGs. 11A-11G show IT MQ833 generates strong antitumor response in MC38 model. For FIGs. 11A and 11B, 5 xlO ⁵ MC38 cells were implanted intradermally into the right flank of WT C57BL/6J mice. Intratumoral injections of MQ832, MQ833, or PBS were given twice weekly once the tumors were established. Tumor volumes and mice survival were monitored. FIG. 11A is a Kaplan-Meier survival curve of mice in each treatment group (n=10). PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (* < 0.05). FIG. 1 IB shows tumor volumes of mice separated by group over days post treatment. For FIGs. 11C-11E, 5 xlO ⁵ MC38 cells were implanted intradermally into the left (IxlO ⁵ cells) and right (5xl0 ⁵ cells) flanks of WT C57BL/6J mice. Intratumoral injections of MQ832, MQ833, or PBS were given twice weekly once the injected tumors reaches 5 mm in diameter. Tumor volumes and mice survival were monitored. FIG. 11C showns tumor volumes on the non-injected and injected sides upon first virus injection in each group. FIG. HD is a Kaplan-Meier survival curve of mice in each treatment group (n=10). PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (***P < 0.001). FIG. HE shows injected (top) and non-injected (bottom) tumor volumes of mice separated by treatment groups over days post treatment. For FIGs. HF and 11G, 5x105 MC38 cells were implanted intradermally into the right flank of WT C57BL/6J and NOS2' ¹ ' mice. Intratumoral injections of MQ833 were given twice weekly once the tumors were established. Tumor volumes and mice survival were monitored. FIG. 1 IF is a Kaplan-Meier survival curve of mice in each treatment group (WT : n=10, Nos2' ^! '-. n = 9). Survival data were analyzed by log rank (Mantel -Cox) test. FIG. 11G shows tumor volumes of mice separated by group over days post treatment.

[0020] FIGs. 12A-12C show IT MQ833 generates abscopal antitumor effects facilitated by combination therapy with ICBs. For FIGs. 12A-12C, B16-F10 cells were implanted intradermally into the left (with IxlO ⁵ cells) and right (with 5xl0 ⁵ cells) flanks of WT C57BL/6J mice. Intratumoral injections of MQ832 (4 x 10 ⁷ pfu), MQ833 (4 x 10 ⁷ pfu), or PBS were given twice weekly once tumors were established on both sides. aPD-1, aPD-Ll, or aCTLA-4 antibodies were given i.p. in combination of MQ833 injection. Tumor volumes and mice survival were monitored. FIG. 12A shows tumor volumes on the non-injected and injected sides upon first virus injection in each group. FIG. 12B is a Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test (n=10, **P < 0.01, ***P < 0.001). FIG. 12C shows injected and non-injected tumor volumes of mice separated by treatment groups over days post treatment.

[0021] FIGs. 13A- 13C show a flow cytometric analysis of tumor-infiltrating T lymphocytes after MQ833 treatment in a bilateral B16-F10 model. WT C57BL/6J mice were implanted intradermally with B16-F10 cells into the right (with 5xl0 ⁵ cells) and left (with 2.5xl0 ⁵ cells) flanks. Once the tumors were established, 2 doses of 4 x 10 ⁷ PFU of MQ833 were intratum orally delivered to the right flanks twice 3 days apart. Injected and non-injected tumors were harvested at day 2 post the second injection and then stained and analyzed by flow cytometry. FIG. 13A shows representative dot plots showing percentages of CD8+ T cells expressing Granzyme B (Gzmb) in the injected (top) and non-injected (bottom) tumors. FIG. 13B shows representative dot plots showing percentages of CD4 ⁺ T cells expressing Foxp3 (Tregs) in the injected (top) and non-injected (bottom) tumors. FIG. 13C show representative dot plots showing percentages of CD8 ⁺ T cells expressing KLRG1 in the injected (top) and non-injected (bottom) tumors.

[0022] FIGs. 14A-14C show scRNA-seq analysis of tumor infiltrating immune cells after IT MQ833 injection. 5xl0 ⁵ of B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice, MdaS'^Sting^^, or Stat2' ¹ ' mice. One dose of IT MQ833 injection was given once the tumors were established. Tumors were collected and processed into single cell suspension, and CD45 ⁺ cells were sorted. scRNA-seq was performed. FIG. 14A shows dot plots of representative differentially expressed genes for individual clusters. FIG. 14B is a dot plot showing percentages of viral gene transcripts per cell in each cluster. One dot represents one cell. FIG. 14C is a bar plot showing percentage of virus infected cells (virus transcript > 0) of total tumor infiltrating CD45 ⁺ cells in each treatment group.

[0023] FIGs. 15A-15C show a flow cytometric verification of antibody depletion efficacy. Tumors were collected from mice treated with different depletion antibodies. Depletion of specific immune cell population was measured by fluorescent staining and flow cytometry. FIG. 15A shows representative dot plots showing percentages of CD8 ⁺ and CD4 ⁺ T cells from tumors injected with control antibody (left), aCD4 antibody (middle), and aCD8 antibody. FIG. 15B shows representative dot plots showing percentages of NK1. U NK cells from tumors injected with control antibody (left) and aNKl.l antibody. FIG. 15C shows representative dot plots showing percentages of Ly6G ⁺ CDl lb ⁺ neutrophils from tumors injected with PBS or MQ833 in combination of aLy6G antibody.

[0024] FIGs. 16A-16G show a single cell RNA-seq analysis of tumor infiltrating CD45 ⁺ cells after rMVAAE3L and MQ833 treatment. 5 xlO ⁵ B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice. Once the tumors were established, rMVAAE3L, MQ833, or PBS injection were delivered intratum orally. Tumors were collected 2 days after the injection. CD45 ⁺ cells were sorted and scRNA-seq was performed. FIG. 16A is a UMAP display of sorted CD45 ⁺ cells from 3 samples combined following 10X Genomics scRNA-seq workflow (n = 28,223 cells). FIG. 16B show UMAP plots of cell clusters separated by sample. FIG. 16C is a stacked bar plot showing the percentage of each cluster across different samples. FIG. 16D is a UMAP display of subclustered CD45 ⁺ CD3 ⁺ cells from 6 samples combined following 10X Genomics scRNA-seq workflow (n = 4,618 cells). FIG. 16E show UMAP plots of cell clusters separated by sample. FIG. 16F is a stacked bar plot showing the percentage of each cluster across different samples. FIG. 16G show heatmaps of the average expression of representative marker genes for each cluster.

[0025] FIGs. 17A-17B show that intratumoral (IT) injection of rMCA elicits strong antitumor immunity. FIG. 17A shows tumor growth curves of mice (Wild type, cGAS' ^/_ , STING ^GT/GT , STAT2' ^/_ , and STAT I ^_/ ') treated with rMVA, alonf with a WT/PBS control, in a unilateral B16-F10 implantation model. FIG. 17B is a Kaplan-Meier survival curve of the mice in FIG. 17A (n=5~10; ***p < 0.001, ****p < 0.0001, Mantel-Cox test).

[0026] FIGs. 18A-18C show that IT rMVA generates strong systemic and local anti-tumor immune responses dependent on cGAS/STING/STAT2 pathways. FIG. 18A is a schematic diagram of IT rMVA or MVAAE5R for ELISpot assay and TIL analysis in a murine Bl 6-F 10 melanoma implantation model. FIG. 18B are representative images of fFN-y ⁺ spots from ELISpot assay. FIG. 18C is a statistical analysis of fFN-y ⁺ splenocytes from MVAAE5R, rMVA or PBS-treated mice. Data are means ± SD (n=5 or 6; **P < 0.01, t test).

[0027] FIGs. 19A-19D show the impact of IT rMVA treatment on injected and noninjected tumor cellular microenvironment. FIG. 19A are representative flow cytometry plots of Granzyme B ⁺ CD8 ⁺ and Granzyme B ⁺ CD4 ⁺ Foxp3‘ cells in the injected tumors. FIG.

19B shows the percentages and absolute number of Granzyme B ⁺ CD8 ⁺ and Granzyme B ⁺ CD4 ⁺ Foxp3‘ cells in the injected tumors. Data are means ± SD (n=5 or 6; *P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001, t test). FIG. 19C are representative flow cytometry plots of Granzyme B ⁺ CD8 ⁺ and Granzyme B ⁺ CD4 ⁺ Foxp3‘ cells in the non-injected tumors. FIG. 19D shows the percentages and absolute number of Granzyme B ⁺ CD8 ⁺ and Granzyme B ⁺ CD4 ⁺ Foxp3‘ cells in the non-injected tumors. Data are means ± SD (n=5 or 6; *P < 0.05, ** < 0.01, **** P < 0.0001, t test).

[0028] FIGs. 20A-20F show that IT rMVA depletes OX40 ¹¹¹ Tregs in the injected tumors to promote anti-tumor therapy and that IT rMVA preferentially depletes OX40 ¹¹¹ Tregs in the injected tumors in a type I IFN signaling dependent manner. FIG. 20A are representative flow cytometry plots of Foxp3 ⁺ CD4 ⁺ cells in the injected tumors and the average OCdO ¹¹¹ percentage. Mice were treated as described in FIG. 18A. FIGs. 20B-20C show the percentages and absolute number of Foxp3 ⁺ CD4 ⁺ cells in the injected (FIG. 20B) and non- injected (FIG. 20C) tumors. Data are means ± SD (n=6-8; **P < 0.01, ***P<0.001, ****P

< 0.0001, t test). FIG 20D are representative flow cytometry plots of 0X40 expression on tumor infiltrating CD8 ⁺ , Tconv (CD4 ⁺ Foxp3‘), and CD4 ⁺ Foxp3 ⁺ T cells in tumors 15 days after implantation. FIGs. 20E-20F are representative flow cytometry plots and statistical analysis of mean fluorescence intensity of 0X40 on tumor-infiltrating CD8 ⁺ , Tconv (CD4 ⁺ Foxp3‘), and CD4 ⁺ Foxp3 ⁺ T cells. Data are means ± SD in FIG. 20F (n=6~8; ****P

< 0.0001, t test).

[0029] FIGs. 21A-21B show that IT rMVA activates CD8 ⁺ T cells and reduces Foxp3 ⁺ CD4 ⁺ T cells in a human extramammary Paget’s disease (EMPD) tumor model. FIG. 21 A is a schematic diagram of ex vivo infection of human EMPD tumors with rhMVA (MVAAE5R-hFlt3L-hOX40L). FIG. 21B are percentages of Granzyme B ⁺ CD8 ⁺ T cells and Foxp3 ⁺ CD4 ⁺ T cells in the rhMVA or PBS-treated tumor tissues. Data are means ± SD (n=7; *P < 0.05, **P < 0.01, t test).

[0030] FIG. 22 is a working therapeutic model which postulates the following. IT injection of rMVA results in the infection of tumor-infiltrating myeloid cells, including macrophages, monocytes, and dendritic cells, as well as tumor cells. This leads to the activation of cGAS/STING-mediated cytosolic DNA-sensing pathway and the production of type I IFN and cytokines and chemokines that are important for CD8 ⁺ and CD4 ⁺ T cell proliferation and activation (as indicated by Granzyme B, TNF, and IFN-y expression). Flt3L expression of the tumor microenvironment facilitates the proliferation of CD103 ⁺ DCs in the tumors.

OX40L expression by myeloid cell populations and tumor cells results in the depletion of OX40 ¹¹¹ Tregs infiltrating the tumors via OX40L-OX40 ligation, which is promoted by type I IFN. This leads to the blunting of their inhibition on tumor-specific effector CD4 ⁺ and CD8 ⁺ T cells. Taken together, IT delivery of rMVA results in the alteration of tumor immunosuppressive microenvironment through activation of innate immunity and boosting of antitumor T cells by depletion of OX40 ¹¹¹ regulatory T cells.

[0031] FIGs. 23A-23D show that IFNAR1 on Tregs is important for rMVA-induced antitumor effects in two murine tumor models. FIG. 23A is a Kaplan-Meier survival curve of MC38-bearing Ifnarl^ and Foxp3 ^Cre Ifnarl^ mice treated with IT rMVA or PBS (n=5 or 10; *P<0.05, **P<0.01, ***P < 0.001, Mantel-Cox test). FIG. 23B shows MC38 tumor volumes over days in Ifnar 1^ and Foxp3 ^Cre Ifnar Invoice treated with IT rMVA or PBS control. FIG. 23C is a Kaplan-Meier survival curve of B I 6-F I 0-bearing / //a/7 ^{// /z} and Foxp3 ^Cre IJharl^ mice treated with IT rMVA (n=9; *P<0.05, Mantel-Cox test). FIG. 23D shows B 16-F 10 tumor volumes over days in Ifnarl^ and FoxpS^Ifnarl^ mice treated with IT rMVA.

[0032] FIG. 24 is an illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 1) given by GenBank Accession No. U94848.1.

[0033] FIG. 25 is a plasmid map of the pUC57-dell86-pH5-huIL12 plasmid (SEQ ID NO: 8).

[0034] FIG. 26 is a plasmid map of the P503 plasmid (SEQ ID NO: 9).

[0035] FIGs. 27A and 27B are the plasmid maps of the pMA-MVA-AE5R-hFlt3L- hOX40L vector (SEQ ID NO: 11; FIG. 27A) and the pMA-MVAAE5R-hFlt3L-mOX40L vector (SEQ ID NO: 12; FIG. 27B).

[0036] FIGs. 28A and 28B are illustrative, non-limiting plasmid maps for generation of MQ832.

[0037] FIG. 29 is an illustrative, non-limiting plasmid design for deletion of WR199 with IL-12 sequence (murine) (SEQ ID NO: 15).

[0038] FIG. 30 is an illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 19) given by GenBank Accession No. AY603355.

DETAILED DESCRIPTION

[0039] 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

[0040] 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. [0041] 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.

[0042] 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.

[0043] As used herein, the term “adaptive immune response” or “adaptive immune system response” refers to an antigen-mediated response by the immune system of a subject. A subject has a “deficient adaptive immune system response” when the subject’s adaptive immune system does not respond to one or more antigens as an active adaptive immune system would (e.g., no T cell response or a diminished T cell response, or no B cell response or a diminished B cell response).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12). For example, an immune checkpoint blockade inhibitor administered in close temporal proximity with one or more engineered poxviruses of the present technology (e.g., MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12). 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12) (by intravenous or intratumoral injection when the MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 is administered intratumorally or systemically as stated above) or before or after the MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 administration. In some embodiments, if the MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 administration and the immune checkpoint blockade inhibitor 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.”

[0049] The term “corresponding wild-type strain” or “corresponding wild-type virus” is used herein to refer to the wild-type MVA from which the engineered MVA was derived. As used herein, a wild-type MVA 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 strain or virus is a strain or virus that has not been engineered to disrupt or delete (knock out) the E5R gene, E3L gene, and/or WR199 gene, and/or not engineered to express additional immunomodulatory proteins as described herein, such as Flt3L, 0X40, and/or IL-12. The term “corresponding MVAAE5R strain” or “corresponding MVAAE5R virus” is used herein to refer to the MVA strain or virus having the same sequence as MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL- 12 except for having an E5R deletion alone (i.e., an MVAAE5R strain or virus that has not been engineered to disrupt or delete (knock out) the E3L and WR199 genes and that has not been engineered to express any of Flt3L, 0X40, or IL-12 but that is otherwise identical to the corresponding MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12).

[0050] As used herein, the terms “delivering” and “contacting” refer to depositing the one or more engineered poxviruses (e.g., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12) 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, MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12) 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.

[0051] The terms “disruption” and “mutation” and “mutant” 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.

[0052] 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL- 12) 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 (z.e., slows down or stops) cancer cell infiltration into peripheral organs; inhibits (z.e., slows down or stops) metastatic growth; inhibits (stabilizes or arrests) tumor growth; allows for treatment of the tumor; induces and promotes an immune response against the tumor; prevents cancer recurrence for a period of time in a subject in need thereof; or alters the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. The TIME includes any immune cell or factor present in the tumor microenvironment, including, but not limited to, cytokines, myeloid cells, and/or lymphocytes. Alterations to the TIME may include, but are not limited to, increased proportions of pro-inflammatory cytokines, increased porportions of inflammatory monocytes and neutrophils, decreased proportions of M2-like macrophages, and/or increased proportion of polarized Ml -like macrophages. 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 IO ¹⁰ plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage is about 10 ⁶ -l 0 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 of the viruses of the present technology 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 the 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.

[0053] 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12) 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, or to preventing cancer recurrence for a period of time in a subject in need thereof, or to alter the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof. 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.

[0054] With particular reference to combination therapy with an immune checkpoint blockade inhibitor, an “effective amount” or “therapeutically effective amount” for an immune checkpoint blockade inhibitor means an amount of an immune checkpoint blockade inhibitor 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 blockade inhibitors 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-Ll (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. [0055] 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 blockade inhibitors block more generally immune suppressive mechanisms within the tumors, elicited by tumor cells, stromal cells, and tumor-infiltrating immune cells.

[0056] For example, the CTLA-4 inhibitor ipilimumab, when administered as adjuvant therapy after surgery in melanoma, can be 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12). 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.

[0057] As another example, pembrolizumab is prescribed for administration as adjuvant therapy in melanoma, for example, diluted to 25 mg/mL. It can be 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, MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL- 12).

[0058] 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, MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12). Nivolumab can be prescribed for administration at 3 mg/kg as an intravenous infusion every two weeks, for example, or at a dose of 240 mg every two weeks, 360 mg every three weeks, or 480 mg every four weeks, for example, with the specific dose and infusion rate depending upon the tumor type. [0059] 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- 0X40 antibody can bind to 0X40 and potentiate T-cell receptor signaling leading to T-cell activation, proliferation and survival. Other examples include agonist antibodies against 4- 1BB (CD137), and GITR.

[0060] 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., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12). Alternatively, the immune stimulating agonist antibodies can be used conjointly with one or more engineered poxviruses of the present technology (e.g., MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12) via intratumoral delivery either simultaneously (i.e., concurrently) or sequentially.

[0061] 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 modified vaccinia Ankara virus” refers to a modified vaccinia Ankara strain that has been manipulated to be genetically altered, modified, or changed. In the present context, “engineered” or “genetically engineered” includes recombinant modified vaccinia Ankara viruses.

[0062] 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., hOX40L, hFlt3L, hIL-12, 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., hOX40L, hFlt3L, hIL-12, a selectable marker, or a combination thereof).

[0063] 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 acids may comprise segments that are a copy of a sequence that is naturally found in the virus into which it has been introduced. [0064] 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. Additionally, where no designation of human or murine is used, the gene may be either human or murine.

[0065] As used herein, “immune checkpoint inhibitor” or “immune checkpoint blocking agent” or “immune checkpoint blockade agent” or “immune checkpoint blockade inhibitor” or “ICB 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 blockade inhibitors 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-Ll (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, MEDL6469, 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.

[0066] 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-a/p) is a critical regulator of the innate immunity (Huber el al., Immunology 132(4):466-474 (2011)). Animal and human studies have shown a role for IFN-a/p 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.

[0067] 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 MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12, an antigen, an adjuvant comprising any one or more of the foregoing engineered viruses, and/or an adjuvant comprising MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12, 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).

[0068] As used herein, the term “inactivated MV A” 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.

[0069] 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.

[0070] As used herein, “UV-inactivated MV A” 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.

[0071] 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.

[0072] 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.

[0073] As used herein, “MV A” 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). An illustrative, non-limiting sequence is 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.

[0074] The term “MVAAE3L” 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. Patent 7,049,145, for example.

[0075] The term “MVAAE5R,” is used herein to refer to a modified vaccinia Ankara (MVA) mutant 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, “MVAAE5R” 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, “MVAAE5R” encompasses a recombinant MVA virus that does not express a functional E5 protein. In some embodiments, the AE5R mutant includes a deletion of all or a majority of the E5R gene sequence. For example, as used herein, “MVAAE5R” encompasses a recombinant MVA nucleic acid sequence, wherein all or a majority of the nucleic acid sequence corresponding to the position of E5R in the MVA genome (e.g., positions 44,180-45,175 set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1) or positions 38,432 to 39,385 set forth in GenBank Accession No. AY603355 (SEQ ID NO: 19)) is deleted. In some embodiments, all or a portion of the E5R gene is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes one or more specific genes of interest and/or a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry). In some embodiments, the MVAAE5R virus encompasses a recombinant MVA virus that does not express a functional thymidine kinase (TK) protein. In some embodiments, the MVAAE5R 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.

[0076] As used herein, the terms “rMVA” or “MQ710” or “MVAAE5R-Flt3L-OX40L” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L. In some embodiments, both of the transgenes are inserted into the E5R locus. In some embodiments, Flt3L and OX40L are linked by a P2A self-cleaving sequence and their expression is driven by the vaccinia synthetic early-late promoter.

[0077] As used herein, the terms “rMVAAE3L” or “MVAAE3LAE5R-Flt3L-OX40L” refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, and a mutant E3L gene. In some embodiments, the rMVAAE3L is an rMVA virus that has been engineered to further comprise a mutant E3L gene. In some embodiments, the mutation is a deletion of the E3L gene.

[0078] As used herein, the terms “rMVAAE3LAWR199” or “MQ832” or “MVAAE3LAE5R-Flt3L-OX40LAWR199” may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, and mutant WR199 gene. In some embodiments, the rMVAAE3LAWR199 is an rMVA or an rMVAAE3L virus that has been engineered to further comprise both a mutant E3L gene and a mutant WR199 gene or a mutant WR199 gene, respectively. In some embodiments, the mutation is a deletion of the E3L and WR199 genes. In some embodiments, AWR199 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 (SEQ ID NO: 19) or position 164,209-165,933 of the sequence set forth in GenBank Accession No. U94848.1 (SEQ ID NO: 1)) is deleted. In some embodiments, all or a portion of the WR199 gene is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes one or more specific genes of interest and/or a selectable marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., gpt, GFP, mCherry).

[0079] As used herein, the terms “MVAAE3LAE5R-Flt3L-OX40LAWR199-hIL-12” or “MVAAE5R-Flt3L-OX40L-AE3L-AWR199-hIL-12” or “MQ833” may be used interchangeably and refer to a modified vaccinia Ankara (MV A) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant WR199 gene, and a third transgene, IL-12. In some embodiments, the IL-12 transgene may be inserted into the WR199 locus. In some embodiments, IL-12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the MQ833 is an MQ832 virus that has been engineered to further comprise an IL-12 transgene. In some embodiments, the IL-12 transgene encodes an extracellular matrix binding domain (e.g.

RRPKGRGKRRREKQRPTDCHL SEQ ID NO: 20) which anchors the IL- 12 to the extracellular matrix. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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: sarcomas including 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), Extramammary Paget Disease (EMPD), head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous 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, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer, 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, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer and the metastases of any of the foregoing. Any of the preceding types of tumors can, in some embodiments, be resistant to immune checkpoint blockade (ICB) inhibitor therapeutics. In some embodiments, the ICB inhibitor resistance is due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to a mutation in the B2M gene and/or reduced B2M activity. The B2M protein plays an important role in MHC-I antigen presentation. In some embodiments, an ICB inhibitor-resistant tumor is resistant to CD8 ⁺ T cell mediated immune responses.

[0085] As used herein, “resistant to immune checkpoint inhibitor,” “resistance to immune checkpoint blockade inhibitors,” or “resistance to immune checkpoint inhibitors,” or “resistance to immune checkpoint blockade inhibitors,” or “immune checkpoint blockade resistant” refers to a tumor that is resistant to immune checkpoint blockade (ICB) inhibitor treatment, wherein the ICB treatment does not elicit an immune response against the resistant tumor to the same degree as the ICB treatment would elicit against a non-resistant tumor of the same type. For example, a tumor that is resistant to immune checkpoint blockade (ICB) inhibitors may either increase or lack reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors. A tumor that is resistant to ICB inhibitors may exhibit no changes or diminished changes to the tumor immune microenvironment despite being treated with an ICB inhibitor. There are multiple possible mechanisms for resistance to immune checkpoint inhibitors, including, but not limited to, deficiency in MHC-I antigen presentation, mutations in the B2M gene, reduced B2M activity, inadequate tumor associated antigens and/or deficient antigen presentation thereof, inadequate cytokine expression, defficieny in cytokine co-stimulation, active immunosuppressive signaling pathways, or combinations thereof. A tumor that is resistant to ICB inhibitor treatment may comprise a mutation in the B2M gene and/or reduced B2M activity. A tumor that is resistant to ICB inhibitor treatment may comprise mutations to one or more genes encoding proteins that are involved in the MHC-I antigen presentation process. Resistance to immune checkpoint inhibitors is a matter of degree, meaning that a tumor can be fully resistant or partially resistant to one or more immune checkpoint inhibitors. Resistance to immune checkpoint inhibitors protects tumors from the immune response. In some embodiments, resistance to immune checkpoint inhibitors protects a tumor from CD8 ⁺ T cell mediated killing, meaning the tumor is resistant to CD8 ⁺ T cell mediated immune responses. Tumors that are resistant to CD8 ⁺ T cell mediated immune responses either do not elicit increased levels of CD8 ⁺ T cells or are not responsive to increased levels of CD8 ⁺ T cells.

[0086] As used herein, “MHC-I antigen presentation” refers to the localization of major histocompatibility class (MHC) I proteins at the surface of a cell, wherein the MHC-I proteins form the MCH-I complex and present an antigenic peptide fragment for recognition by an immune cell. MHC-I antigen presentation is a step in the activation of CD8 ⁺ T cells. “MHC-I antigen presentation deficiency” refers to a complete or partial loss of MHC-I antigen presentation capacity in a cell, cells, or tumor, and is a known mechanism for cancer immune evasion. Additionally, complete or partial loss of MHC-I antigen presentation is a mechanism for cancer resistance to immune checkpoint blockade inhibitor treatment. Cells and/or tumors are deficient in MHC-I antigen presentation when the cells and/or tumors possess diminished amounts of the MHC-I complex at the cell surface membrane and/or diminished amounts of antigen presenting MHC-I complexes at the cell surface membrane.

[0087] 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. [0088] 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.

[0089] “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, z.e., arresting its development; (ii) relieving a disease or disorder, z.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.

[0090] 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.

[0091] 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 “antitumor 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. [0092] 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.

[0093] 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, Thl and Th2, which produce different cytokines.

[0094] 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.

[0095] 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.

[0096] 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: 2. A non-limiting example of a pUC57-hFlt3L-GFP vector according to the present technology is set forth in SEQ ID NO: 3.

[0097] 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

[0098] 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.

[0099] Numerous studies support the importance of the differential presence of immune system components in cancer progression (Jochems et al., Exp. Biol. Med. 236 5'y.56 ^r l-5 ^r 19 (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, naive 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.

[0100] 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-P) 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)).

[0101] 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.

[0102] 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. [0103] Some cancers, including, but not limited to, melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD), can be resistant to treatment with immune checkpoint blockade (ICB) inhibitors. Other cancers not specifically recited here can also be ICB inhibitor-resistant. In some embodiments, the ICB inhibitor resistance is due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICB inhibitor resistance is due to a mutation in the B2M gene and/or reduced B2M activity. The B2M protein plays an important role in MHC-I antigen presentation. In some embodiments, an ICB inhibitor-resistant tumor is resistant to CD8 ⁺ T cell mediated immune responses. In some embodiments, a tumor is diagnosed as being resistant to treatment with ICB inhibitors due to lack of response to administration of one or more ICB inhibitors, such as, for example, either an increase or a lack of reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors.

III. Modified Vaccinia Ankara (MV A) Virus

[0104] Poxviruses are in the forefront as oncolytic therapy for metastatic cancers (Kirn et al., Nature Review Cancer 9:64-71 (2009)). 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., PLoSMed 4:e353 (2007); Thorne et al., J. Clin. Invest. 117:3350-3358 (2007)). 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.,

31 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.

[0105] Modified Vaccinia Ankara (MV A) virus is 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)).

[0106] 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.

[0107] 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)).

[0108] 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 (eDCs) and plasmacytoid dendritic cells (pDCs). The former, especially the CD103 ⁺ /CD8a ⁺ subtype, are particularly adapted to cross-presenting antigens to T-cells; the latter are strong producers of Type I IFN. [0109] 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 (a). 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., r \ et al., J Virol. 84:5314-5328 (2010)).

[0110] An illustrative, non-limiting MVA genome sequence is set forth in SEQ ID NO: 1 and is given by GenBank Accession No. U94848.1.

IV. 0X40 ligand (QX40L)

[OHl] The 0X40 ligand (OX40L) and its binding partner, tumor necrosis factor receptor 0X40, 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- 0X40 interaction provides survival and activation signals for T-cells expressing 0X40. 0X40 additionally suppresses the differentiation and activity of Treg, further amplifying this process. 0X40 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 recombinant viruses of the present technology can be either huOX40L or muOX40L.

[0112] Illustrative human OX40L (huOX40L) nucleic acid (SEQ ID NO: 4) and polypeptide sequences (SEQ ID NO: 5) are provided below. huOX40L-ORF (SEQ ID NO:4):

ATGGAAAGGG TCCAACCCCT GGAAGAGAAT GTGGGAAATG CAGCCAGGCC AAGATTCGAG AGGAACAAGC TATTGCTGGT GGCCTCTGTA ATTCAGGGAC TGGGGCTGCT CCTGTGCTTC ACCTACATCT GCCTGCACTT CTCTGCTCTT CAGGTATCAC ATCGGTATCC TCGAATTCAA AGTATCAAAG TACAATTTAC CGAATATAAG AAGGAGAAAG GTTTCATCCT CACTTCCCAA AAGGAGGATG AAATCATGAA GGTGCAGAAC AACTCAGTCA TCATCAACTG TGATGGGTTT TATCTCATCT CCCTGAAGGG CTACTTCTCC CAGGAAGTCA ACATTAGCCT TCATTACCAG AAGGATGAGG AGCCCCTCTT CCAACTGAAG AAGGTCAGGT CTGTCAACTC CTTGATGGTG GCCTCTCTGA CTTACAAAGA CAAAGTCTAC TTGAATGTGA CCACTGACAA TACCTCCCTG GATGACTTCC ATGTGAATGG CGGAGAACTG ATTCTTATCC ATCAAAATCC TGGTGAATTC TGTGTCCTTT GA huOX40L polypeptide (SEQ ID NO: 5) V Q F T E Y K K E K G F I L T S Q K E D E I MK V Q N N S V I I N C D G F Y L I S L K G Y F S Q E V N I S L H Y Q K D E E P L F Q L K K V R S V N S L MV A S L T Y K D K V Y L N V T T D N T S L D D F H V N G G E L I L I H Q N P G E F C V L Stop

[0113] Illustrative murine OX40L (muOX40L) nucleic acid (SEQ ID NO: 6) and polypeptide sequences (SEQ ID NO: 7) are provided below. muOX40L-ORF (codon optimized)(SEQ ID NO: 6):

ATGGAGGGCGAGGGGGTCCAGCCTCTGGACGAGAACCTCGAAAACGGGTCTCGCCCT CGCTT TAAATGGAAGAAGACTCTTAGGCTCGTTGTAAGCGGCATCAAGGGGGCCGGTATGTTGCT GT GCTTCATATATGTGTGTTTGCAACTTAGCTCTTCACCTGCAAAAGACCCCCCCATACAAC GC CTTCGGGGGGCTGTGACCCGCTGTGAAGATGGTCAATTGTTTATTTCTTCTTACAAGAAC GA G T AT C AGAC GAT G GAAG T C C AGAAT AAC TCCGTAGTGAT T AAG T G T GAG G GAG T G TAG AT GA T C TAG T T GAAAGGAT C T T T T T T CCAGGAGGT CAAAAT T GACC T CCAC T T CAGGGAGGAT GAG AACCCTATCTCAATCCCTATGTTGAACGACGGCAGAAGAATCGTCTTTACTGTAGTCGCT TC ACTGGCCTTCAAGGATAAGGTGTACTTGACCGTAAACGCTCCTGATACCTTGTGCGAGCA TT TGCAAATCAACGATGGAGAACTTATCGTTGTCCAACTCACACCAGGTTACTGTGCTCCTG AG G G GAG T T AT GAG AG TAG AG T GAAG GAAG TGC GAG T G T GA muOX40L polypeptide (SEQ ID NO: 7): S T V N Q V P L Stop

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

[0114] 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 etal., 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 CD8a ⁺ /CD103 ⁺ DCs and pDCs.

[0115] CD103 ⁺ /CD8a ⁺ DCs are required for spontaneous cross-priming of tumor antigenspecific 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 naive 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/p- 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-Ll 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.

[0116] 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.

[0117] 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 etal., Science 348(6230):56-61 (2015)).

VI. Interleukin 12 (IL-12)

[0118] Human Interleukin 12 (IL- 12) is a cytokine molecule that was independently identified by two investigators in 1989 and 1990. The use of IL-12 for its immunostimulatory properties has been heavily investigated, particularly in the cancer context. While systemic IL- 12 treatment can result in a toxic, inflammatory response, multiple investigators have demonstrated that targeted treatment, via lipid nanoparticle delivery, for example, can suppress tumorigenesis without any undesirable toxic effects. Progress continues to be made in understanding the therapeutic potential of IL-12, as both a treatment and an adjuvant, in the anti-cancer context.

[0119] IL-12 consists of a p35 monomer and p40 monomer which are linked by disulfide bonds to form the biologically active p70 heterodimer. IL-12 functions by binding to the IL- 12 receptor (IL-12R), which is predominantly expressed by natural killer (NK) cells and T cells. IL-12R activation results in a signaling cascade via the JAK-STAT pathway that eventually leads to an array of pro-immune responses, including the production of interferon gamma (IFN-y) and tumor necrosis factor-alpha (TNF-a).

[0120] Some of the genetically engineered or recombinant viruses of the present disclosure express a recombinant IL-12 which is bound to the extracellular matrix, thus preventing a systemic delivery of IL-12 and the associated toxicity. Intratumoral delivery of the present genetically engineered or recombinant MVA viruses that comprise recombinant IL-12 allows for efficient, localized delivery of IL-12 to induce an immune response against the target tumor. An illustrative, non-limiting plasmid sequence used to generate engineered or recombinant MVA viruses comprising an hIL-12 transgene and a deletion of the WR199 gene (also known as MVA186) include the sequence shown in Table 1.

VII. E3L

[0121] 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-KB 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 (AE3L) 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 AE3L virus elicits proinflammatory responses that are masked during infection with wildtype 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 AE3L virus triggered NF-KB 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 AE3L virus does induce the production of IFN-P, 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)). [0122] Deletion of E3L sensitizes vaccinia virus replication to IFN inhibition in permissive RK13 cells and results in a host range phenotype, whereby AE3L 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 viruses with deletion of the N-terminal Z- DNA-binding domain are replication competent in HeLa and BSC40 cells (Brandt et al., 2001). An illustrative, non-limiting, plasmid sequence used to generate engineered or recombinant MVA viruses comprising a deletion of the E3L is shown in Table 2.

VIII. E5R

[0123] 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 (MV A), a highly attenuated vaccinia strain, induces IFN production via a cGAS/STING-dependent mechanism. However, MVA infection triggers cGAS degradation. It has been shown that vaccinia E5R is a dominant inhibitor of cGAS and is the key protein mediating cGAS degradation. MVAAE5R 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. MVAAE5R-mediated type I IFN production is dependent on cGAS. Furthermore, MVAAE5R gains replication capability in cGAS' ^/_ skin fibroblasts. As a vaccine vector, skin scarification or intradermal vaccination with MVAAE5R-0VA leads to much higher OVA-specific CD8 ⁺ T cell responses than MVA-OVA in vivo. Intratumoral injection of MVAAE5R leads to stronger anti -tumor immune responses and better survival compared with MVA. E5 is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production.

[0124] An illustrative full-length vaccinia virus E5R host range protein, given by GenBank Accession No. AAB59825.1 (SEQ ID NO: 10) is provided below.

MLILTKVNI YMLI IVLWLYGYNFI ISESQCPMINDDSFTLKRKYQIDSAESTIKMDKKRTKF QNRAKMVKEINQTIRAAQTHYETLKLGYIKFKRMIRTTTLEDIAPS IPNNQKTYKLFSDISA IGKASRNPSKMVYALLLYMFPNLFGDDHRFIRYRMHPMSKIKHKI FSPFKLNLIRILVEERF YNNECRSNKWRI IGTQVDKMLIAESDKYTIDARYNLKPMYRIKGKSEEDTLFIKQMVEQCVT SQELVEKVLKILFRDLFKSGEYKAYRYDDDVENGFIGLDTLKLNIVHDIVEPCMPVRRPV AK ILCKEMVNKYFENPLHI IGKNLQECIDFVSE

IX. Engineered Poxyirus Strains of the Present Technology

[0125] Illustrative, non-limiting examples of OX40L and hFlt3L expression constructs according to the present technology are shown in Table 3. 1151 GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG

1201 GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA

1251 CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGGAG

1301 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG

1351 CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC

1401 TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG

1451 GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTGCCACCCC CATGGAGTCC

1501 CCGGCCCCTG GAGGCCACAG CCCCGACAGC CCCGCAGCCC CCTCTGCTCC

1551 TCCTACTGCT GCTGCCCGTG GGCCTCCTGC TGCTGGCCGC TGCCTGGTGC

1601 CTGCACTGGC AGAGGACGCG GCGGAGGACA CCCCGCCCTG GGGAGCAGGT

1651 GCCCCCCGTC CCCAGTCCCC AGGACCTGCT GCTTGTGGAG CACTGACTCG

1701 AGTTTACTTG TACAGCTCGT CCATGCCGAG AGTGATCCCG GCGGCGGTCA

1751 CGAACTCCAG CAGGACCATG TGATCGCGCT TCTCGTTGGG GTCTTTGCTC

1801 AGGGCGGACT GGGTGCTCAG GTAGTGGTTG TCGGGCAGCA GCACGGGGCC

1851 GTCGCCGATG GGGGTGTTCT GCTGGTAGTG GTCGGCGAGC TGCACGCTGC

1901 CGTCCTCGAT GTTGTGGCGG ATCTTGAAGT TCACCTTGAT GCCGTTCTTC

1951 TGCTTGTCGG CCATGATATA GACGTTGTGG CTGTTGTAGT TGTACTCCAG

2001 CTTGTGCCCC AGGATGTTGC CGTCCTCCTT GAAGTCGATG CCCTTCAGCT

2051 CGATGCGGTT CACCAGGGTG TCGCCCTCGA ACTTCACCTC GGCGCGGGTC

2101 TTGTAGTTGC CGTCGTCCTT GAAGAAGATG GTGCGCTCCT GGACGTAGCC

2151 TTCGGGCATG GCGGACTTGA AGAAGTCGTG CTGCTTCATG TGGTCGGGGT

2201 AGCGGCTGAA GCACTGCACG CCGTAGGTCA GGGTGGTCAC GAGGGTGGGC

2251 CAGGGCACGG GCAGCTTGCC GGTGGTGCAG ATGAACTTCA GGGTCAGCTT

2301 GCCGTAGGTG GCATCGCCCT CGCCCTCGCC GGACACGCTG AACTTGTGGC

2351 CGTTTACGTC GCCGTCCAGC TCGACCAGGA TGGGCACCAC CCCGGTGAAC

2401 AGCTCCTCGC CCTTGCTCAC CATGGTACCA GGCCTAGATC TGTCGACTTC

2451 GAGCTTATTT ATATTCCAAA AAAAAAAAAT AAAATTTCAA TTTTTCTCGA

2501 GTATGAGTAT AGTGTTAAAT GACACTTACT AAATAGCCAA GGTGATTATT

2551 CGTATTTTTT TAAGGAGTAA CCATGTCCGC AATTAGATTT ATTGCATGTC

2601 TATATCTCAT TTCCATCTTC GGAAATTGTC ATGAGGATCC ATATTATCAA

2651 CCATTTGATA AATTAAACAT TACTCTAGAT ATATACACTT ATGAGGATCT

2701 AGTACCATAC ACCGTAGACA ATGACACAAC TTCTTTCGTT AAGATATACT

2751 TTAAAAATTT TTGGATTACG GTTATGACTA AATGGTGTGC TCCGTTTATT

2801 GATACCGTTA GCGTATACAC ATCTCATGAT AATCTGAATA TACAATTTTA

2851 TAGTAGGGAC GAATATGATA CACAAAGCGA GGATAAAATT TGTACCATTG

2901 ATGTTAAAGC ACGATGCAAA CATCTAACAA AACGAGAAGT TACAGTACAA

2951 CAAGAAGCCT ACAGATAATC TAGATGCATT CGCGAGGTAC CGAATCGGAT

3001 CCCGGGCCCG TCGACTGCAG AGGCCTGCAT GCAAGCTTGG CGTAATCATG

3051 GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA

3101 ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG

3151 AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG 3201 AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG

3251 GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG

3301 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT

3351 AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG

3401 CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG

3451 TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC

3501 AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC

3551 CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC

3601 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG

3651 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG

3701 GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT

3751 AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC

3801 AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA

3851 CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA

3901 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG

3951 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG

4001 TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT

4051 TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA

4101 AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT

4151 TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT

4201 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT

4251 CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA

4301 CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG

4351 CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC

4401 CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC

4451 AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT

4501 AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC

4551 GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG

4601 TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT

4651 CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT

4701 GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT

4751 CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG

4801 CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA

4851 TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA

4901 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT

4951 CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG

5001 GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA

5051 CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC

5101 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA

5151 GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC

5201 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG

[0126] An illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 1) given by GenBank Accession No. U94848.1 is provided in FIG. 24. An illustrative, non-limiting MVA virus genome sequence (SEQ ID NO: 19) given by GenBank Accession No. AY603355 is provided in FIG. 30.

MQ710

[0127] The disclosure of the present technology relates to modified vaccinia Ankara (MVA) viruses referred to as “rMVA” or “MQ710” or “MVAAE5R-Flt3L-OX40L,” which may be used interchangeably and refer to a modified vaccinia Ankara (MVA) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, and a mutation of the E5R gene. In some embodiments, the present technology relates to an immunogenic composition comprising MQ710. In some embodiments, both of the transgenes are inserted into the E5R locus. In some embodiments, Flt3L and OX40L genes are linked by a P2A self- cleaving sequence and their expression is driven by the vaccinia synthetic early-late promoter. In some embodiments, plasmid constructs were used to create MQ710 via homologous recombination with the MVA genome. In some embodiments, illustrative, nonlimiting, plasmid constructs used to create MQ710 include those shown in Table 4. The plasmid maps are shown in FIGs. 27A and 27B. MQ832

[0128] The disclosure of the present technology relates to modified vaccinia Ankara (MV A) viruses referred to as “rMVAAE3LAWR199” or “MQ832” or “MVAAE3LAE5R- Flt3L-OX40LAWR199,” which may be used interchangeably and refer to a modified vaccinia Ankara (MV A) mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E3L gene, a mutant E5R gene, and a mutant WR199 gene. In some embodiments, the rMVAAE3LAWR199 is an rMVA or an rMVAAE3L virus that has been engineered to further comprise both a mutant E3L gene and a mutant WR199 gene, or a mutant WR199 gene, respectively. In some embodiments, the mutation is a deletion of the E3L and WR199 genes. In some embodiments, the present technology relates to an immunogenic composition comprising MQ832. In some embodiments, plasmid constructs were used to create MQ832 via homologous recombination with the MVA genome. In some embodiments, illustrative, non-limiting, plasmid constructs used to create MQ832 include those shown in Table 5. Illustrative, non-limiting plasmids for engineering the E3L knockout are shown in FIG. 28A (E3L_mcherryFRT_Kan (SEQ ID NO: 13) and FIG. 26 (P503 (SEQ ID NO: 9)). An illustrative, non-limiting plasmid for engineering the WR199 knockout is shown inTable 5 and in FIG. 28B (WR199_FRT_mCherry (SEQ ID NO: 14).

MQ833

[0129] The disclosure of the present technology relates to modified vaccinia Ankara (MV A) viruses referred to as “MVAAE3LAE5R-Flt3L-OX40LAWR199-hIL-12” or “MVAAE5R-Flt3L-OX40L-AE3L-AWR199-hIL-12” or “MQ833,” which may be used interchangeably and refer to a MVA mutant virus that has been engineered to comprise two transgenes, Flt3L and OX40L, both of which may be inserted into the E5R locus, a mutant E5R gene, a mutant E3L gene, a mutant WR199 gene, and a third transgene, IL-12. In some embodiments, the IL- 12 transgene may be inserted into the WR199 locus. In some embodiments, IL- 12 is inserted into the MVA genome with an expression cassette comprising IL-12b and IL-12a linked by sequences encoding P2A peptides under the control of a modified H5 promoter. In some embodiments, the MQ833 is an MQ832 virus that has been engineered to further comprise an IL-12 transgene. In some embodiments, the IL-12 transgene is inserted into the WR199 locus of MQ832 to generate MQ833. In some embodiments, the IL-12 transgene is engineered such that IL-12 is anchored to the extracellular matrix when the transgene is expressed in a cell. In some embodiments, the present technology relates to an immunogenic composition comprising MQ833. In some embodiments, plasmid constructs were used to create MQ833 via homologous recombination with the MVA genome. In some embodiments, illustrative, non-limiting, plasmid constructs used to create MQ833 include those shown in Table 6. An illustrative, non-limiting plasmid design for deletion of WR199 (also known as MVA186) and insertion of human IL-12 is shown in FIG. 25 (SEQ ID NO: 9). An illustrative, non-limiting plasmid design for deletion of WR199 with IL-12 sequence (murine) is shown in Table 6 and FIG. 29 (Plasmid design for deletion of WR199 with IL-12 sequence (murine) (SEQ ID NO: 15).

[0130] MQ833 is replication incompetent but, as demonstrated herein, can profoundly remodel the TIME to promote both innate and adaptive antitumor immunity. As demonstrated herein, MQ833-induced neutrophil recruitment and activation is independent of mature T and B cells, and IT MQ833 delayed tumor growth in subjects completely or partially lacking an adaptive immune system. Intratumoral (IT) delivery of MQ833 generates potent antitumor responses via CD8+ T cells, neutrophils, and Ml-like macrophages, the nucleic acid-sensing pathways mediated by MDA5/STING, and interferon feedback loop. IT MQ833 promotes the recruitment and activation of neutrophils and inflammatory monocytes into the injected tumors, depletion of M2-like macrophages, and expansion of Ml-like macrophages, generating potent antitumor immunity against tumors resistant to ICB due to MHC-I antigen presentation and B2M deficiencies. IT administration of MQ833 demonstrated antitumor efficacy in B2m' ^/ ‘ tumors resistant to CD8 ⁺ T cell killing. As demonstrated herein, IT MQ833 can be used to treat subjects with primary or acquired resistance to ICB. MQ833 is also efficacious in treating tumors in subjects lacking an adaptive immune response.

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

[0131] Type I IFN plays important roles in host antitumor immunity (Fuertes et al., Trends Immunol. 34:67-73 (2013)). IFNARl-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.

XI. Immune Response

[0132] 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 (TH) 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.

[0133] 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 y (IFN-y), tumor necrosis factor (TNF), IFN-a, IFN-P (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).

XII. Pharmaceutical Compositions and Preparations of the Present Technology

[0134] Disclosed herein are pharmaceutical compositions comprising MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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 affected 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.

[0135] Pharmaceutical compositions and preparations comprising MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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.

[0136] 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.

[0137] Sterile injectable solutions are prepared by incorporating MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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, methods of preparation include 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.

[0138] In some embodiments, the compositions of the present disclosure comprising MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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 compositions comprising MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, l-dodecylhexahydro-2H-azepin-2- one (laurocapran), oleic acid, sodium citrate, Tris HC1, 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)).

[0139] 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.).

[0140] The engineered poxviruses of the present technology can be stored at -80°C with a titer of 3.5 xlO 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.

[0141] For therapy, the lyophilisate can be dissolved in an aqueous solution, such as physiological saline or Tris buffer, and administered either systemically or intratum orally. The mode of administration, the dose, and the number of administrations can be optimized by those skilled in the art.

[0142] 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)). XIII. Therapeutic Methods of the Present Technology

[0143] 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 (MV A) virus comprising a mutant E3L gene (AE3L), a mutant E5R gene (AE5R), a mutant WR199 gene (AWR199), a heterologous nucleic acid molecule encoding human OX40L (hOX40L), a heterologous nucleic acid molecule encoding human Flt3L (hFlt3L), and a heterologous nucleic acid molecule encoding human IL-12 (hIL-12) (MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12), a MVAAE5R- Flt3L-OX40L virus (MQ710), a MVAAE3LAE5R-Flt3L-OX40LAWR199 virus (MQ832), 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 MV A, MVAAE3L, or rMVA; and increased splenic production of effector T-cells as compared to the corresponding MV A, MVAAE3L, or rMVA strain. In some embodiments, the subject is a human. In some embodiments, the composition of the present technology comprising MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof is administered to the subject by intratumoral or intravenous injection or a simultaneous (z.e., concurrent) or sequential combination of intratumoral and intravenous injection.

[0144] In some embodiments, the subject is diagnosed with a cancer such as melanoma, colon carcinoma, breast cancer, prostate cancer, sarcoma, soft-tissue sarcoma, 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, malignant nerve sheath tumor, malignant peripheral nerve sheath tumor, anaplastic thyroid cancer vaginal tumor, and the metastases of any of the foregoing. In some embodiments, the cancer is a sarcoma.

[0145] In some embodiments, the subject is diagnosed with an immune checkpoint blockade inhibitor (ICB) resistant cancer. In some embodiments, the an immune checkpoint blockade inhibitor resistant cancer melanoma, colon cancer, breast cancer, bladder cancer, prostate carcinoma, sarcoma, ovarian cancer, glioblastoma, head-and-neck squamous cell carcinoma, advanced skin squamous cell carcinomas, basal cell carcinomas, angiosarcomas, sebaceous carcinomas, Kaposi sarcoma, malignant peripheral nerve sheath tumors, pancreatic cancer, malignant nerve sheath tumors, malignant peripheral nerve sheath tumors, anaplastic thyroid cancer, pancreatic cancer or Extramammary Paget disease (EMPD). Other cancers not expressly recited here can also be ICB inhibitor-resistant. In some embodiments, the ICB resistant cancer is a solid tumor. In some embodiments, the ICB resistant cancer is a sarcoma. In some embodiments, the cancer is ICB inhibitor resistant due to a deficiency in MHC-I antigen presentation. In some embodiments, the ICD inhibitor resistance is due to a mutation in the B2M gene and/or reduced B2M activity. The B2M protein plays an important role in MHC-I antigen presentation. In some embodiments, an ICB inhibitorresistant tumor is resistant to CD8 ⁺ T cell mediated immune responses. In some embodiments, a tumor is diagnosed as being resistant to treatment with ICB inhibitors due to lack of response to administration of one or more ICB inhibitors, such as, for example, either an increase or a lack of reduction in tumor size or volume, metastasis, nodal invasion, or progressive disease in spite of treatment with one or more ICB inhibitors. In some embodiments, the ICB resistant cancer comprises a solid tumor that is resistant to immune checkpoint blockade inhibitor treatment and/or wherein the tumor comprises tumor cells comprising loss or mutation of the beta-2 -microglobulin (B2M) gene or are characterized by downregulation of B2M, and/or wherein the tumor is MHC-1 antigen presentation deficient. In some embodiments, engineered poxviruses of the present technology, i.e., MVAAE5R- Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof, are not administered in combination with (i.e., separately, sequentially, or simultaneously with) any immune checkpoint blocking inhibitor. In some embodiments, a subject has previously received treatment with an immune checkpoint blockade inhibitor (e.g., has received at least one dose or in some cases has received a full course of treatment with an ICB inhibitor) prior to starting treatment with MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof. In some embodiments, the subject continues to receive treatment with an ICB inhibitor upon starting treatment with MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or a combination thereof. In other cases, treatment with an ICB inhibitor is discontinued upon starting treatment with MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or a combination thereof. In some cases, a subject is treated with MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof, and also with another therapy or treatment such as chemotherapy, hormonal treatment, surgery, or radiation.

XIV. Combination Therapy

[0146] In some embodiments, the engineered poxviruses of the present technology (e.g., MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof) are combined or separately, sequentially, or simultaneously i.e., concurrently) administered with one or more other anti-cancer agents, such as one or more chemotherapy agents, one or more hormonal agents, or one or more immune checkpoint blockade inhibitors. Thus, in some cases for treating an ICB inhibitor resistant cancer, the engineered poxviruses may be administered with one or more ICB inhibitors. In some embodiments, the combined administration of the engineered poxviruses of the present technology (e.g., MVAAE5R- Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof) with one or more immune checkpoint blockade inhibitors (such as anti-PD-1, anti-PD-Ll, or anti- CTLA4 antibodies) may result in a synergistic effect with respect to the treatment of solid tumors.

Immune Checkpoint Blockade Inhibitors

[0147] In some embodiments, MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R- Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof is combined or separately, sequentially, or simultaneously (z.e., concurrently) administered with one or more immune checkpoint blockade (ICB) inhibitors. The one or more immune checkpoint blockade inhibitors 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-Ll, or anti-huCTLA-4 antibodies).

[0148] In some embodiments, the one or more immune checkpoint blockade inhibitors are selected from 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, MEDL6469, 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. [0149] In some embodiments, the combination of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and one or more immune checkpoint blockade inhibitors may result in a synergistic effect. In some embodiments, the combination of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and one or more immune checkpoint blockade inhibitors may result in an enhanced anti -turn or effect. In some embodiments, the combination of MVAAE5R- Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and anti-PD-Ll may result in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and anti-PD-1 may result in a synergistic effect with respect to the treatment of solid tumors. In some embodiments, the combination of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and anti-CTLA-4 may result in a synergistic effect with respect to the treatment of solid tumors.

[0150] It has been reported that the sequential (z.e., serial) administration of anti-OX40 antibody followed by the immune checkpoint blockade 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 0X40 antibody and results in poor treatment outcomes in mice. (See, Shrimali et al., (2017); Messenheimer et al., (2017)). In some embodiments, the combination of the viruses expressing the OX40L transgene of the present technology, e.g., MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and one or more immune checkpoint blockade inhibitors (e.g., anti-PD-Ll antibody, anti-PD-1 antibody, anti-CTLA-4 antibody) may result in an enhanced anti-tumor effect as compared to the combination of an immune checkpoint blockade inhibitor and anti -0X40 agonist antibody. In some embodiments, the combination of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R- Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and one or more immune checkpoint blockade inhibitors (e.g., anti-PD-Ll antibody, anti-PD-1 antibody, anti-CTLA-4 antibody) may result in a synergistic effect with respect to the treatment of solid tumors as compared to the combination of an immune checkpoint blockade inhibitor and anti-OX40 agonist antibody.

XV. Kits Comprising Engineered Poxyiruses of the Present Technology

[0151] The present disclosure provides for kits comprising one or more compositions comprising one or more of the engineered poxviruses, e.g., MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), described hereintogether with instructions for the administration of the engineered poxviruses to a subject to be treated. The instructions may indicate a dosage regimen for administering the composition or compositions as provided below. In some embodiments, the kits contain instructions for methods for treating a solid tumor wherein the solid tumor is resistant to immune checkpoint blockade inhibitor treatment, methods of preventing cancer recurrence for a period of time in a subject in need thereof, methods for treating a tumor in a subject in need thereof wherein the subject has a deficient adaptive immune system response, and methods for altering the tumor immune microenvironment (TIME) in a tumor in a subject in need thereof.

[0152] In some embodiments, the kit may also comprise an additional therapeutic composition, such as, for example, a composition comprising one or more chemotherapeutic agents, one or more hormonal agents, and/or comprising immune checkpoint blockade inhibitors for conjoint administration with the engineered poxvirus, e.g., MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof.

XVI. Effective Amount and Dosage of the Engineered Poxyiruses of the Present Technology

[0153] In general, in some embodiments, the subject is administered a dosage of

MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof in the range of about 10 ⁶ to about 10 ¹⁰ plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage ranges from about 10 ² to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ³ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁴ to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁵ to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁶ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁷ to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁸ to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁹ to about IO ¹⁰ pfu. In some embodiments, dosage is about 10 ⁷ to about 10 ⁹ pfu. The equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, a pfu is equal to about 5 to 100 virus particles and 0.69 pfu is about 1 TCID50. A therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.

[0154] For example, a therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or a combination thereof 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 ability of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or a combination thereof to elicit a desired immunological response in the particular subject (the subject’s response to therapy). In delivering MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof to a subject, the dosage may also vary depending upon such factors as the general medical condition, previous medical history, disease type and progression, tumor burden, the presence or absence of tumor infiltrating immune cells in the tumor, and the like.

[0155] In some embodiments, it may be advantageous to formulate compositions of the present disclosure in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form as used herein” refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.

XVII. Administration and Therapeutic Regimen of the Engineered Poxyiruses of the Present Technology

[0156] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Administration of the engineered poxviruses of the present technology, such as MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof, can be achieved using more than one route. Examples of routes of administration include, but are not limited to parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), intratumoral, intrathecal, intranasal, systemic, transdermal, iontophoretic, intradermal, intraocular, or topical administration. In one embodiment, MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof is administered directly into the tumor, e.g., by intratumoral injection, where a direct local reaction is desired. Additionally, administration routes of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or a combination thereof can vary, e.g., first administration using an intratumoral injection, and subsequent administration via an intravenous injection, or any combination thereof. A therapeutically effective amount of a MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or a combination thereof injection can be administered for a prescribed period of time and at a prescribed frequency of administration. In certain embodiments, MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof can be used in conjunction with other therapeutic treatments. For example, the engineered poxvirus of the present technology can be administered in a neoadjuvant (preoperative) or adjuvant (postoperative) setting for subjects inflicted with bulky primary tumors. It is anticipated that such optimized therapeutic regimen will induce an immune response against the tumor and reduce the tumor burden in a subject before or after primary therapy, such as surgery. Furthermore, MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof can be administered in conjunction with other therapeutic treatments such as chemotherapy, hormonal agents, an ICB inhibitor, surgery, or radiation.

[0157] In certain embodiments, the MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof is administered at least once weekly or monthly but can be administered more often if needed, such as two times weekly for several weeks, months, years, or even indefinitely as long as benefits persist. More frequent administrations are contemplated if tolerated and if they result in sustained or increased benefits. Benefits of the present methods can include but are not limited to the following: reduction of the number of cancer cells, reduction of the tumor size, eradication of tumor, inhibition of cancer cell infiltration into peripheral organs, inhibition or stabilization or eradication of metastatic growth, inhibition or stabilization of tumor growth, and stabilization or improvement of quality of life. Furthermore, the benefits may include induction of an immune response against the tumor, activation of effector CD4 ⁺ T-cells, an increase of effector CD8 ⁺ T-cells, or reduction of regulatory CD4 ⁺ cells.

XVIII. Vectors

[0158] In some embodiments, a pCB plasmid-based vector may be used to insert a specific gene of interest (SG), such as OX40L (murine or human), under the control of the vaccinia synthetic early and late promoter (PsE/L). The methodology for constructing the vector has been described (See M. Puhlmann, C. K. Brown, M. Gnant, J. Huang, S. K. Libutti, H. R. Alexander, D. L. Bartlett, Vaccinia as a vector for tumor-directed gene therapy: Biodistribution of a thymidine kinase-deleted mutant Cancer Gene Therapy 7(1): 66— 73 (2000)). A xanthine-guanine phosphoribosyl transferase (gpt) gene under the control of vaccinia P7.5 promoter may used as a selectable marker. An illustrative pCB-mOX40L-gpt vector nucleic acid sequence is set forth in SEQ ID NO: 2. In some embodiments, a pUC57 plasmid-based vector may be used to insert a specific gene of interest (SG), such as OX40L (murine or human), under the control of the vaccinia synthetic early and late promoter (PsE/L). In some embodiments, a pMA plasmid-based vector may be used to insert a specific gene of interest (SG), such as OX40L (murine or human), under the control of the vaccinia synthetic early and late promoter (PsE/L). An mCherry gene under the control of vaccinia P7.5 promoter may be used as a selectable marker. Illustrative vector nucleic acid sequences of the present technology are shown in Tables 1-6.

[0159] In some embodiments, vectors of the present technology contain expression cassettes that are flanked by a partial sequence of the TK gene on each side (TK-L, TK-R). Homologous recombination that occurs at the TK locus of the plasmid DNA and viral genomic DNA results in the insertion of the expression cassette into the genomic DNA TK locus to generate a TK knockout viral strain. Additionally or alternatively, suitable loci other than the TK locus within the virus could be used. Homologous recombination that occurs at a suitable viral gene locus (e.g., E3L, E5R, WR199, etc.) of the plasmid DNA and viral genomic DNA results in the insertion of one or more specific gene of interest (e.g., OX40L, hFlt3L, IL-12, etc.) and/or selectable marker expression cassettes into the viral genomic DNA viral gene locus to generate recombinant poxviruses such as those described herein.

[0160] In some embodiments, a vector of the present technology contains an expression cassette comprising two transgenes, Flt3L and OX40L. In some embodiments, both of the transgenes are contained in an expression cassette that is flanked by a partial sequence of the E5R gene on each side. In some embodiments, the Flt3L and OX40L genes in the expression cassette are linked by a P2A self-cleaving sequence and their expression is driven by the vaccinia synthetic early-late promoter. In some embodiments, the vector is used to generate an MVA-AE5R-Flt3L-OX40L viral construct via homologous recombination between the vector and the MVA genome (SEQ ID NO: 1) at positions 38,432 to 39,385. The recombinant viruses are enriched by selection and plaque-purified for 4-5 rounds until the appropriate recombinant viruses are obtained.

[0161] In some embodiments, a vector of the present technology contains a selectable marker flanked by a partial sequence of the E3L gene on each side. In some embodiments, the selectable marker comprises mCherry. In some embodiments, the vector is used to generate an MVA-AE5R-Flt3L-OX40L-AE3L viral construct via homologous recombination between the vector and the MVA genome (e.g., SEQ ID NO: 1 at positions 42,697-43,269 or SEQ ID NO: 19 at positions 36,907-37,479). The recombinant viruses are enriched by selection and plaque-purified for 4-5 rounds until the appropriate recombinant viruses are obtained. [0162] In some embodiments, a vector of the present technology contains a selectable marker flanked by a partial sequence of the WR199 gene on each side. In some embodiments, the selectable marker comprises mCherry. In some embodiments, the vector is used to generate an MVA-AE5R-Flt3L-OX40L-AE3L-AWR199 viral construct via homologous recombination between the vector and the MVA genome (e.g., SEQ ID NO: 1 at positions 164,209-165,933 or SEQ ID NO: 19 at positions 158,419-160,143). The recombinant viruses are enriched by selection and plaque-purified for 4-5 rounds until the appropriate recombinant viruses are obtained.

[0163] In some embodiments, a vector of the present technology contains an expression cassette comprising an IL-12 transgene. In some embodiments, the IL-12 transgene is engineered such that expression of the transgene in a cell results in IL-12 that is anchored to the exctracellular matrix. In some embodiments, the IL-12 transgene in the vector is flanked by partial sequences of the MVA genome on each side. In some embodiments, the vector is used to generate an MVA-AE5R-Flt3L-OX40L-AE3L-AWR199-hIL-12 viral construct via homologous recombination between the vector and the MVA genome (e.g., SEQ ID NO: 1 at positions 164,209-165,933 or SEQ ID NO: 19 at positions 158,419-160,143). The recombinant viruses are enriched by selection and plaque-purified for 4-5 rounds until the appropriate recombinant viruses are obtained.

[0164] It will be appreciated, that any other expression vector suitable for integration into the MVA genome could be used as well as alternative promoters, regulatory elements, selectable markers, cleavage sites, and/or nonessential insertion regions of MVA. In some embodiments, the selectable marker is a reporter protein, wherein the reporter protein is a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. In some embodiments, the reporter protein is green fluorescent protein (GFP). In some embodiments, the selectable marker is xanthine-guanine phophoribosyl transferase gene (gpt). In some embodiments, the selectable marker is an mCherry gene. MVA encodes many immune modulatory genes at the ends of the linear genome, including Cl 1, K3, Fl, F2, F4, F6, F8, F9, Fl 1, F14.5, J2, A46, E3L, B18R (WR200), E5R, K7R, C12L, B8R, B14R, NIL, C11R, K1L, C16, MIL, N2L, and WR199 (or their orthologs). These genes (or their orthologs) can be deleted to potentially enhance immune activating properties of the virus, and/or allow insertion of transgenes. XIX. Delivery of the Engineered Poxyirus Strains of the Present Technology as an Adjuvant to a Subject to Treat Cancer

A. Compositions

Immune-Activating Cancer Vaccine Adjuvants

[0165] Recent discoveries of cancer neoantigens have generated a renewed interest in cancer vaccination and the combination of cancer vaccination with immune checkpoint blockade inhibitors to enhance vaccination effects. Developing effective vaccine adjuvants that can maximize antitumor immune responses is critical for the success of cancer vaccines.

[0166] Cancer vaccines comprise cancer antigens and immune adjuvants. Cancer antigens generally include tumor differentiation antigens, cancer testis antigens, neoantigens, and viral antigens in the case of tumors associated with oncogenic virus infection. Cancer antigens can be provided in the form of irradiated tumor cells, dendritic cells (DCs) loaded with tumor cell lysates or peptides, DNA or RNA encoding antigen, as well as oncolytic virus with transgene(s) encoding cancer antigen(s). Dendritic cells (DCs) are professional antigen- presenting cells that are important for priming naive T-cells to generate antigen-specific T- cell responses. Immune adjuvants are agents that promote antigen uptake by DCs and/or DC maturation and activation. Several immune adjuvants, including toll-like receptor (TLR) agonists, poly (I:C) (TLR3 agonist), CpG (TLR9 agonist), Imiquimod (TLR7 agonist), as well as STING agonists, have been shown to improve vaccine efficacy in preclinical models and clinical settings.

Engineered Poxvirus Strains of the Present Technology as Adjuvant Therapy

[0167] The disclosure herein encompasses the use of the engineered poxvirus strains described herein (e.g., MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof) as vaccine adjuvants. In some embodiments, the disclosure of the present technology relates to the use of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as a vaccine adjuvant. In some embodiments, the disclosure of the present technology relates to the use of MVAAE5R- Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof in combination with Heat-inactivated vaccinia (Heat-iMVA, Heat-iMVAAE5R) as a vaccine adjuvant. Heat-iMVA has been shown to induce type I IFN in conventional DCs (eDCs) via the cGAS/STING-dependent pathway and also induces type I IFN in plasmacytoid DCs (pDCs) via the TLR7/MyD88-dependent mechanism. Moreover, intratumoral injection of Heat-iMVA eradicates injected tumors and leads to the generation of systemic antitumor immunity either as monotherapy or in combination with other therapies immune checkpoint blockade (ICB) inhibitors. In some embodiments herein, the use of MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as a vaccine adjuvant is in the context of administration to a subject with an ICB resistant tumor.

Target Antigens

[0168] The compositions and methods disclosed herein are not intended to be limited by the choice of antigen or neoantigen. While numerous examples of antigens and neoantigens are provided, the skilled artisan can easily utilize the adjuvant disclosed herein with an antigen or neoantigen of choice. Exemplary, non-limiting target antigens that may be used in therapeutic regimens of the present technology include 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, pl 5, 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 (gplOO), GnT-V intron V sequence (N- acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), P-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 combinations thereof. In some embodiments, the antigen comprises a neoantigen selected from the group consisting of M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) (SEQ ID NO: 16), M30 (PSKPSFQEFVDWENVSPELNSTDQPFL) (SEQ ID NO: 17), M48 (SHCHWNDLAVIPAGVVHNWDFEPRKVS) (SEQ ID NO: 18), and combinations thereof. The target antigen may also be a fragment or fusion polypeptide comprising an immunologically active portion of the antigens listed above. Immune Checkpoint Blockade (ICB) Inhibitors

[0169] In some embodiments, use of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof may be in combination with one or more immune checkpoint blockade inhibitors. Immune checkpoint blockade (ICB) inhibitors, such as antibodies targeting immune checkpoint proteins for example, have been at the forefront of immunotherapy and have been accepted as one of the pillars of cancer management options, including surgery, radiation, and chemotherapy. Because immune checkpoints have been implicated in the downregulation of antitumor immunity, agents and antibodies targeting immune checkpoint proteins or their ligands (CTLA-4, PD-1, or PD-L1) have been successful in disinhibiting antitumor T-cells, thereby leading to proliferation and survival of activated T-cells.

[0170] Non-limiting examples of immune checkpoint blockade inhibitors include agents or antibodies that modulate the activity of one or more checkpoint proteins including anti -PD-1 antibody, anti-PD-Ll 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.

Pharmaceutical Compositions and Preparations of the Present Technology [0171] Disclosed herein are also, for example, pharmaceutical compositions comprising an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant 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. In some embodiments, the pharmaceutical compositions comprise an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and Heat-iMVA as adjuvants. 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.

[0172] Pharmaceutical compositions and preparations comprising an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate formulating preparations suitable for in vitro, in vivo, or ex vivo use.

[0173] 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.

[0174] Sterile injectable solutions are prepared by incorporating an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant 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.

[0175] In some embodiments, an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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 antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof 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 HC1, 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.

[0176] In some embodiments, the compositions of the present technology can be stored at - 80°C. For the preparation of vaccine shots, e.g., 10 ² - 10 ⁸ or 10 ² - 10 ⁹ viral particles can be lyophilized, for example, 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 recombinant virus 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.

[0177] For therapy, the lyophilisate can be dissolved in an aqueous solution, such as physiological saline or Tris buffer, and administered either systemically or intratum orally. The mode of administration, the dose, and the number of administrations can be optimized by those skilled in the art. [0178] The pharmaceutical compositions comprising an antigen and MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant according to the present disclosure may comprise an additional adjuvant including 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.

Vaccines and Immunogenic Compositions

[0179] In some embodiments, compositions comprising MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant and one or more antigens are formulated into immunogenic compositions or vaccines. In some embodiments, the compositions comprise MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant. In some embodiments, the compositions comprise MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and Heat-iMVA as adjuvants. In some embodiments, the immunogenic compositions or vaccines are tumor antigen-containing whole cell vaccines (e.g., an irradiated whole cell vaccine). In some embodiments, the immunogenic compositions or vaccines are administered to a subject to elicit an immune response against the antigens formulated therewith.

Effective Amount and Dosage ofMVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as a Cancer Vaccine Immune Adjuvant

[0180] In some embodiments, the subject is administered a dosage of MVAAE5R-Flt3L- OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof in the range of about 10 ⁶ to about 10 ¹⁰ plaque forming units (pfu), although a lower or higher dose may be administered. In some embodiments, the dosage ranges from about 10 ² to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ³ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁴ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁵ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁶ to about 10 ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁷ to about IO ¹⁰ pfu. In some embodiments, the dosage ranges from about 10 ⁸ to about IO ¹⁰ pfu.

In some embodiments, the dosage ranges from about 10 ⁹ to about IO ¹⁰ pfu. In some embodiments, dosage is about 10 ⁷ to about 10 ⁹ pfu. The equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, a pfu is equal to about 5 to 100 virus particles and 0.69 PFU is about 1 TCID50. A therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.

[0181] For example, a therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant 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 ability of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof to elicit a desired immunological response in the particular subject (the subject’s response to therapy). In delivering MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof to a subject, the dosage may also vary depending upon such factors as the general medical condition, previous medical history, disease type and progression, tumor burden, the presence or absence of tumor infiltrating immune cells in the tumor, and the like.

[0182] In some embodiments, it may be advantageous to formulate compositions of the present disclosure in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form as used herein” refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.

Administration and Therapeutic Regimen of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as a Cancer Vaccine Immune Adjuvant

[0183] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Administration of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant in an immunogenic composition (e.g, vaccine) can be achieved using more than one route. Examples of routes of administration include, but are not limited to parenteral (e.g, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), intratumoral, intrathecal, intranasal, systemic, transdermal, iontophoretic, intradermal, intraocular, or topical administration. In one embodiment, the pharmaceutical composition of the present technology comprising an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant is administered directly into the tumor, e.g. by intratumoral injection, where a direct local reaction is desired. In some embodiments, the pharmaceutical composition of the present technology comprising an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant is administered peripherally relative to tumor beds. Additionally, the administration routes can vary, e.g., first administration using an intratumoral injection, and subsequent administration via an intravenous injection, or any combination thereof. A therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant in a cancer vaccine injection can be administered for a prescribed period of time and at a prescribed frequency of administration. In certain embodiments, the pharmaceutical compositions of the present technology can be used in conjunction with other therapeutic treatments such as chemotherapy, hormone therapy, surgery, immune checkpoint blockade inhibitor therapy, or radiation. In some embodiments, the pharmaceutical compositions of the present technology comprising a therapeutically effective amount of MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant can be used in conjunction with immune checkpoint blockade therapy. [0184] In certain embodiments, the pharmaceutical composition comprising an antigen and MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant is administered at least once weekly or monthly but can be administered more often if needed, such as two times weekly for several weeks, months, years or even indefinitely as long as benefits persist. More frequent administrations are contemplated if tolerated and if they result in sustained or increased benefits. Benefits of the present methods include but are not limited to the following: reduction of the number of cancer cells, reduction of the tumor size (e.g., tumor volume), eradication of tumor, inhibition of cancer cell infiltration into peripheral organs, inhibition or stabilization or eradication of metastatic growth, inhibition or stabilization of tumor growth, and stabilization or improvement of quality of life. Furthermore, the benefits may include induction of an immune response against the tumor, increased ZFN-y ⁺ CD8 ⁺ T-cells, increased IFN-y ⁺ CD4 ⁺ T-cells, activation of effector CD4 ⁺ T- cells, an increase of effector CD8 ⁺ T-cells, or reduction of regulatory CD4 ⁺ cells. For example, in the context of melanoma, in some embodiments, a benefit may be a lack of recurrences or metastasis within one, two, three, four, five or more years of the initial diagnosis of melanoma.

B. Methods

[0185] In one aspect, the present disclosure provides for a method for treating solid tumor by enhancing an immune response in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising one or more antigens and an adjuvant comprising MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof, thereby treating the tumor by enhancing immune response. In some embodiments, the adjuvant comprises MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12 (MQ833), or combinations thereof. In some embodiments, the adjuvant comprises MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L- OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and Heat-iMVA.

[0186] In some embodiments, the disclosure provides methods comprising administering the immunogenic composition comprising one or more antigens and MVAAE5R-Flt3L-

I l l OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R- hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof as an adjuvant to a subject in order to elicit an immune response against the antigens.

[0187] In some embodiments of the methods disclosed herein, the administration step comprises administering the immunogenic composition in multiple doses.

[0188] In some embodiments, the methods described herein further comprise administering to the subject an immune checkpoint blockade inhibitor selected from the group consisting of anti-PD-1 antibody, anti-PD-Ll 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 immunogenic composition is delivered to the subject separately, sequentially, or simultaneously with the administration of the immune checkpoint blockade inhibitor.

C. Kits

[0189] In some embodiments, kits are provided. In some embodiments, the kit includes a container means and a separate portion of each of: (a) an antigen and (b) an adjuvant comprising MVAAE5R-Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof. In some embodiments, the adjuvant comprises MVAAE3LAE5R-hFlt3L- hOX40LAWR199-hIL-12. In some embodiments, the adjuvant comprises MVAAE5R- Flt3L-OX40L (MQ710), MVAAE3LAE5R-Flt3L-OX40LAWR199 (MQ832), MVAAE3LAE5R-hFlt3L-hOX40LAWR199-hIL-12 (MQ833), or combinations thereof and Heat-iMVA.

EXPERIMENTAL EXAMPLES

[0190] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. General Materials and Methods for Examples 1-14

[0191] Mice. Female C57BL/6J mice between 6 and 8 weeks of age were purchased from the Jackson Laboratory (Strain #000664) and were used for the preparation of bone marrow- derived dendritic cells and for in vivo experiments. These mice were maintained in the animal facility at the Sloan Kettering Institute. All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Sloan Kettering Cancer Institute. Sting ^Gt/Gt mice were generated in the laboratory of Dr. Russell Vance (University of California, Berkeley). eGas' ¹ ' (Strain # 026554), Statl' ¹ ' (Strain # 012606), Stat2' ^G (Strain #023309) and Nos2' ^G (Strain #002609) mice were purchased from Jackson Laboratory. Mda5' ^G mice were generated in Marco Colonna’s laboratory (Washington University). Mda5' ' Sting ^GtlGt were bred in our lab.

[0192] Cell Lines. BHK-21 (baby hamster kidney cell, ATCC CCL-10) cells were cultured in Eagle’s minimal essential medium containing 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, penicillin, and streptomycin. The murine melanoma cell line Bl 6- F10 was originally obtained from I. Fidler (MD Anderson Cancer Center). The MC38 cell line was obtained from ATCC. The B16-F10 cell line was maintained in RPML1640 medium supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, penicillin, and streptomycin. The B 16-F10 cell line lacking B2M gene were generated by using CRISPR- cas9 technology.

[0193] Viruses. The MVA virus was obtained from Gerd Sutter (University of Munich). MVAAE5R, MVAAE5R-hFlt3LmOX40L (rMVA or MQ710), rMVAAE3L, rMVAAE3LAWR199 (MQ832), rMVAAE3LAWR199-IL12 (MQ833), and hMQ833 were generated by transfecting pUC57-based plasmids into BHK-21 cells that were infected with MVA at MOI 0.05. Recombinant viruses were purified after 4~6 rounds of plaque selection based on the fluorescence marker. PCR and DNA sequencing were performed to verify the purity of the recombinant viruses. Viruses were propagated in BHK-21 cells and purified through a 36% sucrose cushion.

[0194] Human monocyte-derived dendritic cell (moDC) generation and T cell isolation. All collection and use of human specimens adhered to protocols reviewed and approved by the Institutional Review and Privacy Board of Memorial Hospital, MSKCC. Buffy coat products were obtained from healthy donor at New York Blood Center and peripheral blood mononuclear cells (PBMCs) were separated by standard centrifugation over Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden). Tissue culture plastic adherent PBMCs comprised the moDCs precursors, which were cultured in complete PRMI 1640-1% human serum supplemented with 1000 lU/ml GM-CSF (PeproTech) and 500 lU/ml IL-4 (PeproTech). Fresh medium and cytokines were replenished every 48 h. T cells were obtained from tissue culture nonadherent PBMCs, then further purified by CD8+ T cell or CD4+ T cell isolation kit (Miltenyi Biotec) following manufacturer’s instruction.

[0195] Tumor processing and flow cytometry staining. Tumor samples were collected in cold RPMI and thoroughly chopped into small pieces with surgical scissors. Then Liberase TL (1.67 Wunsch U/ml) and DNase I (0.2 mg/ml) were added to the samples in a tube and the mixture was incubated at 37 °C on a shaker for 20-30 minutes. Digested tumors were then transferred to GentleMACs C Tubes and homogenized using the Gentle MACs Octo Dissociator (Miltenyi Biotec) according to manufacturer’s instruction. Samples were then filtered through 70 pm filter and quenched with 10 ml cold PBS and spun down. Cells were washed with MACS buffer (Miltenyi Biotec) twice, and stained with Fc block (BD), viability dye eFluor506 (eBioscience), and cell surface antibodies diluted in MACS buffer for 30 minutes on ice in the dark, and subsequently fixed and permeabilized using the Foxp3 fixation and permeabilization kit (Thermo Fisher). Cells were then incubated with intracellular antibodies diluted in permeabilization buffer for 30 minutes or overnight.

[0196] The following antibodies were used for flow cytometric staining: CD45 Pacific blue (clone 30-F11, Biolegend), CD3 BUV395 (clone 145-2C11, BD), CD4 BUV737 (clone RM4-5, BD), CD8Alexa Fluor 700 (clone 53-6.7, Biolegend), 0X40 BV605 (clone OX-86, Biolegend), TIM3 BV711 (clone RMT3-23, Biolegend), PD-1 AF647 (clone RMPI-30, BD), KLRG1 APC-Cy7 (clone 2F1/KLRG1, Biolegend), Foxp3 PerCP-Cy5.5 (clone FJK- 16s, Invitrogen), Ki67 BV786 (clone B56, BD), TOX PE (clone REA473, Miltenyi Biotec), Granzyme B PE-Texas red (clone GB11, Invitrogen), CD1 lb APC-eFluor780 (clone MI/70, eBioscience), Ly6G PE-Cy7 (clone 1A8, BD), F4/80 APC (clone BM8, Biolegend), CD68 BV421 (clone FA-11, Biolegend), and iNOS PE (clone W16030C, Biolegend). After staining, the cells were washed and resuspended with MACS buffer and transferred into FACS tubes with 70 um filter caps. Single cell suspensions were run on Cytek Aurora analyzer, and the data was analyzed with Flowjo. [0197] Generation of bone marrow -derived dendritic cells. Bone marrow cells were extracted from the femur and tibia of mice. After centrifugation, cells were re-suspended in ACK Lysing Buffer (Lonza) for red blood cell lysis by incubating the cells on ice for 1-3 min. Cells were then resuspended in fresh medium and filtered through a 70-pm cell strainer (BD Biosciences). For the generation of Flt3L-BMDCs, the bone marrow cells (5 million cells in each well of 6-well plates) were cultured in complete medium (CM) in the presence of Flt3L (100 ng/ml; R & D systems) for 7-9 days. Cells were fed every 2-3 days by replacing 50% of the old medium with fresh medium. For the generation of GM-CSF- BMDCs, the bone marrow cells (5 million cells in each 15 cm cell culture dish) were cultured in RPML1640 medium supplemented with 10% fetal bovine serum (FBS) in the presence of 30 ng/ml GM-CSF (BioLegend) for 9-12 days. Fresh medium and cytokine were replenished every 2-3 days.

[0198] RNA isolation and Real-time PCR. Cells were infected with various viruses at a MOI of 10 for 1 hour or were mock-infected. The inoculum was removed, and the cells were washed with PBS twice and incubated with fresh medium for 16 hours or overnight. RNA was extracted from whole-cell lysates with RNeasy Plus Mini kit (Qiagen) and was reverse- transcribed with cDNA synthesis kit (Thermo Fisher). Real-time PCR was performed in triplicate with SYBR Green PCR Master Mix (Life Technologies) and Applied Biosystems 7500 Real-time PCR Instrument (Life Technologies) using gene-specific primers. Relative expression was normalized to the levels of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).

[0199] ELISA. For IL-12 transgene expression verification, B16 cells were infected with MVAAE5R or MQ833 at a MOI of 10 for 1 hour. Mock-infected cells were used as a control. The supernatant was removed, and the cells were washed with PBS twice and incubated with fresh medium for 16 hours. Supernatant was collected from the culture, and IL-12 levels were determined using Duoset ELISA kit (R&D) following manufacturer’s instruction. For T cell activation assays, supernatant from the co-culture system was collected and IFNy levels were determined by Duoset ELISA kit (R&D).

[0200] ELISPOT assay. Spleens were harvested from mice treated with different viruses and were mashed through a 70 pm strainer (Thermo Fisher Scientific). Red blood cells were lysed using ACK Lysis Buffer (Life Technology), and the cells were resuspended in RPMI medium. Enzyme-linked ImmunoSpot (ELISPOT) assay was performed to measure IFN-y ⁺ CD8 ⁺ T cells, according to the manufacturer’s protocol (BD Bioscience). 1 x 10 ⁶ splenocytes were co-cultured with 2.5>< 10 ⁵ irradiated Bl 6-F 10 cells in complete RPMI medium overnight. ZFNy ⁺ splenocytes were detected by Mouse IFNy ELISPOT kit (BD Biosciences).

[0201] Western blot analyses. Bl 6-F 10 cells or BMDCs were infected with MQ832 or MQ833 at a MOI of 10 for 1 hour. Mock-infected cells were used as a control. Cells were washed with PBS twice and then cultured with fresh medium. Cells were lysed in RIPA lysis buffer supplemented with lx Halt™ Protease and Phosphatase Inhibitor Cocktail at the indicated time points. Protein samples were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. Primary antibodies specific for phospho-IRF3 (1 :500, CST, 4947), IRF3 (1 : 1000, CST, 4302), cGAS (1 : 1000, CST, 31659), STING (1 : 1000, CST, 13647), phospho-STING (1 :500, CST, 72971), TBK1 (1 : 1000, CST, 3504), MDA5 (1 :500, CST, 5321) and phospho-TBKl (1 :500, CST, 5483) were used. GAPDH antibody (1 :5000, CST, 2118) were used as loading controls. Anti-rabbit HRP -linked IgG antibody was used as a secondary antibody (1 :5000, CST, 7074). Detection was performed using SuperSignal TM Substrates (Thermo Fisher, 34096 or A38555).

[0202] In vitro T cell activation assay. For murine MQ833, splenic CD1 lc+ DCs were sorted from WT mice and subsequently infected with various viruses at a MOI of 10 for 1 hour and pulsed with OVA (1.25 mg/ml). OT-1 T cells were isolated from the spleens OT-1 mice using negative selection with CD8a ⁺ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. OT-1 T cells were then co-cultured with DCs (DC:T = 1 :3) for 3 days. Alternatively, T cells were incubated with supernatant collected from the infected CD1 lc ⁺ DCs for 3 days. IFN-y levels in the supernatants were determined by ELISA. For human MQ833, human moDCs were infected with various viruses at a MOI of 10 for 1 hour and pulsed with OVA (1.25 mg/ml). CD8 or CD4 T cells were purified from human PBMCs using isolation kits (Miltenyi Biotec) following manufacturer’s instruction. T cells and moDCs were co-cultured (DC:T = 1 : 5) for 3 days and supernatant were then collected for IFN-y ELISA.

[0203] Generation ofB2m~ ~, Sling Mda5~ ~Sting~ ~ B16-F10 cells. B16-F10 cells were transfected with plasmids for expression of Cas9 and gRNAs against B2M. Two gRNA expression plasmids targeting exon 2 of B2M were co-transfected. Plasmid backbones used to create these CRISPR constructs were created by the Church group and obtained from Addgene (Mali et al 2013). After transfection, cells were induced to express the complex MHC class I by treating with 100 units of interferon gamma for 2 days. Afterwards, cells were dissociated with trypsin and stained with an antibody against H-2Kb-PE (BD, AF6- 88.5). The MHC class I complex (including H-2Kb) requires B2M to be stable on the surface of cells. Single cells that were negative for surface MHC-I were sorted into 96 well plates. Single cells were then expanded to produce clonal isolates. After outgrowth of clones, PCR using primers specific to the B2M locus was used to confirm which isolates contained a deletion of B2M (between the two gRNA target sites). The same PCR products were also cloned into TopoTA plasmids for sequencing. A subsequent western blot confirmed loss of B2M protein using an antibody against B2M protein (Abeam 75853). PCR, DNA sequencing, and western blot all confirmed loss of B2M. Sting' ⁷ ' and MdaS'^Sting' ⁷ ' B16-F10 cells were generated similarly to the B2m' ^/ ' except that instead of sorting, transfected cells were isolated by limiting dilution. Lesions at the target sites were confirmed by PCR amplification and T7 endonuclease assay.

[0204] Tumor challenge and treatment. For flow cytometric analysis and single cell RNA- seq, 5*10 ⁵ B16-F10 tumor cells were implanted intradermally into the right flanks of the mice for the unilateral tumor implantation model. For bilateral implantation, 5* 10 ⁵ and 2.5* 10 ⁵ B16-F10 tumor cells were implanted intradermally into the right and left flanks of mice. At 8-11 days after implantation when tumors were established, the tumors at the right flank were injected with 4 * 10 ⁷ PFU of MQ832, MQ833, or PBS once, or twice with 3 days apart. Tumors were harvested two days after second injection.

[0205] For survival experiments, 5* 10 ⁵ B16-F10 cells or l >< 10 ⁷ B2m' ^/ ' B16 cells were implanted intradermally into the shaved skin on the right flanks of WT C57BL/6J mice or age-matched eGas' ¹ ', Sting ^{G lG} Mda5~ ^l Sting^^MdaS' ¹ ' , and Stat2' ¹ ' mice. Once the tumors were 3 mm in diameter or larger, they were injected with 4* 10 ⁷ PFU of MQ832, MQ833 or PBS when the mice were under anesthesia. Viruses were injected twice weekly as specified in each experiment and tumor sizes were measured twice a week. For combination therapy with ICBs, the following antibodies were injected interperitoneally twice weekly: anti- CTLA-4 (100 pg per mouse), anti-PD-Ll (200 pg per mouse), anti-PD-1 (200 pg per mouse), or isotype control antibody. Tumor volumes were calculated according to the following formula: 1 (length) x _w (width) x h (height)/2. Mice were euthanized for signs of distress or when the diameter of the tumor reached 10 mm. In the bilateral tumor implantation model, B16-F10 cells were implanted intradermally into right (5x 10 ⁵ ) and left (1 x 10 ⁵ ) flanks of C57BL/6J mice. At 7 days after implantation when the tumors were established, the tumors at the right flank were injected with 4* 10 ⁷ PFU of MQ832, MQ833 or PBS twice weekly.

[0206] For the tumor rechallenge study, the surviving mice (more than 60 days after initiation of intratumoral virotherapy) were rechallenged with intradermal delivery of a lethal dose of B16-F10 or B2m KO B16-F10 (I MO ⁵ cells) at the contralateral side.

[0207] Purification of anti-CSF-1 monoclonal antibody. The anti -CSF-1 monoclonal antibody (clone 5A1) was purified from cell supernatant of a hybridoma (ATCC, #CRL- 2702)61, 62. Briefly, hybridoma cells were cultured in Hybridoma-SFM medium (Thermo #12045076) for 3 weeks to produce antibodies. Cell supernatant was collected for antibody purification using a LigaTrap Rat IgG purification column (LigaTrap Technologies #LT-138) according to the manufacturer’s procedure. Antibody quality and concentration were examined by SDS-PAGE gel electrophoresis and nanodrop, respectively.

[0208] In vivo antibody depletion experiment. Immune cell subsets were depleted by administering 200 pg of the listed monoclonal antibodies intraperitoneally twice weekly starting 1 day prior to the first viral injection as indicated and continuing until the animals either died, were euthanized, or were completely clear of tumors: anti-CD8-a for CD8+ T cells (clone 2.43, BioXCell), anti-CD4 for CD4+ T cells (clone GK1.5, BioXCell), anti- NK1.1 for NK cells (clone PK136, BioXCell). 500 pg of anti-Ly6G (clone 1A8, BioXCell) was given for neutrophil depletion. 500 pg of anti-CSFl (provided by Ming Li’s laboratory) was given every 5 days for macrophage depletion. Depletion efficacies of CD8 T cells, CD4 T cells, NK cells and neutrophils were confirmed by flow cytometry of peripheral blood.

(FIG. 15A)

[0209] Cell sorting for tumor infiltrating immune cells. Tumors were harvested and single cell suspensions were prepared. Cells were then stained with Cell Viability Dye eFluor506 (eBioscience) and anti-CD45 antibody Pacific blue (clone 30-F11, Biolegend). CD45+ live cells were then sorted using BD Aria cell sorter.

[0210] Single-cell RNA-sequencing and data analysis: Library preparation and sequencing. CD45+ populations from tumors were purified via FACS sorting and single-cell 5' gene expression profiling was performed on single-cell suspensions using a Chromium Single Cell V(D)J Solution from lOx Genomics according to the manufacturer’s instructions. Cell- barcoded 5' gene expression libraries were sequenced on an Illumina NovaSeq6000 sequencer with pair-end reads.

[0211] Single-cell RNA-sequencing and data analysis: Gene expression UMI counts matrix generation. The sequencing data was primarily analyzed by the 10x cellranger pipeline (v3.0.2) in two steps. In the first step, cellranger mkfastq demultiplexed samples and generated fastq files; and in the second step, cellranger count aligned fastq files to the reference genome and extracted gene expression UMI counts matrices. In order to measure both human and viral gene expression, a custom reference genome was built by integrating the MVA virus genome into the 10x pre-built mouse reference using cellranger mkref. The MVA virus genome was downloaded from NCBI.

[0212] Single-cell RNA-sequencing and data analysis: Single-cell RNA-seq data analysis. All cells expressing <200 or >6,000 genes were removed as well as cells that contained >5% mitochondrial counts. Samples were merged and normalized. The default parameters of Seurat were used, unless mentioned otherwise. Briefly, 2,000 variable genes were identified from the clustering of all cell types, and principal component analysis (PCA) was applied to the dataset to reduce dimensionality after regressing for the number of UMIs (counts). The top 12-15 most informative principal components (PCs) were used for clustering and Uniform Manifold Approximation and Projection for dimension reduction (UMAP). To characterize each cluster, both the FindAUMarkers and FindMarkers procedure in Seurat were applied, which identified markers using log fold changes (FC) of mean expression. To identify differentially expressed genes between two groups of clusters, the FindMarkers functions in Seurat and Enhanced Volcano R package (vl.8.0) were used. For subsequent T cell analysis, the CD3+ cells were first extract from the original dataset and then the clustering procedures were repeated in the T cell subset.

[0213] Single-cell RNA-sequencing and data analysis: Gene set enrichment analysis (GSEA). The GSEA analysis was done and visualized using the clusterProfiler (v4.0.5) and enrichplot (vl.12.3) packages, which use predefined gene sets from the Molecular Signatures Database (MSigDB v7.4) 63. For the GSEA analysis the hallmark gene sets collection was used. Genes were ranked by the test statistic value obtained from differential expression analysis and the pre-ranked version of the tool was used to identify significantly enriched biological pathways. [0214] Statistical analysis. Two-tailed unpaired Student’s t test was used for comparisons of two groups in the studies. Survival data was analyzed by log-rank (Mantel-Cox) test. The p values deemed significant are indicated in the figures as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The numbers of animals included in each study is indicated in the Detailed Description of the Drawings.

Example 1: Design of a Highly Immunogenic Second-Generation Recombinant MVA (MQ833).

[0215] The E5R gene of the MVA genome encodes a potent inhibitor of the DNA sensor cGAS. Infection of mouse bone marrow-derived dendritic cells (BMDCs) with MVA lacking the E5R gene (MVAAE5R) generates high levels of IFN-P production and promotes DC maturation, which is dependent on cGAS. Recombinant MVA (rMVA or MQ710), MVAAE5R-hFlt3L-mOX40L, was previously generated by inserting an expression cassette containing two transgenes encoding membrane-bound human Flt3L and murine OX40L linked by a P2A self-cleaving sequence into the E5R gene. Flt3L encodes a growth factor for DCs, and OX40L encodes a co-stimulatory ligand for OX40-expressing T cells. Intratumoral (IT) delivery of rMVA (MQ710) generates strong antitumor immunity in multiple tumor models, which is dependent on CD8 ⁺ T cells, the cGAS/STING pathway, and IFNAR feedback loop. IT rMVA (MQ710) also leads to depletion of intratumoral OX40 ¹¹¹ regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. To further potentiate the antitumor efficacy of the virus, a second-generation recombinant MVA (MQ833) was designed by removing two more viral immune evasion genes, E3L and WR199, and inserting the gene encoding interleukin 12 (IL- 12) anchored to extracellular matrix to prevent systemic toxicity (FIGs. 1A-1B).

Example 2: Vaccinia E3 and E5 synergize to inhibit the MDA5/STING-mediated nucleic acid-sensing pathways in tumor cell lines

[0216] The vaccinia E3L gene encodes a viral immune evasion factor that attenuates the cytosolic dsRNA sensing pathway. Deleting both the E5R and E3L genes in MVA synergistically boosted type I IFN production in multiple tumor cell lines (FIG. 8A). To assess whether this effect is dependent on the MDA5 and STING signaling pathways, Mda5' 'xi&MdaS' ¹ ' Sting' ¹ ' B16-F10 cell lines were generated using CRISPR-Cas9.

MVAAE3LAE5R infection resulted in elevated p-IRF-3 production in WT B16-F10 cells. However, in Mda5' ' tumor cells, p-IRF-3 induction was dramatically reduced. MVAAE3LAE5R failed to induce p-IRF-3 in MdaS' ¹ ' Sting' ¹ ' B16 cells (FIG. 8B). Additionally, MVAAE3LAE5R induced IFN-P secretion from B16-F10 cells was attenuated in MdaS' ¹ ' av6. Mda5' ' Sting' ¹ ' B16-F10 cells (FIG. 8C). These results indicate that MVAAE3LAE5R-induced IFN production in melanoma cells depends on MDA5 and STING-mediated pathways.

Example 3: Deletion of the E3L gene from MQ710 improves the generation of tumorspecific T cells in vivo

[0217] rMVA (MQ710) was sequentially depleted to generate rMVAAE3L and rMVAAE3LAWR199 (MQ832). rMVAAE3L and rMVAAE3LAWR199 (MQ832) were assessed for the capacity to generate antitumor T cells in vivo. In a bilateral B16-F10 murine melanoma model, 5 x 10 ⁵ and 2.5 x 105 B16-F10 cells were intradermally implanted into the right and left flanks, respectively. When the tumors were established, the larger tumors were injected twice, three days apart, with either heat-inactivated MVA (Heat-iMVA), rMVA (MQ710), or rMVAAE3L. PBS was used as a control (FIG. 9A). IT rMVA (MQ710) and rMVAAE3L generated higher levels of Gzmb ⁺ CD8 ⁺ T cells in the injected and non-injected tumors compared with heat-iMVA or PBS (FIG. 9B). IT rMVA (MQ710) and rMVAAE3L reduced 0X40+ Tregs in the injected tumors, whereas IT heat-iMVA failed to do so (FIG. 9C). ELISPOT results showed that rMVAAE3L treatment generated stronger antitumor T cell responses in the spleens than rMVA (MQ710) or Heat-iMVA (FIG. 9D).

[0218] The vaccinia gene WR199, also known as the MVA186 gene, encodes a 68-kDa protein consisting of N-terminal ankyrin repeats and a C-terminal F-box-like domain. WR199 protein interacts with the SCF (Skpl, cullinl, F-box-containing complex) via the F- box-like domain and is reported to be involved in poxvirus genome uncoating and DNA replication in mammalian cells. In addition to E5, WR199 protein was also identified as a cGAS inhibitor, although its mechanism of action is still under investigation. rMVAAE3LAWR199 (MQ832) was generated by deleting WR199 from rMVAAE3L. IT MQ832 generated slightly higher levels of Gzmb ⁺ CD8 ⁺ T cells and lower levels of OX40 ^hl Tregs than rMVAAE3L (FIG. 9E-9F).

[0219] Interleukin 12 (IL-12) is a well-studied cytokine for its stimulatory effect on T and NK cells and local delivery of IL-12 expressed by oncolytic viruses enhanced antitumor potential. IL-12 is a disulfide-linked heterodimer composed of p40 subunit encoded by the 1112b gene and p35 subunit encoded by the 1112a gene. To further potentiate the immunogenicity of our virus, an expression cassette with 1112b and 1112a linked by sequences encoding the P2A peptides under the control of a modified H5 promoter was inserted into the WR199 locus to generate the final product MVAAE3LAE5R-hFlt3L-hOX40LAWR199-IL- 12 (MQ833) (FIGs. 1A-1B). In addition, the p35 subunit of IL-12 is tagged with a matrixbinding sequence at the C-terminus to prevent its systemic release and associated toxicities (FIGs. 1A-1B)

Example 4: MQ833 activates the cGAS/STING mediated DNA-sensing pathway and promotes IFN production

[0220] The expression of the hFlt3L, hOX40, and hIL-12 transgenes was verified by flow cytometry and ELISA (FIGs. 1C-1D). Infection of B16-F10 murine melanoma cells with MQ833 results in the robust expression of hFlt3L and mOX40L on the surface of infected cells at 16 h post-infection (FIG. 1C). MQ833 infection of B16-F10 cells also resulted in the secretion of mIL-12 as determined by ELISA of mIL-12p40 (FIG. ID). Infection of MQ832 and MQ833 resulted in strong induction of Ifnb gene expression and IFN-P production in B16-F10 cells (FIGs. 1E-1F). Western blot analyses showed stronger induction of phosphorylation of STING and IRF3 in MQ832 and MQ833 -infected B16-F10 cells compared with MVA (FIG. 1G). These findings indicate that the triple deletion of E5R, E3L, and WR199 caused dramatic enhancement of the ability of the virus to induce type I IFN production and proinflammatory cytokines and chemokines from infected tumor cells in vitro. In addition, both MQ832 and MQ833 infection in BMDC led to high levels of IFN-P production in a STING-dependent manner (FIG. 1H). MQ833 infection in BMDCs resulted in activation of the cGAS/STING pathway and elevated p-IRF3 expression (FIG. II).

[0221] To test whether IL-12 expression by MQ833 promotes antigen-specific T cell activation, CD1 lc ⁺ splenic DCs isolated from WT or cGAS' ^/_ mice were infected with MVA, MVAAE5R, or MQ833 at a MOI of 3. Cells were then pulsed with OVA and washed after three hours, and then incubated with OT-1 cells at a DC:T cell ratio of 1 :3. Supernatants were collected and IFN-y levels in the supernatants were determined by ELISA (FIG. 1 J). MQ833 infection of DCs resulted in much higher levels of IFN-y production compared with MVAAE5R (FIG. IK). Whereas MVAAE5R-induced OT-1 T cell activation was largely dependent on cGAS, MQ833 -induced OT-1 cell activation was slightly reduced when co- cultured with cGAS' ^/_ DCs (FIG. IK). These results demonstrated that the IL- 12 in MQ833 is functional, and MQ833-induced antigen cross-presentation is only partially dependent on cGAS. Flt3L-cultured BMDCs were also generated and sorted for CD103 ⁺ DCs for an antigen cross-presentation assay. As observed in splenic CD1 lc ⁺ DCs, infection of cultured CD103 ⁺ DCs with MQ833 promoted antigen cross-presentation and antigen-specific T cell activation at a much higher level than MVAAE5R (FIGs. 1J-1L). To test whether MQ833- induced T cell activation requires DC and T cell contact, supernatants were collected from infected BMDCs and then incubated with OT-1 cells for three days. Supernatants from BMDCs infected with MQ833 stimulated OT-1 T cells to produce IFN-y, but the OT-1 T cells produced much lower levels of IFN-y without DCs when compared with DC/T cell coculture (FIGs. 1J, IM).

Example 5: Validation of hMQ833 (MVAAE5R-hFlt3L-hOX40L-AE3L-AWR199-hIL- 12)

[0222] A humanized MQ833 (hMQ833) was generated by substituting mOX40L and mlL- 12 with their human counterparts. Infection of human monocyte-derived DCs (moDCs) with hMQ833 resulted in the expression of hFlt3L and hOX40L on the cell surface of infected cells (FIG. 10A) at similar levels as hMQ710 (MVAAE5R-hFlt3L-hOX40L). ELISA analysis of the supernatants revealed high levels of IL-12p70 protein secreted by moDCs infected by hMQ833 but not by hMQ710 (FIG. 10B). Infection of moDCs with hMQ833 resulted in stronger induction of IFNB and CXCL10 gene expression than hMQ710 determined by RT-PCR analysis (FIG. 10C).

[0223] To test whether human IL-12 (hIL-12) produced by hMQ833 -infected moDCs is functional, a co-culture experiment was performed in which human moDCs were infected with either MV A, hMQ710, mMQ833, or hMQ833 for 1 h, then cells were washed to remove the viruses and co-cultured with either allogenic or autologous CD4 or CD8 T cells at a DC:T ratio of 1 :5. Supernatants were collected at 72 h post co-culture. IFN-y levels in the supernatants were measured by ELISA (FIG. 10D). In the allogenic DC and T cell coculture conditions, hMQ833 infection of moDCs resulted higher levels of IFN-y secretion by activated T cells compared with mMQ833. Allogenic CD4 T cells secreted more IFN-y than CD8 T cells. Neither MVA nor hMQ710 promoted IFN-y secretion. These results indicate that both hIL-12 produced by moDCs infected with hMQ833 and mIL-12 produced by moDCs infected with mMQ833 were able to stimulate allogenic CD4 or CD8 T cells to produce IFN-y, with the hIL-12 being more potent than mIL-12.

[0224] In the autologous DC and T cell co-culture conditions, IFN-y levels were lower than those observed in the allogeneic DC and T cell co-culture, and autologous CD4 and CD8 T cells, once stimulated by DCs, produced similar levels of IFN-y. hIL-12 was more potent than mIL-12 in stimulating autologous CD4 and CD8 T cells to produce IFN-y (FIG. 10E).

Example 6: Intratumoral delivery of MQ833 expressing IL-12 generates more potent antitumor effects than MQ832

[0225] The antitumor effects of MQ833 and MQ832 were compared in a murine B16-F10 melanoma model. Briefly, B16-F10 murine melanoma cells were implanted intradermally in C57BL/6J mice. MQ833 or MQ832 was injected into the tumors twice weekly (4 x 10 ⁷ pfu per mouse per injection) and tumor sizes were measured, and mouse survival was monitored (FIG. 2A). Intratumoral delivery of MQ833 generated a 100% cure in B16-F10 tumorbearing mice, significantly higher than the 40% survival rate generated by MQ832 treatment (FIGs. 2B-2D) IT MQ833 also resulted in 100% cure in a MC38 murine colon cancer model, compared with 60% cure in the group of mice treated with MQ832 (FIGs. 11A-11B). These results indicate that in a unilateral tumor model, IT MQ833 treatment resulted in better antitumor efficacy than MQ832. In a large tumor, bilateral MC38 tumor implantation model, IT MQ833 was found to be more effective than MQ832 in delaying tumor growth and extending median survival time (FIGs. 11C-11E).

[0226] A bilateral B16-F10 implantation model was used to test the abscopal antitumor response induced by IT MQ833 and whether that response could be enhanced with the combination of systemic delivery of ICB. Briefly, 7.5 x 10 ⁵ and 1.5 x 10 ⁵ B16-F10 cells were implanted intradermally into the right and left flanks of C57BL/6J mice. MQ833 or MQ832 was injected into the tumors on the right flank twice weekly. IT MQ833 eradicated most of injected tumors, significantly delayed distal non-injected tumor growth, and generated a 40% cure in the bilateral model, whereas tumor-bearing mice treated with IT MQ832 had no survivors (FIGs. 12A-12C). Combination therapy of IT MQ833 with intraperitoneal delivery of anti-PDl, anti-PD-Ll, and anti-CTLA-4 antibodies resulted in a survival rate of 60% (FIGs. 12A-12C). Collectively, these results indicate that IT MQ833 generates stronger abscopal effects in noninjected tumors than MQ832, and the treatment effect of MQ833 can be synergized with systemic delivery of ICB.

[0227] Modulation of tumor-infiltrating T cells was assayed for using a bilateral B16-F10 tumor implantation model, wherein MQ833 or MQ832 was intratumorally delivered into the larger tumors on the right flanks twice, three days apart. Both the injected and non-injected tumors were harvested two days after the second injection. Both MQ833 and MQ832 treatment resulted in the increase of Granzyme B ⁺ CD8 ⁺ and KLRG1 ⁺ CD8 ⁺ T cells and reduction of Foxp3 ⁺ regulatory CD4 ⁺ T (Tregs) cells. IT MQ833 was more effective in reducing Tregs in both injected and non-injected tumors than MQ832 (FIGs. 2E-2F and FIGs. 13A-13C)

[0228] Accordingly, these results demonstrate that the engineered poxviruses of the present technology are effective in methods for treating both directly injected and distant solid tumors in a subject in need thereof.

Example 7: MQ833-induced antitumor efficacy is dependent on STING/MDA5- mediated nucleus acid-sensing pathways and STAT2-mediated type I IFN signaling pathway

[0229] To test whether MQ833 antitumor effects would be reduced in mice deficient for cytosolic DNA and dsRNA sensing pathways, B16-F10 melanoma cells were implanted into the right flanks of WT, Sting ^GlG Mda5' and Sting^^MdaS' ¹ ' mice. IT MQ833 treatment resulted in 81% cure in WT mice and 45%, 46%, and 40% cure in Sting ^{G IG} MdaS' ¹ ', and Sting^^MdaS' ¹ ' mice, respectively (FIGs. 2G-2H). These results indicate that both the cytosolic DNA and dsRNA-sensing pathways in the tumor-bearing mice are important for IT MQ833-induced antitumor effects.

[0230] The type I IFN signaling pathway is important for rMVA (MQ710)-induced antitumor effects. Tumor-bearing Stat2' ^! ' mice also responded poorly to IT MQ833 treatment (FIGs. 2G-2H) with only an 11% cure rate, confirming the importance of IFNAR/STAT2 pathway in viral -induced antitumor immunity. Example 8: Single-cell RNA sequencing (scRNA-seq) approach to interrogate the remodeling of tumor immune microenvironment (TIME) after IT delivery of MQ833

[0231] Several scRNA-seq experiments were conducted with the following objectives: (i) to investigate the impact of IT MQ833 on TIME (tumor immune microenvironment); (ii) to elucidate the roles of nucleic acid-sensing and IFNAR/STAT2 signaling pathways in regulating the transcriptomes of the myeloid and T cell populations; and (iii) to examine the influence of IL- 12 expressed in MQ833 -injected tumors on modulating innate and adaptive immune responses.

[0232] B16-F10 tumors were implanted intradermally in WT, Stin^^MdaS' ¹ ', and Stat2' ^! ' mice and then harvested two days after one dose of IT MQ833. CD45 ⁺ immune cells were sorted from the tumors and scRNA-seq analysis was performed (FIG. 3A). Unsupervised clustering revealed a total of 19 distinct clusters among the tumor-infiltrating CD45 ⁺ immune cells, including four T and NK cell clusters (Cl -4), seven monocyte-macrophage clusters (C5-11), one neutrophil cluster (C12), four DC clusters (C13-16), and one B cell cluster (Cl 7). Small populations of undefined cells (Cl 9) and melanoma cell contamination (Cl 8) were observed (FIGs. 3B-3C). To verify the annotation for each cluster, the expression score of a set of known cellular markers was computed for each cell type. Expression of markers for T cells (Cd3d, Cd3g, Trbc2 regulatory T cells (Foxp3, Tnfrsf4, Tirfrsfl8\ NK cells (Ncrl, Nkg7, Prfl, Xcll, Gzma . DCs (Itgax, H2-Ebl, Ccr7, Siglech, Wdfy4 M2-like macrophages (Apoe, Clqa, Mrcl, Pf4), Ml -like macrophages (CxcllO, Chil3, Plac8, Ly6c2 inflammartoy monocytes (Fcgrl, Ill8bp, Ccll2, Ccl7), and neutrophils (S100a9, S100a8, Cxcl2) all correlated well with the cluster annotation (FIG. 3C, FIG. 14A).

[0233] At two days post MQ833 injection, rapid recruitment of inflammatory monocytes (C5) and neutrophils (C12) into the injected tumors was observed. The percentages of C5 and C12 out of CD45+ immune cells expanded from 1% to 29% and 0.3% to 12%, respectively (FIGs. 3D-3E). The percentages of M2-like resident macrophages decreased from 13% to 0.6%, whereas the percentages of Ml -like macrophages increased from 8.3% to 17% (FIGs. 3D-3E). Four DC clusters were identified, cDCl, cDC2, pDC, and migrating DCs. The percentages of cDCls in the tumors was reduced from 4.7% to 0.5% after IT MQ833 treatment, suggesting that activation of cDCl facilitates its migration to the draining lymph nodes (FIGs. 3D-3E). [0234] IT MQ833 treatment of tumors in Sting^^MdaS' ¹ ' (DKO) mice resulted in a dramatic reduction of recruited neutrophils compared with the treatment result in WT mice (2% in DKO vs. 12% in WT mice), a modest reduction of inflammatory monocytes recruitment (20% in DKO vs. 29% in WT mice), and a less efficient depletion of M2-like macrophages (6% in DKO vs. 0.6% in WT mice), although Ml-like macrophage polarization/activation was similar to that in WT mice (FIGs. 3D-3E). IT MQ833 treatment resulted in reduced cDCl cells from 6.2% to 2.1% in DKO mice, suggesting less efficient activation of cDCl in DKO mice compared with WT mice (FIGs. 3D-3E).

[0235] Tumor-infiltrating CD45 ⁺ immune cells in Stat2' ¹ ' mice showed some unique features compared with WT mice. Two M2-like resident macrophage (CIO) and M2-like proliferating macrophage clusters (Cl 1) were observed that were unique to Stat2' ^! ' mice in PBS mock-treated tumors. Upon IT MQ833 treatment, these two populations decreased from 21% to 0.1% and from 8.5% to 2% respectively. By contrast, IT MQ833 treatment resulted in a dramatic increase of Ml-like macrophage (C9) in Stat2' ^! ' mice from 0.7% to 46%, as well as an increase of neutrophils (C12) from 0.17% to 16.5% (FIGs. 3D-3E).

[0236] Gene set enrichment analysis (GSEA) of differentially expressed genes in CD45 ⁺ immune cells sorted from MQ833-treated tumors and from PBS mock-treated tumors revealed strong induction of genes involved in the IFNA and IFNG responses and TNF signaling post MQ833 treatment, followed by IL6/JAK/STAT3 signaling, apoptosis, hypoxia, complement, and IL2/STAT5 signaling pathways (FIGs. 3F-3G). On the other hand, genes involved in oxidative phosphorylation and Myc targets were downregulated after MQ833 injection (FIGs. 3F-3G).

[0237] A heatmap of the expression of genes involved IFNA and IFNG responses showed that IT MQ833 induced the expression of a large array of IFN-stimulated genes in CD45 ⁺ immune cells from WT mice and that such responses were dramatically attenuated in DKO mice and absent in Stat2' ¹ ' mice (FIG. 3H). For example, Ifihl (which encodes MDA5) was confirmed to be absent in CD45 ⁺ cells from DKO mice. IT MQ833 induced the expression of Ifihl in WT mice, but not in Stat2' ¹ ' mice.

[0238] Similarly, Zbpl and Adar expressions were attenuated in DKO mice and absent in Stat2' ^! ' mice. Other examples with similar differential expression patterns include transcription factors Stat2 x\&Irf7, cytokines and chemokines such as CxcHO, Ccl7, 116, and III 5, and ISGs including Rsad2, Isgl5, Ifit3, and Ifitm3. B2m expression was reduced in DKO cells and absent in Stat2' ¹ ' cells (FIG. 3H).

[0239] Among all the cell clusters, neutrophils were one of the most heavily infected cells by MQ833 injection (FIGs. 14B-14C). Therefore, the analysis was refined on the neutrophil cluster. Differential gene expression was analyzed in recruited neutrophils from WT mice after IT MQ833 vs. PBS treatment. B2m, Isgl5, Gbp2, IfH3, CxcllO, and Irf7 were among the top regulated genes (FIGs. 3I-3J). The comparison of gene expression between neutrophils from WT and Stat2' ^! ' mice showed that neutrophils were sensitive IFN stimulation and expressed innate immune sensors, cytokines and chemokines, ISGs and activation markers (FIGs. 3I-3J). Interestingly, MQ833 induced expression of antitumor- related genes from neutrophils such as Ifrig, Nos2, Fas, III 2a, Cxcll, and CxcllO, with said induction partially dependent on the MDA5/STING signaling pathway and IFNAR/STAT2 positive feedback loop.

[0240] Collectively, these results demonstrate that IT MQ833 reprograms the tumorinfiltrating myeloid cell compartment by recruiting inflammatory monocytes and neutrophils, depleting M2-like macrophages, and polarizing Ml-like macrophages. IT MQ833 also activates neutrophils to express antitumor genes, including Nos2, in a MDA5/STING and STAT2-dependent fashion.

[0241] Accordingly, these results demonstrate that the engineered poxviruses of the present technology are effective in methods for altering the TIME in a tumor in a subject in need thereof.

Example 9: IT MQ833 delivery remodels the T cell compartment of TIME

[0242] A sub-clustering analyses of CD3 ⁺ T cells from the same scRNA-seq dataset was performed, which identified eight T-cell clusters including Tcf7 ⁺ Stem-like CD4 ⁺ T cells (Cl), Tcf7 ⁺ progenitor T cells (C2), Tcf7 ⁺ ISGhiCD8 (C3), Early effector CD8 (C4), Effector CD8 (C5), Proliferating T cells (C6), Thl CD4 T cells (C7), and Tregs (C8) (FIGs. 4A-4B). Cl expressed Tcfl, Il7r, Lars2, and low CD4, but no activation markers, representing stemlike CD4 ⁺ T cells in the tumors (FIG. 4C). Cl was enriched in PBS mock-treated tumors from Stat2' ¹ ' mice, which was reduced after IT MQ833 (FIGs. 4A-4B). C2 expressed Tcf7, Cd7, Klra7, Gzma, but lacked CD4 and CD8 expression (FIGs. 4A-4C) and was designated as Tcf7 ⁺ progenitor T cells or CD8 Naive-like. C3 was designated as TCF ⁺ ISGhiCD8 because it expressed Tcf7, Isgl5, Ifitl, Ifit3, but not Gzma, Gzmb or Ifng (FIGs. 4A-4C). Cl- C3 had the highest Tcf7 expression and had stem-like features. C4 was designed as early effector CD8, because it not only expressed CD8a, Ifgl5, fu3. but also low levels of Gmzb and Ifng. C5 was designed as effector CD8 because it had the highest expression of Gzma, Gzmb and Ifng among all of T cell subsets. C6 was designated as proliferating CD8 T cells because it had the highest expression of Mki67. Top2a, and Histlhib. C7 expressed CD4, H7r, Cxcr6. and CD40lg and was designed as Thl CD4 ⁺ T cells. Finally, C8 was designated as Tregs due to its expression of CD4, Foxp3, Tnfrsf4 (encoding 0X40), and Tnfrsfl8 (encoding GITR) (FIGs. 4A-4C).

[0243] In WT mouse tumors treated with IT MQ833, Cl, C2, and C3 populations expressing Tcf7 decreased from 5%, 37%, and 19% in the PBS mock-treated tumors to 1%, 10%, and 10%, respectively (FIGs. 4B-4D). In contrast, Early effector CD8 (C4), Effector CD8 (C5) and Proliferating CD8 (C6) increased from 6.6%, 3.2%, 9.0% in PBS mock-treated tumors to 10%, 20%, and 28%, respectively in IT MQ833 treated tumors (FIGs. 4B-4D). In tumors from DKO mice, an increase of Cl from 2% to 5%, a decrease of C3 from 21% to 15%, and no significant change of C2 (at 14%) was observed upon MQ833 treatment (FIGs. 4B-4D). Early effector CD8 (C4) decreased from 28% to 12% upon MQ833 treatment, while Effector CD8 (C5) and Proliferating CD8 (C6) increased from 1.5% and 6.2% to 4.8% and 20.4% in DKO mice (FIGs. 4B-4D). These results indicate that MQ833-induced differentiation of Tcf7 ⁺ stem-like T cells and generation of Early effector CD8+ (C4) and Effector CD8 ⁺ T cells (C5) was attenuated in DKO mice, whereas the generation of proliferating CD8 ⁺ T cells were not severely affected in DKO mice.

[0244] In PBS mock-treated tumors from Stat2' ^! ' mice, the Tcf7 ⁺ Stem-like CD4 T cells (Cl) and Tcf7 ⁺ progenitor T cells (C2) made up 51% and 15% of all CD3+ T cells respectively (FIGs. 4B-4D). By contrast, Tcf7 ⁺ ISGhiCD8 (C3), Early effector CD8 (C4), and Effector CD8 (C5) were nearly absent in tumors from Stat2' ^! ' mice, indicating the essential role of type I IFN signaling in the differentiation of Tcf7 ⁺ stem-like T cells into Tcf7 ⁺ ISGhiCD8 (C3), Early effector CD8 (C4), and Effector CD8 (C5) (FIGs. 4B-4D). Upon MQ833 -treatment, the Cl population decreased from 51% to 11% in Stat2-/~ mice, the proliferating CD8+ T cells (C6) population increased from 6% to 27%, while C3, C4 and C5 were still absent, suggesting that in the absence of IFNAR/STAT2 signaling, (FIGs. 4B-4D), IL-12 alone cannot drive the differentiation of Tcf7 ⁺ progenitor T cells (C2) into transitory effector (C3 and C4) and effector CD8 T cells (C5). In addition, pseudotime and cell trajectory analysis revealed that MQ833 treatment induced a gradual shift of the T cell transcriptomic profile towards decreased sternness and increased effector function and early exhaustion (FIGs. 4E-4F).

[0245] Accordingly, these results demonstrate that the engineered poxviruses of the present technology are effective in methods for altering the TIME in a tumor in a subject in need thereof.

Example 10: scRNA-seq in bilateral B16-F10 tumor implantation model

[0246] scRNA-seq was performed using the bilateral tumor implantation model in which the larger tumors were injected with MQ833 twice three days apart, while the smaller tumors were not injected. Two days after the second injection, both the injected and non-injected tumors were harvested, and scRNA-seq of tumor-infiltrating CD45 ⁺ cells was performed (FIG. 5A). Unsupervised clustering revealed a total of 14 distinct clusters, including four T and NK cell clusters (Cl -4), five monocyte-macrophage clusters (C5-9), one neutrophil cluster (CIO), three DC clusters (Cl 1-13), and one B cell cluster (C14) (FIGs. 5B-5C).

[0247] The neutrophil population (CIO) increased from 0.65% in PBS mock-treated tumors to 15% in tumors after two injections of MQ833 and to 0.5% in the contralateral non-injected tumors (FIGs. 5D-5E). The inflammatory monocyte population (C5) increased from 1.8% in PBS mock-treated tumors to 10.4% in the injected tumors and 12% in the non-injected tumors (FIGs. 5D-5E). The Ml -like macrophage population (C6) increased from 11% in PBS mock-treated tumors to 16% in the injected tumors and 12% in the non-injected tumors, whereas the ISG+ macrophage cluster (C7) increased from 0.3% to 4.8% in the injected tumors and 28% in the non-injected tumors (FIGs. 5D-5E). By contrast, the M2-like macrophage population (C8) decreased from 14.5% in PBS mock-treated tumors to 5.3% in the injected tumors and 2.5% in the non-injected tumors (FIGs. 5D-5E). These results indicate that IT MQ833 extensively remodeled the TIME, with recruitment of neutrophils to the injected but not to the non-injected tumors, expansion of ISG ⁺ macrophages and depletion of M2 -like macrophages in both the injected and non-injected tumors.

[0248] Sub-clustering analyses of CD3 ⁺ T cells revealed a total of 9 clusters (FIGs. 5F- 5G). Cluster 1, representing Tcf7 ⁺ stem-like or progenitor T cells, expressed 7b 7, IL7r, Klf2, and Ikzf2 and lacked Cd4 and Cd8a expression. Cluster 2 expressed Cd8bl, Ccl5, Isgl5, Ifit3. Tc , and Zfp36l2, representing Tcf7 ⁺ ISGhiCD8 ⁺ T cells. Cluster 3 expressed Cd8a, Ilgbl. I)ennd4a. Ccr5, and H2rb. representing resident memory CD8 T cells. Cluster 4 expressed Cd8bl, Gzmb. Gzma. Gzmk. If ng. Ccl5, and Klrgl, representing effector CD8 T cells. Cluster 5 expressed CD8a, Tax. Pdcdf Haver 2. Nkg7, Klrdl. and If ng, representing stimulated CD8 ⁺ T cells expressing exhaustion markers. Cluster 6 expressed CD8a, Mki67, Top2a, His Hi lb, and Ifhg, representing proliferating CD8 ⁺ T cells. Cluster 7 expressed CD4, Tcf7, Il7r, IfH3, and Cd40lg, representing Thl CD4 T cells. Cluster 8 expressed Cd4, Foxp3, Tnfrsf4, Tnfrsfl8, representing Tregs. Cluster 9 expressed Lyz2, CD74, IfilmS. Feer 1 g, Ms4a6c, H2-Abl, H2-Aa, Cxcl2, representing activated CD3 ⁺ macrophages (FIGs. 5F-5G).

[0249] Upon IT MQ833 treatment, the Tcf7 ⁺ stem-like or progenitor T cells population (Cl) decreased from 35% in the PBS mock-treated tumors to 11.5% in MQ833 -injected tumors and to 17% in MQ833-non-injected tumors (FIGs. 5H-5I). Tcf7 ⁺ ISGhiCD8 ⁺ T cells (C2) population decreased from 8.2% to 0.8% in the injected tumors and to 2.4% in the noninjected tumors (FIGs. 5H-5I). In addition, Resident memory CD8 ⁺ T cells (C3) increased from 3.4% in the PBS -treated tumors to 7.3% in the injected tumors and to 4% in the noninjected tumors (FIGs. 5H-5I). By contrast, Effector CD8+ cells (C4) increased from 0.8% in PBS mock-treated tumors to 9% in injected tumors and to 26% in the non-injected tumors, whereas Stimulated CD8+ T cells expressing exhaustion markers (C5) increased from 1.1% to 27% in the injected tumors and to 2% in the non-injected tumors (FIGs. 5H-5I).

Proliferating CD8+ T cells (C6) also increased from 2.2% to 12.5% in the injected tumors and 9% in the non-injected tumors (FIGs. 5H-5I). On the other hand, Thl CD4+ T cells expressing 7b 7, H7r, and Cd40lg (C7) decreased from 33% to 7.4% in the injected tumors and to 16% in the non-injected tumors (FIGs. 5H-5I). In addition, Tregs (C8) decreased from 11.8% to 4% in the injected tumors and 7.5% in the non-injected tumors (FIGs. 5H-5I). Finally, the unique CD3+ population expressing macrophage markers (C9) expanded from 4.2% to 20% in the injected tumors and to 16% in the non-injected tumors (FIGs. 5H-5I). These results indicate that after two injections with MQ833, effector CD8 ⁺ T cells (C4) undergo expansion in the injected tumors and perhaps traffick to the non-injected tumors to elicit abscopal effects. Stimulated CD8 ⁺ T cells expressing exhaustion markers (C5) became the largest population in the injected tumors after IT MQ833. Example 11: MQ833-induced antitumor efficacy is mediated by both innate and adaptive immune systems

[0250] To dissect the role of different immune cells in the MQ833-induced antitumor response, an antibody depletion study using the unilateral B16-F10 tumor implantation model was performed. Briefly, B16-F10 melanoma cells were implanted intradermally into the right flanks of C57BL/6J mice. After the tumors were established, depleting antibodies for CD8 (anti-CD8), CD4 (anti-CD4), NK (anti-NKl. l), neutrophils (anti-Ly6G), and macrophages (anti-CSF) were delivered intraperitoneally twice weekly, one day prior to IT MQ833 treatment. Tumor sizes were measured and survival was monitored for treated mice over time (FIG. 6A).

[0251] Flow cytometric analysis of peripheral blood confirmed the efficacy of antibody depletion efficacy for anti-CD4, CD8, NK1.1, and Ly6G (FIGs. 15A-15C). Anti-CSFl antibodies have been shown to deplete macrophages in the tumors. MQ833 treatment alone yielded a 55% survival rate. CD8 ⁺ T cell depletion abrogated the antitumor effects resulting in no survivors, but treatment still extended the median survival from 11.5 days in PBS mock-treated mice to 30.5 days in MQ833 + aCD8 group (p < 0.0001), suggesting that other immune cells also contribute to virus-induced antitumor efficacy (FIGs. 6B-6C).

Surprisingly, neutrophil depletion significantly impaired the overall therapeutic efficacy, resulting in 10% survival with a median survival of 24 days (p < 0.0001; PBS vs. MQ833 + aLy6G) (FIGs. 6B-6C). Macrophage depletion with aCSFl also resulted in attenuated antitumor effects from MQ833 treatment, with a 10% survival rate and a median survival of 34.5 days (p < 0.0001; PBS vs. MQ833 + aCSFl) (FIGs. 6B-6C). Depleting CD4 ⁺ T cells or NK cells did not negatively impact the antitumor efficacy (FIGs. 6B-6C), although it has previously been shown that CD4 ⁺ T cells are critical for generating memory T cell responses for virus-induced antitumor immunity. Collectively, these findings demonstrate that MQ833- induced antitumor immunity is mediated by both innate and adaptive immunity, and that CD8 ⁺ T cells, neutrophils, and macrophages are the most crucial players.

Example 12: Nos2 is required for MQ833-induced antitumor effects

[0252] The role of inducible nitric oxide synthase (iNOS or Nos2) in promoting or restricting tumor growth is complicated and context-dependent. iNOS protein expression was analyzed in neutrophils and macrophages in tumors harvested two days after a second injection with MQ833 using flow cytometry analysis (FIG. 6D). Intracellular iNOS protein levels were strongly induced in tumor-infiltrating neutrophils and Ml-like macrophages and were reduced in CD206 ⁺ M2-like macrophages upon MQ833 treatment (FIG. 6E-6F).

[0253] B16-F10 melanoma cells were implanted into the right flanks of NOS2' ¹ ' mice and age-matched WT C57BL/6J mice to analyze the importance of Nos2 for MQ833-induced antitumor effects. Tumors were treated with IT MQ833 twice weekly or PBS as a control. IT MQ833 resulted in a 50% cure rate in WT mice and only 10% cure rate in NOS2' ¹ ' mice (FIGs. 6G-6H). IT MQ833 extended the median survival from 14 days in PBS mock-treated mice to 61 days in WT mice (p < 0.0001). However, in Nos2' ^! ' mice, IT MQ833 extended the median survival from 17 days to in PBS mock-treated mice to 40 days (p = 0.0003). In PBS mock-treated mice, Nos2' ^! ' mice died 3 days later than their WT controls (p = 0.029). Our results indicate that Nos2 is required for MQ833-induced antitumor effects, but in the absence of viral treatment, Nos2 plays a pro-tumor role. To rule out that the Nos2 requirement is specific for B 16-F10 melanoma model, the therapeutic efficacy of IT MQ833 for MC38 murine colon cancer in Nos2' ^! ' and age-matched WT mice was compared. It was observed that IT MQ833 generated 100% cure in WT mice but had reduced efficacy in Nos2' ^! ' mice (FIG. 11F-11G)

[0254] To determine whether IL-12 expression by MQ833 contributes to Nos2 gene expression, scRNA-seq analysis was performed on CD45 ⁺ immune cells sorted from tumor samples harvested at 2 days post one injection of MQ833, rMVAAE3L, or PBS. Unsupervised clustering analysis revealed a total of 14 clusters. MQ833 and rMVAAE3L treatment reduced M2-like macrophages from 16% to 3.4% and 5%, respectively and increased inflammatory monocytes from 0.7% to 25% and 30%, respectively. MQ833 and rMVAAE3L treatment also increased neutrophils from 0.3% to 13% and 18%, respectively (FIGs. 16A-16C)

[0255] CD3 ⁺ T cell sub -clustering analysis revealed that upon MQ833 and rMVAAE3L treatment, TCF7 ⁺ Stem-like T cells (Cl) decreased from 32% to 2% and 7% respectively (FIGs. 16D-16G). The most striking difference is that the C5 effector memory T cell population (expressing I.ars2. Vps37b. Salbl. Plac8, Emb, Dennd4a) increased from 0.2% to 40% in MQ833 -treated tumors and remained unchanged in rMVAAE3L-treated tumors. In addition, the C7 effector CD8 ⁺ T cell population (expressing Nkg7, Lag3, Ifrig, Cxcr6, Cd-l, Gzmb) also increased from 6% to 13.6% in MQ833-treated tumors and decreased to 4% in rMVAAE3L-treated tumors. By contrast, the C6 population (expressing Mki67 and other cell proliferation markers) increased from 4.7% to 12% and 10.5% after MQ833 and rMVAAE3L treatment, respectively. These results suggest that IL12-expressing MQ833 is more efficient in promoting the expansion of effector memory T cells and effector CD8 ⁺ T cells than rMVAAE3L.

[0256] Indeed, Nos2 expression was upregulated in neutrophils and Ml-like macrophages and monocytes after IT MQ833 treatment (FIG. 61). Heatmap of differential gene expression involved in antitumor activities showed that MQ833 treatment was more effective than rMVAAE3L in inducing Fas, Tnf, Ifng, Nos2, and CxcllO in CD45, neutrophils, Ml-like macrophages and monocytes in the tumor microenvironment (FIG. 6J). Flow cytometry analysis revealed that iNOS expression in neutrophils was reduced in MQ833-treated tumors from Statl' ¹ ' mice compared with WT controls, suggesting that IFNGR signaling might also play a role in iNOS induction (FIGs. 6J-6L). Taken together, these results suggest that the nucleic sensing pathways, and IFNAR and IFNGR signaling on neutrophils might be important for promoting iNOS expression and ultimately the production of nitric oxide for tumor cell killing.

[0257] To gain deeper insights into the dynamics of neutrophils and monocytes in B16-F10 tumors following MQ833 treatment, tumor samples were collected at Days 1, 2, and 3 post IT MQ833 and flow cytometry analyses were conducted. Neutrophils were observed to be recruited on Day 1 after MQ833 injection and exhibited activation on both Day 1 and Day 2 (FIG. 6M). On the other hand, more monocytes were recruited to the treated tumors on Day 2, with activation occurring on Day 1 and Day 2 (FIG. 6M). These findings indicate that neutrophils act as first responders to MQ833 infection, while monocytes are recruited subsequently. Understanding these temporal dynamics provides crucial insights into the early immune response triggered by MQ833 in the tumors. A tumor killing assay was performed by stimulating bone marrow neutrophils from WT or NOS2' ¹ ' mice with IFN-beta (100 ng/ml), IFN-gamma (100 ng/ml), or both, for 24 h. Cells were washed to remove cytokines and then co-incubated to B 16-F10 melanoma cells, with a neutrophil to B 16 ratio of 10: 1. Tumor cells were collected at 48 h and plated to determine colony forming units. WT neutrophils exhibited potent tumor killing after they were stimulated with IFN-beta, IFN-gamma, or both (FIG. 6N). By contrast, Nos2' ^! ' neutrophils had weak tumor killing function only when they were stimulated with IFN-beta, IFN-gamma (FIG. 6N). Example 13: IT MQ833 delays tumor growth in the absence of adaptive immune system

[0258] A Ragl' ¹ ' mouse model was used to test whether IT MQ833 generates antitumor effects in the absence of mature T and B cells. Briefly, B16-F10 melanoma cells were implanted intradermally into the right flanks of Ragl' ¹ ' and age-matched WT controls. When the tumors grew to 3 mm in diameter, they were injected with MQ833 (4 x 10 ⁷ pfu) twice weekly or with PBS as a mock treatment control (FIG. 7A). IT MQ833 generated 10% cure in Ragl' ¹ ' mice and extended the median survival from 11 days in the PBS mock-treatment control to 42 days in the treatment group (p < 0.0001; FIGs. 7B-7C). Flow cytometry analysis of tumor-infiltrating myeloid cells in Ragl' ¹ ' mice revealed that more than 55% CD45 ⁺ cells in the MQ833 -injected tumors were neutrophils compared with 1.8% in PBS mock-injected tumors, and 31% of the enutrophils expressed iNOS (FIGs. 7D-7E). These results indicate that MQ833-induced neutrophil recruitment and activation is independent of mature T and B cells and IT MQ833 delays tumor growth in mice lacking an adaptive immune system.

[0259] Accordingly, these results demonstrate that the engineered poxviruses of the present technology are effective in methods for treating a tumor in a subject in need thereof, wherein the subject has a deficient adaptive immune system response.

Example 14: MQ833 cures B16-F10 tumor lacking MHC-I

[0260] Mutations in MHC Class I antigen presentation machinery have been observed in the tumors of patients that relapse after initial response to immune checkpoint blockade (ICB) inhibitors. Beta-2 microglobulin (B2M) is a component of the MHC-I molecule and is essential for MHC-I antigen presentation and B2M loss or downregulation is associated with acquired resistance to ICB therapy in melanoma patients. B2M-loss tumors are resistant to CD8 ⁺ T-cell mediated killing. To test whether IT MQ833 generated an antitumor effect independent of CD8 ⁺ T cells, cell line was generated using CRISPR-cas9. 7>2/7 ’ Bl 6-F I 0 cells were implanted intradermally into the right flanks of WT C57BL/6J mice. After the tumors were established, they were treated with either MQ833, MQ832, or PBS. IT MQ833 treatment eradicated 70% of the B2m' ¹ ' tumors, whereas IT MQ832 resulted no cure (FIGs. 7F-7G). When survivors were rechallenged with a lethal dose of WT B16 tumor cells two months later, all of the mice rejected the tumor rechallenge, indicating a long-lasting memory response induced by MQ833 treatment (FIG. 7H). These results highlight the potential application of MQ833 in treating ICB-resistant tumors in patients.

[0261] Accordingly, these results demonstrate that the engineered poxviruses of the present technology are effective in methods for treating a solid tumor in a subject in need thereof, wherein the tumor is resistant to immune checkpoint blockade inhibitor treatment and/or comprises a mutation or loss of the B2M gene, and/or is deficient in MHC-I presentation. These results further demonstrate that the engineered poxviruses of the present technology are effective in methods for preventing cancer recurrence for a period of time in a subject in need thereof.

Example 15: Materials and Methods for Examples 16-21

[0262] Animals were assigned to various experimental groups in random. For survival studies, sample sizes of 8 to 10 mice were used, and the experiments were performed for at least twice. For experiments designed to evaluate the tumor immune cell infiltrates, three to five mice were used for each experiment and the experiments were performed for at least two to three times.

[0263] Cell lines: BHK-21 (baby hamster kidney cell, ATCC CCL-10) cells were cultured in Eagle’s minimal essential medium containing 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, penicillin, and streptomycin. The murine melanoma cell line Bl 6- F10 was originally obtained from I. Fidler (MD Anderson Cancer Center). The A20 B cell lymphoma cell line were obtained from ATCC. Both B16-F10 and A20 were maintained in RPMI-1640 medium supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, penicillin, and streptomycin. B16 cell line expressing murine OX40L (mOX40L) or human Flt3L (hFlt3L) were created by transduction into B16 cells with vesicular stomatitis virus (VSV) G protein-pseudotyped murine leukemia viruses (MLV) containing pQCXIP-mOX40L or pQCXIP-hFlt3L. Cells were selected and maintained in growth media including 2 pg/ml puromycin for selection of stably transduced cells.

[0264] Viruses: The MVA virus was provided by G. Sutter (University of Munich). MVAAE5R, MVAAE5R-hFlt3L, MVAAE5R-mOX40L, rMVA (MVAAE5R-hFlt3L- mOX40L) and rhMVA (MVAAE5R-hFlt3L-hOX40L) were generated by transfecting pUC57-based plasmids into BHK-21 cells that were infected with MVA at MOI 0.05. Recombinant viruses were purified after 4~6 rounds of plaque selection based on the fluorescence marker. Viruses were propagated in BHK-21 cells and purified through a 36% sucrose cushion. PCR and DNA sequencing were performed to verify the purity of the recombinant viruses. Viral titers were determined using BHK-21 cells.

[0265] Mice: Female C57BL/6J mice and BALB/cJ between 6 and 10 weeks of age were purchased from the Jackson Laboratory (stock #000664 and stock #000651) were used for the preparation of BMDCs and for in vivo experiments. These mice were maintained in the animal facility at the Sloan Kettering Institute. All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Sloan Kettering Cancer Institute. Stin f ^/gt mice were generated in the laboratory of Dr. Russell Vance (University of California, Berkeley)(Sauer et al., 2011). Foxp3^ ^p , Foxp3 ^DTR , Foxp3 ^rFP ~ ^cre mice were generated in the laboratory of Dr. Alexander Y. Rudensky (Memorial Sloan Kettering Cancer Center) (Fontenot et al., 2005; Kim et al., 2007; Rubtsov et al., 2008). MMTV-PyMT mice were provided by Ming Li (Memorial Sloan Kettering Cancer Center)(Franklin et al., 2014). cGas~ ~ mice were generated in Herbert (Skip) Virgin’s laboratory (Washington University)(Schoggins et al., 2014). Ifnarl ¹ , Stall' Stall" and Ox40~ ~ were purchased from Jackson Laboratory. OX40~ ^/ 'Foxp3 ^sfp , Foxp3 ^cre Ifnar mice were bred in lab.

[0266] TIL isolation and flow cytometry: For TIL or myeloid cells analysis, tumors were minced prior to incubation with Liberase (1.67 Wiinsch U/ml) and DNasel (0.2 mg/ml) for 30 min at 37°C. Tumors were then homogenized by gentleMACS dissociator and filtered through a 70-pm nylon filter. Cell suspensions were washed and resuspended with complete RPMI. For cytokine production analysis, cells were restimulated with Cell Stimulation Cocktail (Thermo Fisher) and GolgiPlug (BD Biosciences) in complete RPMI for 6 h at 37°C. Cells were incubated with appropriate antibodies for surface labeling for 30 min at 4°C after staining dead cells with LIVE/DEAD™ Fixable Aqua Stain (Thermo Fisher). Cells were fixed and permeabilized using Foxp3 fixation and permeabilization kit (Thermo Fisher) for 1 hour at 4°C and then stained for Granzyme B, Foxp3, IFNy and TNFa. To analyze transgene expression, cells were infected with various viruses at a MOI of 10 or mock- infected. At 24 h post infection, cells were collected, and the cell viability was determined by labeling with LIVE/DEAD™ Fixable Aqua Stain (Thermo Fisher) 15 min at 4°C. Cells were then sequentially stained with hFlt3L primary antibody, PE-conjugated goat-anti-mouse IgG antibody and AF647-conjugated anti-mOX40L antibody at 4°C, 15 min for each step. For dendritic cell maturation assay, cells were infected with virus at a MOI of 10 and collected at 16 h post infection. Then cells were stained with anti-CD86 antibody for surface labeling for 30 min at 4 °C. LIVE/DEAD™ Fixable Aqua Stain (Thermo Fisher) was used to stain dead cells. Cells were analyzed using the BD LSRFortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Treestar).

[0267] Tumor challenge and treatment: For tumor immune cells analysis, B16-F10 cells were implanted intradermally into right and left flanks of the mice (5xl0 ⁵ to the right flank and 2.5x 10 ⁵ to the left flank). At 7 to 9 days after implantation, the tumors at the right flank were injected with 4xl0 ⁷ PFU of rMVA (MVAAE5RhFlt3L-mOX40L), MVAAE5R or PBS twice, 2 or 3 days apart. Tumors, spleens and/or tumor draining lymph nodes were harvested two days after second injection. In some experiments, 50 pg of aIFNAR-1 antibody (MARISAS, BioXcell) were injected into the tumors together with rMVA. For survival experiments, 2xl0 ⁵ B16-F10 cells were implanted intradermally into the shaved skin on the right flank of WT C57BL/6J mice or age-matched cGas~ Sting ^/gt , Stat2~ and Statl~ ~ mice. In some experiments, 2xl0 ⁵ A20 cells were implanted intradermally into the right flank of WT BALB/cJ mice. At 6 to 9 days after implantation, tumor sizes were measured and tumors that are 3 mm in diameter or larger were injected with 4x 10 ⁷ PFU of rMVA or PBS when the mice were under anesthesia. Viruses were injected twice weekly as specified in each experiment and tumor sizes were measured twice a week. Tumor volumes were calculated according to the following formula: 1 (length) x _w (width) x h (height)/2. Mice were euthanized for signs of distress or when the diameter of the tumor reached 10 mm. For depletion of T cells, depletion antibodies for CD8 ⁺ and CD4 ⁺ cells (200 pg of clone 2.43 and GK1.5, BioXcell) were injected intraperitoneally twice weekly starting 1 day before viral injection, and they were used until the animals either died, were euthanized, or were completely clear of tumors. For depletion of Tregs, 2xl0 ⁵ B16-F10 cells were implanted intradermally into the shaved skin on the right flank of Foxp3 ^DTR mice. Three doses of diphtheria toxin (DT) (200 ng each per mouse) were administered to tumor-bearing mice at - 2, -1, and +1 day relative to IT MVAAE5R or PBS injection at day 0. MVAAE5R were injected intratumorally twice weekly. In the bilateral tumor implantation model, B16-F10 cells were implanted intradermally into right and left flanks of C57BL/6J mice (5xl0 ⁵ to the right flank and 1 x 10 ⁵ to the left flank). At 7 days after implantation, the tumors at the right flank were injected with 4xl0 ⁷ PFU of rMVA (MVAAE5R-hFlt3L-mOX40L) or PBS. 250pg aPD-Ll antibody (10F.9G2, BioXcell) was injected intraperitoneally twice weekly. For the tumor rechallenge study, the survived mice (more than 40 days after initiation of intratumoral virotherapy) were rechallenged with intradermal delivery of a lethal dose of B16-F10 (IxlO ⁵ cells) at the contralateral side.

[0268] ELISpot assay: Spleens were mechanically disrupted by gentleMACS™ dissociator and red blood cells were lysed by ACK lysing buffer. IxlO ⁶ splenocytes were co-cultured with 2.5xl0 ⁵ irradiated Bl 6-F 10 in complete RPMI medium overnight. ZFNy ⁺ splenocytes were detected by Mouse IFNy ELISPOT kit (BD Biosciences)

[0269] Human tumor specimens: Fresh biopsy samples from patients with Extramammary Paget’s disease were obtained at the dermatology service in the Department of Medicine of Memorial Sloan Kettering Cancer Center. Written informed consents were obtained from patients enrolled in the protocol approved by Memorial Sloan Kettering Cancer Center Institutional Review Board (IRB). Studies were conducted in accordance with National Institutes of Health and institutional guidelines for human subject research. Tumor tissues were cut into small pieces using a pair of fine scissors. They were infected with rhMVA (MVAAE5R-hFlt3L-hOX40L) or mock-infected. Cells were collected after 24 h and processed for FACS analyses.

[0270] Statistical analysis: Two-tailed unpaired Student’s t test was used for comparisons of two groups in the studies. Survival data were analyzed by log-rank (Mantel-Cox) test. The p values deemed significant are indicated in the figures as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The numbers of animals included in the study are discussed in each figure legend.

Example 16: Intratumoral injection (IT) of rMVA elicits strong antitumor immune responses that are dependent on cGAS-STING-mediated DNA sensing and STATl/STAT2-mediated IFNAR-signaling pathway

[0271] To test whether cGAS/STING and STAT1/STAT2 are important for IT rMVA- induced antitumor immunity, eGas' ¹ ', Sting ^gt/gt (lacking function STING), Stat I' ¹ ', Stat2' ¹ ' or age-matched WT C57BL/6J mice were implanted with B16-F10 melanoma intradermally. When the tumors were established, they were injected with rMVA (MVAAE5R-hFlt3L- mOX40L) twice weekly. Whereas IT rMVA resulted in tumor eradication or delayed tumor growth in WT mice, it failed to induce antitumor effects in Stat I' ¹ ' and StafZ^’mice (FIGs. 17A-17B). IT rMVA treatment of eGas' ¹ ' or Sting ^Gt/Gt mice extended median survival from 11 days in PBS control group to 18.5 days (p = 0.0002). However, all of the eGas' ¹ ' or Sting ^Gt/Gt mice died from tumor progression (FIGs. 17A-17B). These results demonstrated that activation of the cGAS/STING-mediated cytosolic DNA-sensing pathway, as well as the IFNAR/STAT1/STAT2 signaling, by IT rMVA, is critical for the generation of antitumor immunity.

Example 17: IT rMVA (MVAAE5R-hFlt3L-mOX4QL) generates stronger systemic and local anti-tumor immune responses compared with MVAAE5R in a bilateral B16-F10 murine melanoma implantation model.

[0272] To determine the immunological mechanism of rMVA-induced antitumor immune responses, a bilateral murine Bl 6-F 10 tumor implantation model was used. B16-F10 cells were intradermally implanted into both flanks of C57BL/6J mice. After tumors were established, they were injectedwith MVAAE5R, rMVA, or PBS as a control, to the right-side tumors twice, three days apart. Spleens and both tumors were harvested 2 days after the second injection (FIG. 18A). IT rMVA generated the highest numbers of tumor-specific IFN-y ⁺ T cells in the spleens compared with those treated with MVAAE5R or with PBS as determined by ELISpot assay (FIGs. 18B-18C). In the injected tumors, IT rMVA resulted in stronger T cell activation with higher percentages and absolute numbers of granzyme B ⁺ CD8 ⁺ and granzyme B ⁺ Foxp3‘ CD4 ⁺ cells compared with MVAAE5R, or PBS control groups (FIGs. 19A-19B). In the non-injected tumors, IT rMVA also induced more granzyme B ⁺ CD8 ⁺ and granzyme B ⁺ Foxp3' CD4 ⁺ T cells (FIGs. 19C-19D), demonstrating that IT rMVA enhances T cell activation both locally and systemically. Taken together, these results demonstrate that IT rMVA results in the activation of both CD8 ⁺ and CD4 ⁺ T cells in the injected and non-injected tumors and the generation of systemic antitumor immunity.

Example 18: IT rMVA (MVAAE5R-hFlt3L-mOX4QL) depletes QX40 ^hi Tregs in the injected tumors and preferentially depletes QX40 ^hl Tregs via OX40L-OX4Q interaction and IFNAR signaling.

[0273] In addition to enhanced CD8 ⁺ and CD4 ⁺ T cell activation, it was also observed that IT rMVA treatment resulted in a significant reduction of Tregs in the injected tumors (FIGs. 20A-20B). The mean percentages of Tregs (Foxp3 ⁺ CD4 ⁺ ) out of CD4 ⁺ T cells were 23%, 45%, and 51% in rMVA, MVAAE5R, or PBS-treated tumors, respectively (FIGs. 20A-20B). The absolute numbers of Tregs in rMVA-injected tumors were significantly reduced compared with the PBS-treated group (FIGs. 20A-20B). In the non-injected tumors, however, there was no reduction of the percentages of Tregs out of CD4 ⁺ T cells after rMVA treatment (FIGs. 20C). These results support that rMVA treatment triggers apoptosis in tumor infiltrating Tregs.

[0274] To determine whether OX40L expressed by rMVA-infected myeloid and tumor cells might be important in mediating the reduction of OX40 ¹¹¹ Tregs the surface expression of 0X40 in various T cell populations within the tumor microenvironment was compared. The mean percentages of OX40 ¹¹¹ Tregs among CD4 ⁺ Tregs were 51% compared with 5.6% of OX40 ¹¹¹ CD4 ⁺ Foxp3‘ conventional T (Tconv) cells and 1% of OX40 ¹¹¹ CD8 ⁺ T cells (FIG.

20D). The mean fluorescence intensity (MFI) of 0X40 was higher in CD4 ⁺ Tregs than those in CD4 ⁺ Foxp3‘ Tconv and CD8 ⁺ T cells (FIGs. 20E-20F).

Example 19: Clinical candidate rhMVA (MVAAE5R-hFlt3L-hOX4QL) induces innate immunity and promotes maturation of human monocyte-derived DCs (moDCs).

[0275] To test whether ex vivo infection of human tumor samples with rhMVA could induce phenotypic changes of TILs, skin biopsy samples from patients with Extramammary Paget’s Disease (EMPD) were obtained, processed, infected with rhMVA, and TILs were analyzed 24 h later (FIG. 21A). rhMVA-infected samples exhibited upregulation of granzyme B on CD8 ⁺ T cells and reduction of Tregs compared with the paired control samples (FIG. 21B). These results support rhMVA as a potential clinical candidate for the treatment of human cancers.

Example 20: IFNAR1 signaling on Tregs is important in mediating rMVA (MVAAE5R-hFlt3L-mOX4QL)-indnced antitumor effects.

[0276] To test whether IFNAR signaling on Tregs plays a role in rMVA-induced antitumor effects, MC38 murine colon cancer cells were intradermally implanted into Ifnar 1^ and Foxp3 ^Cre Ifaarl ^/ mice. When the tumors were 3-4 mm in diameter, they were injected with rMVA or PBS twice weekly. A 30% cure in Ifnar 1^ mice and no cure in FoxpS^Ifnarl^ mice was observed after rMVA treatment (FIGs. 23A-23B). The median survival in

Ifnar mice was extended from 14 days in PBS-treated mice to 30 days in rMVA-treated mice, whereas the median survival in FoxpS^Ifnarl^ mice was extended from 14 days in PBS-treated mice to 21 days (FIGs. 23A-23B). In a B16-F10 melanoma model, IT rMVA resulted in eradication of tumors in 8 out of 9 Ifnar 1^ mice, but only in 3 out 9 of the I''()xp3 ^cr "Ifnarl' ^I 'mcQ (FIGs. 23C-23D). These results indicate that IFNAR1 signaling on Tregs plays an important role in mediating rMVA-induced antitumor effects, likely through induction of apoptosis in Tregs and reprogramming of Tregs.

Example 21: Working Model for rhMVA (MVAAE5R-hFlt3L-hOX4QL).

[0277] A working model for rMVA treatment is shown in FIG. 22. Briefly, IT injection of rMVA results in the infection of tumor-infiltrating myeloid cells, including macrophages, monocytes, and dendritic cells, as well as tumor cells. This leads to the activation of cGAS/STING-mediated cytosolic DNA-sensing pathway and the production of type I IFN and cytokines and chemokines that are important for CD8 ⁺ and CD4 ⁺ T cell proliferation and activation (as indicated by Granzyme B, TNF, and IFN-gamma expression). Flt3L expression of the tumor microenvironment facilitates the proliferation of CD103 ⁺ DCs in the tumors. OX40L expression by myeloid cell populations and tumor cells results in the depletion of OX40 ¹¹¹ Tregs infiltrating the tumors via OX40L-OX40 ligation, which is promoted by type I IFN. This leads to the blunting of their inhibition on tumor-specific effector CD4 ⁺ and CD8 ⁺ T cells. Taken together, IT delivery of rMVA results in the alteration of tumor immunosuppressive microenvironment through activation of innate immunity and boosting of antitumor T cells by depletion of OX40 ¹¹¹ regulatory T cells.

EQUIVALENTS

[0278] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0279] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0280] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. [0281] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0282] Other embodiments are set forth within the following claims.

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