Viral vectors for treating cancer in immune checkpoint inhibitor-refractory subjects

Abstract

The invention relates generally to replication defective HSV-1 vectors and their use for treating cancer in immune checkpoint inhibitor (ICI)-refractory subjects by delivering one or more genes encoding transgenic proteins that stimulate immune destruction of tumors.

Description

VIRAL VECTORS FOR TREATING CANCER IN IMMUNE CHECKPOINT INHIBITOR-REFRACTORY SUBJECTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 544,734, filed on October 18, 2023; U.S. Provisional Patent Application No.63 / 574,364, filed on April 4, 2024; and U.S. Provisional Patent Application No. 63 / 700,851, filed on September 30, 2024; the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes. FIELD OF THE INVENTION

[0002] The disclosure relates generally to a method of treating cancer in an immune checkpoint inhibitor-refractory subject with a replication-defective HSV-1 vector delivering one or more genes encoding transgenic proteins that stimulate immune destruction of tumors. BACKGROUND

[0003] Developments in immuno-oncology have provided new therapies directed to treating cancers that have evaded successful treatment because the cancers have evolved mechanisms for evading a subject’s immune system. Despite the significant advances made to date in treating various cancers with immune-stimulating therapies such as immune checkpoint inhibitors (ICI) and CAR T cells, significant numbers of patients remain unresponsive to immunotherapy treatment. For example, 40-60% of patients having skin melanoma do not achieve any significant therapeutic response to ICI, and a substantial proportion of responders experience tumor relapse within 2 years of treatment. Furthermore, solid tumors may be harder to treat using immune-oncologic methods, given the complexity of the tumor microenvironment (TME) of a solid tumor, which contains epithelial cells, endothelial cells, mesenchymal cells, stromal cells, cancer cells, and immune cells. In certain solid tumors, the cancer cells influence the activity or inactivity of the immune cells in the TME. Viral vectors have been developed to deliver therapeutics to cells. However, despite the advances made to date, there is still a need for methods and compositions that can be used to treat immunotherapy-refractory subjects, either alone or in combination with other cancer drugs.SUMMARY OF THE INVENTION

[0004] The invention is based, in part, upon the discovery that oncolytic HSV-1 vectors can be useful in treating cancer in an immune checkpoint inhibitor (ICI)-refractory subject in need thereof. Among other things, the treatment may result in, among other things, a reduction in tumor size. It is contemplated that the HSV-1 vectors described herein are useful for treating cancer in ICI-refractory patients based upon their ability to induce tertiary lymphoid structures (TLS), e.g., in a tumor bed, and activate T and B cell survival and proliferation and, alternatively or additionally, by activating pathways supportive of ICI response. Furthermore, the vectors can induce delayed oncolysis, allowing for sustained expression of a therapeutic payload (e.g., a therapeutic polypeptide) and exhibit increased immunogenicity, increasing immune activity toward tumor cells. The therapeutic payload can include, for example, factors known to regulate TLS.

[0005] The invention also is based, in part, upon the discovery that particular combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) affecting the ability of immune cells to kill cancer cells, thereby reducing the size of a tumor. For example, therapeutic polypeptides that target the stroma of a tumor, support T cell survival (e.g., a CAR T cell) and / or NK cell in a tumor microenvironment, induce TLS in a tumor bed, promote phagocytic innate immune surveillance, disrupt the Sirpα / CD47 axis, promote upregulation or downregulation of one or more proteins associated with a response to an ICI, and / or reduce cancer cell viability can be expressed from a viral vector, e.g., a viral vector lacking functional ICP4 and ICP47 proteins to increase the ability of immune cells to kill tumor cells, for example, to treat cancer in an ICI-refractory subject in need thereof. Vectors can be designed to include a specific combination of therapeutic polypeptides that will be effective to treat a given tumor type, including choosing one or more therapeutic polypeptides that reduce inhibitory features of a specific tumor microenvironment that may prevent the immune system from effectively attacking the tumor.

[0006] Accordingly, in one aspect, the disclosure relates to a method of treating cancer in an ICI-refractory subject, the method including administering a vector including a herpes simplex virus (HSV) genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein.

[0007] In another aspect, the disclosure relates to a method of expressing a therapeutic polypeptide in an ICI-refractory subject, the method including administering to the ICI- refractory subject a vector including a HSV genome, wherein the HSV genome comprisesone or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein.

[0008] In another aspect, the disclosure relates to a method of reducing the size of a tumor in an ICI-refractory subject, the method including administering to the ICI-refractory subject a vector including a HSV genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein, and wherein the vector includes a nucleic acid encoding a therapeutic polypeptide that functions to reduce the size of the tumor.

[0009] In certain embodiments, the HSV-1 genome comprises one or more alterations that prevent the expression of two ICP4 proteins. In certain embodiments, the HSV genome comprises one or more alterations that prevent the expression of two ICP4 proteins and an ICP47 protein. In certain embodiments, the two ICP4 proteins are characterized as having the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 (ICP4). In certain embodiments, the ICP47 protein is characterized as having the amino acid sequence of SEQ ID NO: 5 (ICP47).

[0010] In certain embodiments, the HSV genome is an HSV-1 genome.

[0011] In certain embodiments, the HSV-1 genome is a McKrae genome.

[0012] In certain embodiments, the vector, when administered to the subject, results in one or more of: (a) delayed oncolysis; (b) increased immunogenicity; (c) increased immune activation; (d) formation of TLS in a tumor bed; (e) upregulation or downregulation of one or more proteins associated with a response to ICI; (f) cancer cell viability reduction; and (g) a combination of any one or more of (a) – (f).

[0013] In certain embodiments, the vector includes a nucleic acid encoding one or more therapeutic polypeptides, and wherein the one or more therapeutic polypeptides: (a) target the stroma of a tumor; (b) support T cell survival in a tumor microenvironment; (c) induce TLS in a tumor bed; (d) promote phagocytic innate immune surveillance (e.g., disrupts the Sirpα / CD47 axis); (e) promote upregulation or downregulation of one or more proteins associated with a response to ICI; (f) reduce cancer cell viability; or (g) a combination of any one or more of (a) – (f).

[0014] In certain embodiments, the one or more therapeutic polypeptides target the stroma of a tumor by degrading an extracellular matrix protein. Such a therapeutic polypeptide that targets the stroma of a tumor by degrading an extracellular matrix protein can include, for example, MMP-9, or an inhibitor of lysyl oxidase.

[0015] In certain embodiments, the one or more therapeutic polypeptides target the stroma of a tumor by activating local endothelium to increase T cell infiltration. Such a therapeutic polypeptide that target the stroma of a tumor by activating local endothelium to increase T cell infiltration can include, for example, TNF, IL-1β, IL-6, or IL-18.

[0016] In certain embodiments, the one or more therapeutic polypeptides support T cell survival in the tumor microenvironment by enhancing recruitment of T cells to a site of a tumor. Such a therapeutic polypeptide that supports T cell survival in the tumor microenvironment by enhancing recruitment of T cells to a site of a tumor can include, for example, CCL19 or CCL21

[0017] In certain embodiments, the one or more therapeutic polypeptides support T cell survival in the tumor microenvironment by supporting T cell function. Such a therapeutic polypeptide that supports T cell survival in the tumor microenvironment by supporting T cell function can include, for example, IL-7, IL-12, IL-15, IL-18, or IFN-γ

[0018] In certain embodiments, the T cell is a CAR T cell. Such a therapeutic polypeptide that supports CAR T cell survival in the tumor microenvironment by supporting CAR T cell function can include, for example, soluble TGFβRII. Such a therapeutic polypeptide that supports CAR T cell survival in the tumor microenvironment by supporting CAR T cell function can also include, for example, an antigen recognized by the CAR T cell, such as mesothelin.

[0019] In certain embodiments, the one or more therapeutic polypeptides include a co- stimulatory molecule, such as CD40L and OX40L.

[0020] In certain embodiments, the one or more therapeutic polypeptides induce TLS in a tumor bed. Such a therapeutic polypeptide that induces TLS in a tumor bed can include, for example, CCL19, lymphotoxin β, CXCL13, or TNF.

[0021] In certain embodiments, the one or more therapeutic polypeptides promote phagocytic innate immune surveillance. Such a therapeutic polypeptide that promotes phagocytic innate immune surveillance can, in certain embodiments, disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene or an anti-CD47 antibody.

[0022] In certain embodiments, the one or more therapeutic polypeptides support anti-tumor macrophage polarization. Such a therapeutic polypeptide that supports anti-tumor macrophage polarization can include, for example, TNF, IL-1, IL-12, IL-17, or IFN-γ.

[0023] In certain embodiments, the one or more therapeutic polypeptides include hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19,CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene, an anti-CD47 antibody, IL-1, or IL- 17.

[0024] In certain embodiments, the one or more therapeutic polypeptides include IL-21, LTA, LTB, CXCL12, or VEGF-C.

[0025] In certain embodiments, the one or more therapeutic polypeptides include hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene, an anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, or VEGF-C.

[0026] In certain embodiments, the one or more therapeutic polypeptides include: a) LTA, CCL19, TNF, IL-17, and IL-7; b) LTB, CCL19, CCL21, CXCL13, and IL-7; c) IFN-γ and IL-12; d) IFN-γ and IL-15; e) IL-12 and IL-15; f) IL-15 and IL-21; g) IFN-γ, IL-12, and IL- 15; h) IFN-γ, IL-12, IL-15, and IL-21; or i) CCL19, IL-7, TNF, and IL-17.

[0027] In certain embodiments, the method further includes administering an ICI.

[0028] In certain embodiments, the ICI is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

[0029] In certain embodiments, the ICI includes a PD-1 inhibitor, such as an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab, or cemiplimab); a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab); a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab); or a LAG-3 inhibitor (e.g., relatlimab).

[0030] In certain embodiments, the method includes administering a plurality of ICIs selected from the group consisting of a PD-1 inhibitor, such as an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab, or cemiplimab) and a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab).

[0031] In certain embodiments, the one or more proteins associated with a response to ICI include IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0032] In certain embodiments, inducing TLS in a tumor bed includes promoting the number, size, and / or organization of TLS structures.

[0033] In another aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method including administering a vector comprising a HSV genome, wherein the HSV genome comprises one or more alterations that prevent the expression of afunctional ICP4 protein and / or a functional ICP47 protein and expresses an effective amount of IL-12 and IL-15, thereby to treat the cancer.

[0034] In certain embodiments, the method further includes administering an ICI. The ICI can include a PD-1 inhibitor, such as an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab, or cemiplimab); a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab); a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab); or a LAG-3 inhibitor (e.g., relatlimab).

[0035] In certain embodiments, the method includes administering a plurality of ICIs selected from the group consisting of a PD-1 inhibitor, such as an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab, or cemiplimab) and a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab).

[0036] In certain embodiments, the administration of the vector comprising the herpes simplex virus (HSV) genome that expresses IL-12 and IL-15 results in synergistic tumor regression as compared to administration of vector comprising a herpes simplex virus (HSV) genome (e.g., an otherwise identical vector) that expresses (a) IL-12 and not IL-15 or (b) only IL-15 and not IL-12.

[0037] These and other aspects and features of the invention are described in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The foregoing and other objects, features, and advantages of the disclosure will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings.

[0039] FIG.1 is a bar chart showing the effect in breast cancer-derived Hs578 cells of infection with a variant of a herpes simplex virus (HSV) type-1 (HSV-1) strain vector expressing an interferon γ (IFN-γ) payload and whose HSV-1 genome contains mutant ICP4 and ICP47 proteins (“HSV-1 mICP4 / mICP47”) (“HSV-1 mICP4 / mICP47 IFNγ”), as compared to an HSV-1 mICP4 / mICP47 green fluorescent protein (GFP) vector or uninfected control group, on the fold-change in expression levels of genes involved in the IFN-γ response signature, including ISG15, IRF9, CCL2, and CXCL10. *p < 0.05, ****p < 0.0001.

[0040] FIG.2 is a bar chart showing the luminescence of breast cancer-derived Hs578 cells infected with 0, 3, or 10 plaque forming units (PFU) / cell of an HSV-1 mICP4 / mICP47 IFN-γ vector, with lower luminescence indicative of greater oncolytic activity.

[0041] FIG.3 is a set of bar charts showing a quantification of the amount of murine (m)CXCL13, mCCL19, or mIL-7 payload production in the conditioned media of breast cancer-derived Hs578T cells infected with 3 or 10 PFU / cell of an HSV-1 strain vector expressing the respective payload and whose HSV-1 genome contains mutant ICP4 and ICP47 proteins.

[0042] FIG.4 is a bar chart showing the normalized luminescence of breast cancer-derived Hs578T cells 6 days after infection with the HSV-1 vectors described in FIG.3, with lower luminescence indicative of greater oncolytic activity.

[0043] FIG.5 is a schematic representation depicting activation of innate immune surveillance of a cancer cell by macrophages through interference of the SIRPα / CD47 pathway with an HSV-1 mICP4 / mICP47 vector encoding an extracellular domain of SIRPα and an Fc fragment of human IgG1 (“macro1”) payload.

[0044] FIGs.6A-6D are a set of flow cytograms depicting the quantity of phagocytosis, as measured by cells positive for CD14 and carboxyfluoroscein succinimidyl ester (CFSE), in lymphoma-derived Raji cells cultured in a conditioning media (CM) from breast cancer- derived Hs578T cells treated with an isotype control (IgG) (FIG.6A), an anti-CD47 antibody (FIG.6B) or infected with 3 PFU / cell of a control HSV-1 mICP4 / mICP47 GFP vector (FIG. 6C) or an HSV-1 mICP4 / mICP47 macro1 vector (encoding an extracellular domain of SIRPα and an Fc fragment of human IgG1) (FIG.6D) prior to co-culture with M1 macrophages for 3 hours.

[0045] FIG.7 is a bar chart showing the percentage of phagocytosis in lymphoma-derived Raji cells from the experiment described in FIGs.6A-6D. N = 3; *p < 0.05.

[0046] FIG.8 is a schematic representation of an experimental dosing regimen, where Balb / c nude mice were subcutaneously inoculated in the rear flank with 5 x 106pulmonary carcinoma-derived A549 cells, and after A549-derived tumors reached approximately 100 mm3, were intratumorally (i.t.) treated on Days 1, 4, 7, 10, and 13 with 3 x 107PFU of an HSV-1 mICP4 / mICP47 macro1 vector or 10 mg / kg intraperitoneally (i.p.) of an anti-CD47 control antibody.

[0047] FIGs.9A-9B are graphs depicting the effect of treatment on tumor volume of the Balb / c nude mice described in the experiment in FIG.8. FIG.9A is a graph showing tumor volume in mice infected with a control vector, an HSV-1 mICP4 / mICP47 GFP vector, or an HSV-1 mICP4 / mICP47 macro1 vector. FIG.9B is a graph showing tumor volume in mice treated with a control IgG isotype control antibody or an anti-CD47 antibody.

[0048] FIG.10 is a heat map chart depicting an in silico prediction of the statistical association between gene expression from RNA-seq of subject populations of tumor microenvironment (TME) ICI-treated samples (rows) versus TME survival. A score was calculated by combining the TME-to-survival and TME-to-payload p values as -log10 meta p values to generate a “score” to identify potential payloads (columns) for HSV-1 mICP4 / mICP47 vectors described herein. An exemplary payload, IFN-γ, is highlighted by the black vertical rectangle, which was present across all patient populations (rows) of the analysis.

[0049] FIGs.11A-11B are graphs showing a comparison of the top pathways identified in a Gene Set Enrichment Analysis (GSEA) of RNA-seq data. FIG.11A is a graph showing top pathways identified from stable / progressive (SD / PD) or partial / completely responding (PR / CR) subjects from a SU2C-MARK cohort treated with an immune checkpoint inhibitor (ICI). FIG.11B is a graph showing top pathways identified from cells infected with HSV-1 mICP4 / mICP47 for 72 hours.

[0050] FIG.12 is a graph showing the correlation of all pathways identified in the GSEA experiments of FIGs.11A-11B in cells infected with HSV-1 mICP4 / mICP47, as compared to subjects treated with an ICI. Rank: NES*(-log10(p value)).

[0051] FIG.13 is a graph showing the volume of tumor size in Balb / c mice injected with 5 x 105mammary carcinoma EMT6 cells into the rear flank over time following treatment with a vehicle control, control antibody (10 mg / kg), HSV-1 mICP4 / mICP47 GFP vector (3 x 107PFU), and anti-PD-1 antibody clone RMP1-14 (10 mg / kg). N = 5-8 mice per group.

[0052] FIGs.14A-14B are graphs depicting the effect of treatment on tumor volume in Balb / c nude mice subcutaneously inoculated in the rear flank with mammary carcinoma EMT6 cells. Once EMT6-derived tumors reached approximately 100 mm3, mice were intratumorally infected on Day 1, 4, 7, and 10 (FIG.14A) with 3 x 107of a control HSV-1 mICP4 / mICP47 GFP vector; five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL- 7, mTNF, mIL-17a, and GFP, respectively (6 x 106PFU for each vector; total dose 3 x 107PFU; “Multiplex A”); or five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL-7, mCCL21, mCXCL13, and mLTB, respectively (6 x 106PFU for each vector; total dose 3 x 107PFU; “Multiplex B”), and tumor volume was measured (FIG.14B). N = 8 per group, *p < 0.05, **p < 0.01.

[0053] FIG.15 is a bar chart showing the percentage of CD45-positive cells in the submandibular glands of mice transduced with vehicle (control) or 3 x 107PFU of HSV-1 mICP4 / mICP47 GFP.

[0054] FIG.16 is a bar chart showing the effect of treatment on the number of live breast cancer-derived Hs578T tumor cells uninfected or infected in vitro with 1 PFU / cell of a control HSV-1 mICP4 / mICP47 GFP vector (“GFP”), an HSV-1 mICP4 / mICP47 IFN-γ vector, or an HSV-1 mICP4 / mICP47 vector encoding IL-12 (“A”), IL-15 (“B”), IL-21 (“C”), CD40L (“D”), IFN-γ + IL-12 (“E”), IFN-γ + IL-15 (“F”), IL-12 + IL-15 (“G”), IL-15 + IL-21 (“H”), IFN-γ + IL-12 + IL-15 (“I”), or IFN-γ + IL-12 + IL-15 + IL-21 (“J”), respectively, and co-cultured with peripheral blood mononuclear cells (PBMC) for 72 hours (“+ T cell activator;” groups A-J co-cultured). In experimental groups where IL-15 or IL-21 was present in the payload, a recombinant form of the protein was provided as well as HSV-1 mICP4 / mICP47 GFP, rather than an HSV-1 mICP4 / mICP47 encoding IL-15 and / or IL-21, respectively.

[0055] FIG.17 is a graph depicting a lasso regression analysis of immune cell populations positively (right of 0.0 on x-axis) or negatively (left of 0.0 on x-axis) correlated with PBMC- mediated tumor cell killing of the breast cancer-derived Hs578T tumor cells described in the experiment of FIG.16.

[0056] FIG.18 is a graph showing the volume of tumors in mice injected with 5 x 105mammary carcinoma EMT6 cells into the rear flank over time following treatment with a vehicle control; five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL-7, mTNF, mIL-17a, and GFP, respectively (6 x 106PFU for each vector; total dose 3 x 107PFU; “Multiplex A”); or five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL-7, mCCL21, mCXCL13, and mLTB, respectively (6 x 106PFU for each vector; total dose 3 x 107PFU; “Multiplex B”), and anti-PD-1 antibody clone RMP1-14 (10 mg / kg).

[0057] FIG.19 is a graph showing the count of MHC class I (MHC-I)-positive breast cancer- derived Hs578T cells infected with 10 PFU / cell of a control vector or HSV-1 mICP4 / mICP47 GFP as measured by flow cytometry 24 hours after infection.

[0058] FIG.20 is a graph showing a gene set enrichment analysis performed on RNAseq data showing the top pathways enriched in cells infected with HSV-1 mICP4 / mICP47.

[0059] FIG.21 is a set of bar charts showing a quantification of the amount of interferon gamma (IFN-γ) and IL-12 payload production in the conditioned media of breast cancer-derived Hs578T cells infected with 3 or 10 PFU / cell of an HSV-1 mICP4 / mICP47 expressing the respective payload.

[0060] FIGs.22A-22B are a graph and heatmap showing the cytotoxic effect of breast cancer-derived Hs578T tumor cells uninfected or infected in vitro with 1 PFU / cell of a control HSV-1 mICP4 / mICP47 GFP vector (“GFP”); an HSV-1 mICP4 / mICP47 IFN-γ vector (“IFN-γ”); an HSV-1 mICP4 / mICP47 IL-12 vector (“IL-12”); an HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 (“IL-15”); an HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 (“IL-21”); or a combination of HSV-1 mICP4 / mICP47 vectors encoding IFN-γ or IL-12 (“D1”); GFP or IFN-γ and recombinant human IL-15 (“D2”); GFP or IL-12 and recombinant human IL-15 (“D3”); GFP, IFN-γ, or IL-12 and recombinant human IL-15 (“T”); or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 (“Q”); or co-cultured with PBMC (“activator”) analyzed by flow cytometry (left panel) and ordinary least squares (OLS) regression (right panel).

[0061] FIG.23 is a heatmap showing the composition, as measured by flow cytometry, of control PBMC or PBMC co-cultured with Hs578T cells uninfected (“Uninf.”) or infected with a control HSV-1 mICP4 / mICP47 GFP vector (“GFP”); an HSV-1 mICP4 / mICP47 IL-12 vector (“IL-12”); an HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 (“IL- 21”); an HSV-1 mICP4 / mICP47 IFN-γ vector (“IFN-γ”); a combination of HSV-1 mICP4 / mICP47 vectors encoding IFN-γ or IL-12 (“D1”); an HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 (“IL-15”); or an HSV-1 mICP4 / mICP47 vector encoding GFP or IFN-γ and recombinant human IL-15 (“D2”); IL-12 and recombinant human IL-15 (“T”); GFP or IL-12 and recombinant human IL-15 (“D3”); or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 (“Q”); or co-cultured with PBMC (“Activ.”)

[0062] FIG.24 is a graph depicting a lasso regression analysis of immune cell populations positively (right of 0.0 on x-axis) or negatively (left of 0.0 on x-axis) correlated with PBMC- mediated tumor cell killing of the breast cancer-derived Hs578T tumor cells described in the experiment of FIG.23.

[0063] FIG.25 is a set of graphs showing the total number of dendritic cells (DC) and Ki67- positive / Gzmb-positive CD8-positive T cells of control PBMC or tumor cells uninfected or infected in vitro with a control HSV-1 mICP4 / mICP47 GFP vector (“GFP”); an HSV-1 mICP4 / mICP47 IFN-γ vector (“IFN-γ”); an HSV-1 mICP4 / mICP47 IL-12 vector (“IL-12”); an HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 (“IL-15”); an HSV-1mICP4 / mICP47 GFP vector and recombinant human IL-21 (“IL-21”); or a combination of HSV-1 mICP4 / mICP47 vectors encoding IFN-γ or IL-12 (“D1”); GFP or IFN-γ and recombinant human IL-15 (“D2”); GFP or IL-12 and recombinant human IL-15 (“D3”); GFP, IFN-γ, or IL-12 and recombinant human IL-15 (“T”); or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 (“Q”); or co-cultured with PBMC (“activator”).

[0064] FIG.26A is a graph showing the volume of EMT6 tumor growth in Balb / c mice injected with 5 x 105mammary carcinoma EMT6 cells into the rear flank and infected with 3 x 107PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. FIG.26B is a graph showing the volume of EMT6 tumor growth in individual Balb / c mice injected with 5 x 105mammary carcinoma EMT6 cells into the rear flank and infected with 3 x 107PFU of a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. FIG.26C is a graph showing the weight of EMT6 tumors in Balb / c mice injected with 5 x 105mammary carcinoma EMT6 cells into the rear flank and infected with 3 x 107PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. FIG.26D is a graph showing the weight of LLC1 tumors in Balb / c mice injected with 5 x 105tumorigenic LLC1 cells into the rear flank and infected with 3 x 107PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12.

[0065] FIG.27A is a set of graphs showing the percentage of Ki67-positive and PD1- positive CD8-positive T cells (right and left panels, respectively) found in PBMC in mice injected with mammary carcinoma EMT6 cells into the rear flank and infected with a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12, as measured by flow cytometry. FIG.27B is a graph showing the percentage of Ki67-positive CD8-positive T cells found in the tumors of mice injected with tumorigenic LLC1 cells into the rear flank and infected with a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12, as measured by flow cytometry.DETAILED DESCRIPTION

[0066] The invention is based, in part, upon the discovery that oncolytic HSV-1 vectors can be useful in treating cancer in an immune checkpoint inhibitor (ICI)-refractory subject in need thereof. The treatment may result in, among other things, a reduction in tumor size. It is contemplated that the HSV-1 vectors described herein are useful for treating cancer in ICI- refractory patients based upon their ability to induce tertiary lymphoid structures (TLS), e.g., in a tumor bed, and activate T and B cell survival and proliferation and, alternatively or additionally, by activating pathways supportive of ICI response. Furthermore, the vectors can induce delayed oncolysis, releasing tumor-associated or tumor-specific antigens, allowing for sustained expression of a therapeutic payload (e.g., a therapeutic polypeptide) and exhibit increased immunogenicity, increasing immune activity toward tumor cells. The therapeutic payload can include, for example, factors known to regulate TLS formation in the tumor microenvironment or increasing lymphocyte migration in the tumor.

[0067] The invention also is based, in part, upon the discovery that particular combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of immune cells to kill cancer cells, thereby increasing their cytotoxic activity and reducing the size of a tumor. For example, therapeutic polypeptides that target the stroma of a tumor, support T cell survival (e.g., a CAR T cell) and / or NK cell in a tumor microenvironment, induce TLS in a tumor bed, promote phagocytic innate immune surveillance, disrupt the Sirpα / CD47 axis, promote upregulation or downregulation of one or more proteins associated with a response to an ICI, and / or reduce cancer cell viability can be expressed from a viral vector, e.g., a viral vector lacking functional ICP4 and ICP47 proteins to increase the ability of immune cells to kill tumor cells, for example, to treat cancer in an ICI-refractory subject in need thereof. Vectors can be designed to include a specific combination of therapeutic polypeptides that will be effective to treat a given tumor type, including choosing one or more therapeutic polypeptides that reduce inhibitory features of a specific tumor microenvironment that may prevent the immune system from effectively attacking the tumor. I. Definitions

[0068] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Unless defined otherwise, all technical and scientific terms havethe same meaning as is commonly understood by one of ordinary skill in the art to which the embodiments disclosed belongs.

[0069] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0070] As used herein, the term “agent” refers to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, or combinations thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. Some particular embodiments of agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA / RNA hybrids, antisense oligonucleotides, and ribozymes), peptides, peptide mimetics, etc.

[0071] The terms “about” and “approximately” indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. In one non-limiting embodiment, the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. When values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself.

[0072] As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously, sequentially, or in overlapping dosing regimens.

[0073] As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and / or 3′ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0074] As used herein, the term “immune checkpoint inhibitor-refractory subject” or “ICI- refractory subject” refers to a subject who has received one or more ICI treatments and who has failed to respond or has failed to continue to respond to the one or more ICI treatments. A subject considered to have failed to continue to respond to an ICI treatment may have initially responded to an ICI treatment for a period of time and then ceased responding to the ICI. The one or more ICI treatments can include any dose or administration regimen of an ICI. Exemplary ICIs include a PD-1 inhibitor (e.g., an anti-PD-1 antibody e.g., pembrolizumab, nivolumab, or cemiplimab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab), a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab), and a LAG-3 inhibitor (e.g., relatlimab).

[0075] As used herein, the terms “marker” or “marker protein” refer to a detectable or selectable agent. In some embodiments, a “marker” or “marker protein” is a detectable or selectable nucleic acid sequence. In some embodiments a “marker” or “marker protein” is an expression product (e.g., RNA or protein) whose presence or absence is detectable and / or selectable in cells. In some embodiments, an expression product is or comprises an enzyme. In some embodiments, an expression product is a fluorophore.

[0076] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0077] As used herein, the term “pharmaceutically acceptable” applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0078] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0079] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0080] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated to a viral genome or portion thereof. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors, and herpes simplex virus (HSV) vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0081] The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and / or” unless stated otherwise or clear from context to be disjunctive.

[0082] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0083] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0084] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0085] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0086] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes andmethods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0087] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention. II. Viral Vectors and Herpes Simplex Virus Type 1 (McKrae)

[0088] Viral vectors can be used to facilitate the transfer of nucleic acids into cells. Herpes simplex virus (HSV) type-1 (HSV-1) vectors can typically accommodate up to 25 kb of foreign DNA sequences. HSV-1 has an approximate 152 kb double-stranded linear DNA genome that can be maintained episomally in the nucleus of cells. The HSV-1 virion is enveloped and approximately 110 nm in diameter.

[0089] At least 17 strains of HSV-1 have been isolated, including, but not limited to, McKrae, strain 17, strain F, H129, HF10, MacIntyre, Strain HF, ATCC 2011, and KOS (for review, see Watson et al., (2012) VIROLOGY 433(2):528-537). A. McKrae

[0090] A McKrae strain was isolated from a patient with herpes simplex keratitis and subsequently passaged in tissue culture. A partial genome sequence of McKrae is provided at SEQ ID NO: 1 (GenBank Accession No.: JQ730035.1).

[0091] Inter-strain differences in HSV-1 peripheral replication and virulence are observed after injection into animals. For example, McKrae undergoes spontaneous or induced reactivation at a higher frequency than other known strains and is among the most virulent HSV-1 strains.

[0092] HSV genes influence viral characteristics and phenotype. There are at least 9 genes and several non-coding sequences unique to McKrae strain. In addition to those associated with pathogenesis and latency reactivations, such as RL1, RSI, and RL2, three UL genes (UL36, UL49A, and UL56) and three US genes (US7, US10, and US11) are unique toMcKrae strain. In addition to gene variations, non-coding sequences such as LAT, ‘a’ sequence, and miRNAs contain variations unique to McKrae.

[0093] One or more of the following gene and non-coding sequences can be considered characteristic of McKrae strain. In McKrae, RLl (ICP34.5) has an extended P-A-T repeat between residues 159 and 160 that results in 8 iterations, while other strains contain only 3-5 iterations. The P-A-T repeat is thought to influence cellular localization of the ICP34.5 protein (Mao et al. (2012) J. BIOL. CHEM.277(13): 11423-31). ICP34.5 is thought to be a neurovirulence factor involved in viral replication and anti-host response.

[0094] McKrae strain also contains an extended repeat element of six iterations of the internal tandem repeat STPSTTT (SEQ ID NO: 38) located within the coding sequence of US07 (gl). Additionally in McKrae, UL 36 contains a premature stop codon introduced due to a G nucleotide deletion in a mononucleotide string encoding amino acid residue 2,453 (nucleotide 72,535), and UL 56 (at amino acid 180) contains a single base pair insertion at nucleotide 115,992 (at amino acid 97). McKrae strain also contains an extended ORF in US10 resulting from a single bp insertion at nucleotide 143,416 and the frameshift causes a stop codon loss in McKrae and a unique C-terminal protein sequence. McKrae also has amino acid differences at UL49A at residues 28 and 51 compared to other strains. McKrae has histidine and threonine at residues 28 and 51, respectively, whereas strain 17 has arginine and threonine and other strains (e.g., KOS) have histidine and alanine. Also, McKrae strain contains reduced tandem repeats found at the UL-RL junction (49 bp in McKrae as opposed to 181 bp in strain 17 and KOS) and approximately 330 nucleotides missing immediately following the UL-RL junction repeat. McKrae also contains unique variation within the ‘a’ sequence direct repeat 2 (DR2) array. Instead of a series of unbroken tandem repeats, the McKrae DR2 repeats are interrupted twice by identical guanine-rich sequences.

[0095] Major variation within the LAT intron between strains is due to differences in a repeat element (GCACCCCCACTCCCAC) (SEQ ID NO: 39) that varies in iteration number beginning at nucleotide 119,482 in McKrae strain, with McKrae containing 13 repeats while strains F, H129, and 17 contain 9 repeats and KOS contains 15 repeats. Also, tandem repeat variation between strains is found beginning in McKrae at base 125,520. McKrae repeat elements include twelve iterations of CCCCAGCCCTCCCCAG (SEQ ID NO: 40) and eight iterations of CCCCTCGCCCCCTCCCG (SEQ ID NO: 41). The first repeat unit is unique from other strains in that it contains a G-A transition, and strain McKrae contains three iterations more than any other strain. The McKrae strain second repeat element is collapsed,missing 188 nucleotides relative to all other strains, and separated from the upstream repeat by a 100% conserved sequence of 105 bp containing miR-H5.

[0096] McKrae further contains a unique coding sequence for ICP4 that is not found in other known strains (Watson et al., (2012) supra). ICP4 is an immediate early transcriptional regulator and has been implicated in reactivation. Whereas other strains contain an alanine rich region (AASAPDAADALAAA) (SEQ ID NO: 42) between residues 707 and 720, in McKrae the alanine rich region is replaced by a serine rich sequence (GPRRSSSSSGVAA) (SEQ ID NO: 43). The serine rich block of substitutions present in McKrae is adjacent to the nuclear localization signal (NLS) (amino acids 728-734). A change in conformation of this region may alter the NLS and, in turn, affect localization of not only ICP4, but also other viral proteins (e.g., ICP0 and ICP8) that are affected by ICP4 localization (Knipe and Smith (1986) MOLECULAR AND CELLULAR BIOLOGY 6(7), 2371-2381). Thus, this region may influence viral phenotype in part by altering the localization of proteins to the nucleus. B. Modifications of HSV-1 Vectors

[0097] Vectors comprising an HSV-1 genome (e.g., from a McKrae strain) may have one or more HSV genes necessary for replication rendered nonfunctional. For example, a vector comprising an HSV genome may contain an alteration that prevents expression of one or more functional ICP0, ICP4, ICP22, ICP27, and ICP47 genes. HSV genes necessary for replication include, for example, immediate early genes such as ICP4 and ICP47. ICP4 is a viral transcription factor which is expressed soon after infection and sustains the HSV-1 viral cycle. ICP47 of HSV-1 is an 87-amino acid cytosolic polypeptide or 88 residues if the initiation methionine is included. It binds to the TAP1-TAP2 heterodimer in human but not mouse cells and prevents transport of peptides through blockade of the peptide binding site of TAP. As a consequence, MHC class I molecules fail to be loaded with peptides. The resultant empty MHC class I molecules are retained in the endoplasmic reticulum and presentation of epitopes to cytotoxic T lymphocytes is abolished in HSV-infected human cells. An alteration that prevents expression of one or more functional HSV genes (e.g., ICP4 and ICP47) can include a mutation (e.g., a missense mutation, a nonsense mutation, an insertion, a deletion, etc.) in the coding sequence of the gene or in a regulatory sequence affecting expression of the gene (e.g., a promoter). ICP47 mutations include, but are not limited to, mutations in A4, D27, K31, R32, R34, or R41 relative to the amino acid sequence of SEQ ID NO: 5 and combinations thereof (as described in Mozzie et al. (2022) MOLBIOLEVOL.39(7):msac142). ICP4 mutations and deletion variants that disrupt its ability to activate transcription include, but are not limited to, n208, d8-10, nd8-10, ΔSER, d120, n12, ΔSERn7, d3-8, nd3-8, m20, m20n7, m90, m90n7, d143, d143n7, and nd3-10 (as described in Wagner et al. (2012) J VIROL.86(12):6862-74). Deletion of the region between amino acids 30-210 in the N-terminus of ICP4 is sufficient to eliminate transcriptional activation (as described in Wagner et al. (2013) J VIROL.87(2): 1010-1018). In some embodiments, the disclosure provides replication defective HSV vectors with alterations in one or more of ICP0, ICP4, ICP22, ICP27, and ICP47.

[0098] HSV-1 IE promoters contain one or more copies of an IE-specific regulatory sequence of consensus TAATGARAT (where R is a purine). These motifs are normally located within a few hundred nucleotides of the proximal IE promoter sequences, but in conjunction with their flanking sequences they are discrete functional entities which can confer IE-specific regulation to other proximal promoter elements of different temporal class. In some embodiments, replication-defective viruses are created by deleting nucleotides in an IE- specific regulatory sequence, e.g., in an IE-specific regulatory sequence affecting expression of one or more of ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, an IE- specific regulatory sequence contains an internal deletion. In some embodiments, an IE- specific regulatory sequence contains a terminal deletion. In some embodiments, an IE- specific regulatory sequence is completely deleted.

[0099] In some embodiments, the disclosure provides HSV vectors with nonfunctional ICP4 and ICP47 genes. In certain embodiments, the ICP4 gene or a regulatory sequence affecting expression of ICP4 comprises a mutation and the ICP47 gene or a regulatory sequence affecting expression of ICP47 comprises a mutation. For example, the ICP4 gene can comprise a mutation and the ICP47 gene can comprise a mutation. In another example, the regulatory sequence affecting expression of ICP4 includes a mutation and a regulatory sequence affecting expression of ICP47 includes a mutation. In one specific example, the ICP4 gene or a regulatory sequence affecting expression of ICP4 includes a deletion (e.g., a complete deletion or a partial deletion sufficient to abolish activity) and the ICP47 gene or a regulatory sequence affecting expression of ICP47 includes a deletion (e.g., a complete deletion or a partial deletion sufficient to abolish activity). The ICP4 gene can include a deletion and the ICP47 gene can include a deletion. Furthermore, combinations of the foregoing are contemplated, e.g., deletion of ICP4 and a mutation in a regulatory sequence for ICP47.

[0100] In some embodiments, the disclosure provides HSV vectors or HSV strains with a nonfunctional ICP47 gene. In some embodiments, the disclosure provides HSV vectors or HSV strains with nonfunctional ICP4 and ICP47 genes. In some embodiments, the disclosure provides an HSV vector or an HSV strain with ICP4 and ICP47 genes deleted. In some embodiments, the gene encoding ICP4 and the gene encoding ICP47 is fully or partially deleted, without disrupting expression of any additional immediate early genes.

[0101] HSV-1 vectors that have altered (e.g., mutated) HSV genes can be produced in cell lines that express the deficient protein in trans. In some embodiments, HSV-1 vectors are produced in a mammalian cell line, e.g., in a mammalian cell line of Vero lineage. In some embodiments, the cell line expresses ICP4. In some embodiments, the cell line expresses ICP47. In some embodiments, the cell line expresses ICP4 and ICP47. In some embodiments, the cell line expresses one or more of ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and UL55. In some embodiments, the cell line expresses ICP4, ICP27, and UL55. In some embodiments, the cell line comprises a nucleic acid molecule having a simian virus 40 polyadenylation signal (SV40 pA). Depending upon the circumstances, the viral vectors can be produced in Vero 6-5C cells or Vero D cells.

[0102] In certain embodiments, the viral vectors of the disclosure are McKrae HSV-1 viral vectors. Wild-type McKrae strain HSV-1 comprises two copies of the gene encoding ICP4 and one copy of the gene encoding ICP47. In some embodiments, the disclosure provides HSV vectors with nonfunctional ICP4 and ICP47 genes. In certain embodiments, at least one ICP4 gene (i.e., one or both copies of the gene) or a regulatory sequence affecting expression of ICP4 comprises a mutation and the ICP47 gene or a regulatory sequence affecting expression of ICP47 comprises a mutation. For example, at least one copy of the ICP4 gene (i.e., one or both copies of the ICP4 gene) can comprise a mutation and the ICP47 gene can comprise a mutation. In another example, a regulatory sequence affecting expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) includes a mutation and a regulatory sequence affecting expression of ICP47 includes a mutation. In one specific example, at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) or a regulatory sequence affecting expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) includes a deletion and the ICP47 gene or a regulatory sequence affectingexpression of ICP47 includes a deletion. At least one ICP4 gene (i.e., one or both copies of the ICP4 gene) can include a deletion and the ICP47 gene can include a deletion.

[0103] In some embodiments, the disclosure provides HSV vectors or HSV strains with a nonfunctional ICP47 gene. In some embodiments, the disclosure provides HSV vectors or HSV strains with nonfunctional ICP4 and ICP47 genes. In some embodiments, the disclosure provides an HSV vector or an HSV strain with one or both copies of ICP4 deleted and ICP47 deleted. In some embodiments, the one or both copies of the gene encoding ICP4 is fully or partially deleted and the gene encoding ICP47 is fully or partially deleted, without disrupting expression of any additional immediate early genes. C. Applications of HSV-1 Vectors

[0104] The viral vectors (e.g., HSV-1 vectors) described herein can exhibit features that are advantageous for use in treating disease in a subject, e.g., in an ICI-refractory subject, e.g., in treating cancer.

[0105] In certain embodiments viral vectors as described herein exhibit (e.g., when administered to a subject) delayed oncolysis. Delayed oncolysis allows for viral persistence in target (e.g., cancer) cells, sustained payload expression, exposure of tumor-associated antigens within a payload-primed tumor microenvironment, and limited non-specific inflammation and tissue damage. Delayed oncolysis allows for viral persistence in target cells, sustained payload expression, exposure of tumor-associated antigens within a payload- primed tumor microenvironment, and limited non-specific inflammation and tissue damage.

[0106] In certain embodiments, the viral vector exhibits (e.g., when administered to a subject) increased immunogenicity. Immunogenicity can be measured, for example, by detecting increased Tap1 gene expression and / or increased MHC class I expression (HLA). As described in the examples herein, methods for measuring Tap1 gene expression are known in the art and include, for example, RNA-seq analysis. Methods for measuring HLA expression are known in the art, and include, for example, flow cytometry.

[0107] In certain embodiments, the viral vector exhibits (e.g., when administered to a subject) increased immune activation. Immune activation can be measured, for example, by detecting the increase or decrease in expression of RNAs in pathways relevant to immune cell activation, including, but not limited to, IFN-α response pathways, inflammatory response pathways, myc signaling pathways, and / or MtorC1 signaling pathways. Genes involved in such pathways are described at the Gene Set Enrichment Analysis (GSEA) website. See, forexample, gsea-msigdb.org / gsea / msigdb / collections.jsp. Immune activation can also be detected by detecting increased expression of TNF super family members, including tumor necrosis factor-alpha (TNF), lymphotoxin beta (LTB), APRIL (TNFSF13), and LIGHT (TNFSF14) and / or detecting altered expression of immunogenic cell death regulators indicative of an increase pyroptosis and / or apoptosis, such as detecting an increase in expression of PYCARD and Caspase 10 (CASP10).

[0108] In certain embodiments, administration of a viral vector induces formation of tertiary lymphoid structures (TLS) in a tumor bed. TLS can be measured, for example, by detecting a localized increase in expression of CD11c-positive dendritic cells, CD4-positive T cells, and B220-positive B cells.

[0109] In certain embodiments, administration of a viral vector causes a reduction in cancer cell viability. Cancer cell viability can be determined, for example, by measuring tumor volume or counting live cancer cells. III. Payload

[0110] Viral vectors in accordance with the present disclosure comprise a nucleic acid molecule comprising the payload of the vector. A payload comprises a nucleic acid molecule that encodes one or more polypeptides. It is contemplated that the payload can comprise a nucleic acid molecule that comprises a sequence complementary to a nucleic acid sequence that encodes a polypeptide. The payload can encode a nucleic acid molecule that has a regulatory function, e.g., a small interfering RNA (siRNA) polynucleotide or a micro RNA (miRNA) polynucleotide.

[0111] The payload can be a nucleic acid molecule that encodes a protein that is exogenous to the target tissue or subject to which the vector is administered. For example, the payload can be a nucleic acid molecule that encodes a protein that is endogenous to the target tissue or subject to which the vector is administered. In some embodiments, a nucleic acid molecule is codon optimized.

[0112] The nucleic acid comprising the payload of the vector can encode, for example 1, 2, 3 ,4, 5, 6, 7, 8, 9, or 10 therapeutic polypeptides. In certain embodiments, the payload of the vector encodes 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, or 5 to 10 therapeutic polypeptides.

[0113] The therapeutic polypeptides encoded on the vector can have one or more of the following functional attributes: (1) target the stroma of a tumor; (2) support T cell (e.g., CAR T cell) survival in a tumor microenvironment; (3) induce TLS in a tumor bed; (4) promote phagocytic innate immune surveillance (e.g., by disrupting the Sirpα / CD47 axis); (5) promote upregulation or downregulation of one or more proteins associated with a response to ICI (e.g., IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21); and / or (6) reduce cancer cell viability.

[0114] One or more therapeutic polypeptides from one or more categories (1-6 above) can be encoded on the same vector to target multiple pathways for increasing immune activity against a tumor. For example, a vector can encode 1, 2, or 3 therapeutic polypeptides from category 1 and 1, 2, or 3 therapeutic polypeptides from category 2, 3, 4, 5, or 6. Alternatively, a vector can encode 1, 2, or 3 therapeutic polypeptides from category 2 and 1, 2, or 3 therapeutic polypeptides from category 3, 4, 5, or 6. Alternatively, a vector can encode 1, 2, or 3 therapeutic polypeptides from category 3 and 1, 2, or 3 therapeutic polypeptides from category 4, 5, or 6. Alternatively, a vector can encode 1 or 2 therapeutic polypeptides from category 1 and 1 or 2 therapeutic polypeptides from category 2, 3, 4, 5, and / or 6. Alternatively, a vector can encode 1 or 2 therapeutic polypeptides from category 2 and 1 or 2 therapeutic polypeptides from category 3, 4, 5, and / or 6. Similarly, a vector can encode 1 or 2 therapeutic polypeptides from category 3 and 1 or 2 therapeutic polypeptides from category 4, 5, and / or 6.

[0115] In certain embodiments, the therapeutic polypeptide includes TNF, IL-1β, IL- 6 IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene (e.g., an extracellular domain of SIRPα and an Fc fragment of human IgG1), an anti-CD47 antibody, IL-1, and / or IL-17.

[0116] In certain embodiments, the therapeutic polypeptide includes IL-21, LTA, LTB, CXCL12, and / or VEGF-C.

[0117] In certain embodiments, the therapeutic polypeptide includes hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19, CCL21, IL-7, IL-12,IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene (e.g., an extracellular domain of SIRPα and an Fc fragment of human IgG1), an anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, and / or VEGF-C.

[0118] In certain embodiments, the therapeutic polypeptide includes LTA, CCL19, TNF, IL-17, and / or IL-7.

[0119] In certain embodiments, the therapeutic polypeptide includes LTB, CCL19, CCL21, CXCL13, and / or IL-7.

[0120] In certain embodiments, the therapeutic polypeptide includes IL-12 and IL-15.

[0121] The therapeutic polypeptide(s) can be encoded on the same vector or different vectors.

[0122] The therapeutic polypeptides in the payload can be cloned in tandem and driven from the same promoter, with a linker sequence separating the individual polypeptides. In certain embodiments, the linker sequence comprises a ribosomal skipping site (sequence) such as a 2A peptide. In an expression construct, when such a ribosomal skipping site is included in a linker positioned between two polypeptide sequences, translation of the transcribed mRNA results in production of the two polypeptides separately, rather than as a fusion protein. The 2A peptide sequences share a core sequence motif of DXEXNPGP (SEQ ID NO: 45), wherein X is any amino acid. Non-limiting examples of suitable 2A peptide sequences include T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 46), P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 49), E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 47) and F2A (VKQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 48). Additionally, an optional Gly-Ser- Gly (GSG) tripeptide can be added to the N-terminal end of a 2A peptide to enhance efficiency. A. Targeting the Tumor Stroma

[0123] A viral vector can encode one or more therapeutic polypeptides that target the tumor stroma and / or activate the local endothelium to increase T cell infiltration of the tumor, thereby to reduce the viability of cancer cells within the tumor and decrease the size of the tumor.

[0124] The tumor stroma can be targeted by different mechanisms. For example, the extracellular matrix (ECM) can be dissolved by proteins involved in ECM degradation,including, but not limited to, hyaluronidase, MMP-9, and an inhibitor of lysyl oxidase. Accordingly, in certain embodiments, a viral vector encodes one or more therapeutic polypeptides involved in ECM degradation, including, but not limited to, hyaluronidase, MMP-9, and an inhibitor of lysyl oxidase.

[0125] Additionally, cancer-associated fibroblasts can be targeted by proteins interfering with the TGFβ pathway. Accordingly, in certain embodiments, a viral vector encodes one or more therapeutic polypeptides that interfere with the TGFβ pathway, including, for example, a polypeptide that includes an external domain (e.g., non- transmembrane domain) of TGFβ. In certain embodiments, the one or more therapeutic polypeptides includes a fusion protein comprising an external domain of TGFβ, an IgG domain (e.g., an Fc domain), and an external domain of TGFβRII.

[0126] The local endothelium can be activated to favor T cell infiltration by expressing proinflammatory cytokines, including, but not limited to, TNF, IL-1β, IL-6, and IL-18. Expression of enzymes that degrade the tumor ECM will simultaneously disrupt physical barriers within the tumor to allow for immune cell infiltration. Accordingly, in certain embodiments, a viral vector encodes one or more proinflammatory cytokines, including, for example, TNF, IL-1β, IL-6, and IL-18.

[0127] Tumor stroma targeting and local endothelium activation can be modulated together or separately by localized expression of these therapeutic polypeptides.

[0128] In certain embodiments, one or more genes encoding hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, a one or more therapeutic polypeptides interfering with the TGFβ pathway, TNF, IL-1β, IL-6, or IL-18 are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, e.g., using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0129] In certain embodiments, the effects on the tumor stroma or local endothelium by cancer cells secreting factors that dissolve the tumor stroma or activate the local endothelium for T cell infiltration can be assessed in vitro or in vivo using methods known in the art. B. Supporting T Cell (e.g., CAR T Cell) Survival in the Tumor Microenvironment

[0130] In certain embodiments, a viral vector encodes one or more therapeutic polypeptides that support T cell survival in the tumor microenvironment (TME). In theembodiments described herein, the T cell can be a native T cell (e.g., a T cell existing in the subject to whom a viral vector is administered) or a CAR T cell (e.g., a CAR T cell that has been administered to the subject). In certain embodiments, the viral vector is administered with a T cell (e.g., a CAR T cell), for example, to treat a tumor.

[0131] T cells in the TME can be supported by different mechanisms. For example, the recruitment of T cells, such as native or CAR T cells, to the tumor site can be improved by the presence of T cell recruitment factors such as the chemokine ligands CCL19 or CCL21 in the TME.

[0132] The local T cells can be supported to elicit a strong and durable anti-tumor response. For example, the local expression of T cell trophic factors such as IL-7, IL-12, IL- 15, IL-18, and IFN-γ can elicit a strong and durable anti-tumor response from the local or recruited T cells. Accordingly, in certain embodiments, a viral vector encodes one or more T cell trophic factors, including, for example, IL-7, IL-12, IL-15, IL-18, and IFN-γ. In certain embodiments, a viral vector encodes IL-12 and IL-15.

[0133] Additionally, TGFβ signaling in cells within the tumor can be suppressed by soluble TGFβRII expression. Accordingly, in certain embodiments, the one or more therapeutic polypeptides comprises soluble TGFβRII.

[0134] Local expression of a gene encoding a co-stimulatory molecule such as CD40L or OX40L, depending on the target TME, can stimulate local or recruited T cells. Accordingly, in certain embodiments, the one or more therapeutic polypeptides comprises CD40L or OX40L.

[0135] T cell support or T cell recruitment can be modulated together or separately by these therapeutic polypeptides.

[0136] In certain embodiments, one or more genes encoding CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFN-γ, soluble TGFβRII, CD40L, or OX40L are cloned into a non- replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, e.g., using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0137] Effects on the T cell recruitment or T cell function by cancer cells secreting these factors can be assessed in vitro or in vivo using methods known in the art.C. Supporting NK Cell (e.g., CAR NK Cell) Survival in the Tumor Microenvironment

[0138] In certain embodiments, a viral vector encodes one or more therapeutic polypeptides that support NK cell survival in the tumor microenvironment (TME). In the embodiments described herein, the NK cell can be a native NK cell (e.g., a NK cell existing in the subject to whom a viral vector is administered) or a CAR NK cell (e.g., a CAR NK cell that has been administered to the subject). In certain embodiments, the viral vector is administered with an NK cell (e.g., a CAR NK cell), for example, to treat a tumor.

[0139] NK cells in the TME can be supported by different mechanisms. For example, the recruitment of NK cells, such as native or CAR NK cells, to the tumor site can be improved by the presence of NK cell recruitment factors such as the chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, and CXCL11 in the TME.

[0140] The local NK cells can be supported to elicit a strong and durable anti-tumor response. For example, the local expression of NK cell trophic factors such as IL-2, IL-15, IL-18, and IFN-α can elicit a strong and durable anti-tumor response from the local or recruited NK cells. Accordingly, in certain embodiments, a viral vector encodes one or more NK cell trophic factors, including, for example, IL-2, IL-15, IL-18, and IFN-α.

[0141] Additionally, TGFβ signaling in cells within the tumor can be suppressed by soluble TGFβRII expression. Accordingly, in certain embodiments, the one or more therapeutic polypeptides comprises soluble TGFβRII.

[0142] NK cell support or NK cell recruitment can be modulated together or separately by these therapeutic polypeptides.

[0143] In certain embodiments, one or more genes encoding CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11 IL-2, IL-15, IL-18, IFN-α, or soluble TGFβRII, are cloned into a non-replicating McKrae strain HSV-1 vector. The McKrae strain HSV-1 vector can be packaged into a McKrae strain HSV-1 virus, e.g., using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0144] Effects on the NK cell recruitment or NK cell function by cancer cells secreting these factors can be assessed in vitro or in vivo using methods known in the art.D. Inducing Tertiary Lymphoid Structures (TLS) in a Tumor Bed

[0145] The tumor microenvironment (TME), which supports tumor growth and resistance to treatment by providing a physical and functional barrier to tumor infiltrating lymphocytes (TILs), includes a heterogeneous collection of cells, signaling molecules, blood vessels, and ECM. TME components are often non-responsive to classical immunotherapy, as TILs fail to be effectively recruited and remain activated within the immunosuppressive TME. Increase of TILs within or in the proximity of immune-infiltrated tumors, termed immunologically ‘hot’ tumors, is associated with the formation of TLS, which are ectopic lymphoid formations that occur in non-lymphoid tissue and are structurally and functionally similar to secondary lymphoid organs (e.g., lymph nodes). TLS contain spatially-defined B and T cell zones, high endothelial venules for naïve T cell migration and, in some cases, fully formed germinal centers, hubs of B cell affinity maturation, and clonal development. Functionally, TLS act as a functional niche for the maturation of the T and B cell response against locally-displayed tumor antigens, and TLS support the organization of a robust immune response against solid tumors (Johansson-Percival et al. (2017) NATIMMUNOL., 18(11): 1207-1217; and Tang et al. (2016) CANCER CELL, 30(3): 500).

[0146] TLS formation is associated with improved efficacy of immunotherapy in mice and humans (Sautès-Fridman et al. (2019) NAT REV CANCER, 19(6): 307-325), such as by providing ICI-activated TILs with recruitment and survival signals within the tumor itself. Three independent research groups have concluded that the presence of TLS in subjects treated with ICI therapies were associated with a strong B cell response and increased patient survival in both melanoma and colorectal cancer, underlying the relevance of TLS in cancer progression and response to therapy (Cabrita et al. (2020) NATURE, 577(7791): 561-565; and Helmink et al. (2020) NATURE, 577(7791): 549-555). However, a therapeutic treatment to induce TLS in ‘cold,’ ICI-refractory tumors is an unmet need.

[0147] The formation and functionality of TLS during disease development and / or progression has been shown to be dependent on antigen exposure in the context of the generation of specific responses against antigens or autoantigens (Barone et al. (2016) FRONT IMMUNOL., 7: 477). Factors regulating TLS include secreted and cell-surface expressed proteins in the context of an inflamed microenvironment rich in cytokines such as TNF, IL-6, IL-17, and IL-22. Additionally, it has been demonstrated that delivery of adenovirus alone is able to induce transient TLS formation (Bombardieri et al. (2012) J IMMUNOL.189(7): 3767- 76), highlighting the natural response against antigens released in the early phases of the viralinfection as priming elements for TLS formation. However, it is not well understood which combination of factors are sufficient to initiate TLS establishment.

[0148] Provided herein, in certain embodiments, are viral vectors encoding one or more therapeutic polypeptides that induce TLS within the tumor bed. In certain embodiments, inducing TLS within the tumor bed comprises promoting the number, size, and / or organization of TLS structures.

[0149] As such, a viral vector encoding therapeutic polypeptides that induce TLS formation can be used according to the methods described herein. In certain embodiments, the viral vector is used in combination with additional solid tumor therapeutic agents including, but not limited to, CAR T cell therapy and ICI.

[0150] In certain embodiments, one or more genes encoding one or more therapeutic polypeptides involved in TLS establishment and maintenance, including, but not limited to, CCL19, lymphotoxin β, CXCL13, and TNF, are cloned into an HSV-1 vector (e.g., comprising an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins). The HSV-1 vector can be packaged into an HSV-1 virus, e.g., using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting HSV-1 virus.

[0151] Effects on the TLS within the tumor bed by cancer cells secreting these factors can be assessed in vitro or in vivo using methods known in the art. E. Promoting Phagocytic Innate Immune Surveillance, For Example, by Disrupting the Sirpα / CD47 Axis

[0152] In certain embodiments, a viral vector encodes one or more therapeutic polypeptides that promote phagocytic innate immune surveillance and elimination of tumor cells, for example, by disrupting the signal-regulatory protein α (SIRPα) / CD47 axis.

[0153] CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with SIRPα on macrophages, enabling the escape of these cancer cells from macrophage-mediated phagocytosis. Promotion of the phagocytic innate immune surveillance and elimination of tumor cells can be induced by the disruption of the Sirpα / CD47 axis, for example, by the presence of a Sirpα- IgG fusion transgene. Disruption of the Sirpα / CD47 axis can promote phagocytic innate immune surveillance and elimination of tumor cells by unmasking the tumor ‘eat me’ signals. Accordingly, in certain embodiments, a viral vector encodes a one or more therapeuticpolypeptides that disrupts the Sirpα / CD47 axis, thereby promoting phagocytic innate immune surveillance and elimination of tumor cells. In certain embodiments, the one or more therapeutic polypeptides is an anti-CD47 antibody. In certain embodiments, the viral vector encodes a Sirpα-IgG fusion polypeptide (e.g., extracellular domain of SIRPα and an Fc fragment of human IgG1).

[0154] Additionally, the vector can encode therapeutic polypeptides to support anti- tumor macrophage polarization, including, but not limited to, a Sirpα-IgG fusion transgene, TNF, IL-1, IL-12, IL-17, and IFN-γ.

[0155] In certain embodiments, one or more therapeutic polypeptides involved in TLS establishment and maintenance in the tissue, including, but not limited to, a Sirpα-IgG fusion transgene, TNF, IL-1, IL-12, IL-17, and IFN-γ, can be cloned into a non-replicating McKrae strain HSV-1 vector.

[0156] Effects on phagocytic innate immune surveillance by cancer cells secreting these factors can be assessed in vitro or in vivo using methods known in the art. F. Promoting Regulation of Proteins Associated with a Response to an Immune Checkpoint Inhibitor

[0157] The data provided herein (see, for example, Example 3 and FIG.12) demonstrate that administration of an HSV-1 vector comprising an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins, even without a functional payload, promotes upregulation or downregulation of one or more proteins associated with a response to ICI. Accordingly, it is contemplated that an HSV-1 vector comprising an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins is useful for treating an ICI-refractory subject. It is further contemplated that an HSV-1 vector comprising an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins can be used in combination with an ICI to treat cancer in a subject.

[0158] The viral vectors described herein can include one or more therapeutic polypeptides predicted to drive therapeutic response to ICI, including factors with reported roles in TLS formation including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0159] Effects on the expression of proteins, such as those associated with a response to an ICI, can be assessed using methods known in the art, such as the use of Gene Set Enrichment Analysis (GSEA) of RNA-seq data.IV. Regulatory Elements

[0160] The inclusion of non-native regulatory sequences, gene control sequences, promoters, non-coding sequences, introns, or coding sequences in a nucleic acid of the present disclosure are contemplated herein. The inclusion of nucleic acid tags or signaling sequences, or nucleic acids encoding protein tags or protein signaling sequences, are further contemplated herein. Typically, the coding region is operably linked with one or more regulatory nucleic acid components.

[0161] A promoter included in a nucleic acid of the present disclosure can be a tissue- or cell type-specific promoter, a promoter specific to multiple tissues or cell types, an organ- specific promoter, a promoter specific to multiple organs, a systemic or ubiquitous promoter, or a nearly systemic or ubiquitous promoter. Promoters having stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, inconstant, or unpredictable expression are also included within the scope of the present disclosure. A promoter of the present disclosure may include any of the above characteristics or other promoter characteristics known in the art.

[0162] Examples of known promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, CMV / human beta 3 globin promoter, GFAP promoter, chicken beta actin (CBA) promoter, the β-glucuronidase (GUSB) promoter, and ubiquitin promoters, such as those isolated from human ubiquitin A, human ubiquitin B, and human ubiquitin C.

[0163] In some embodiments, a promoter is a neuron-specific promoter in that it is a promoter having specific expression in neurons, or that typically drives expression of an associated coding sequence in a subset of neurons but not in one or more other tissues or cell types. Examples of such promoters include calcitonin gene-related peptide (CGRP), synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase, microtubule-associated protein IB (MAP1B), and platelet-derived growth factor beta chain promoters, as well as derivatives thereof. In some embodiments, the promoter is a calcitonin gene-related peptide (CGRP) promoter or derivative thereof.

[0164] Other regulatory elements may additionally be operatively linked to the payload, such as an enhancer and a polyadenylation site. In some embodiments, an enhancer comprises a human cytomegalovirus (HCMV) sequence. In some embodiments, a polyadenylation site comprises a bovine growth hormone (BGH) polyadenylation signal.

[0165] In some embodiments, a promoter is a chimera of one or more promoters or regulatory elements found in nature. In some embodiments, the viral vectors comprise a payload whose expression is driven by a CGRP promoter with an HCMV enhancer sequence. V. Preparation of Vectors

[0166] The present disclosure relates particularly to McKrae strain viral vectors that are replication-defective. Viral vectors can be generated by mutation (e.g., deletion) of one or more immediate early genes or regulatory sequences that affect the expression thereof. Viral genes can be mutated using methods of recombinant technology known in the art. A viral vector of the present disclosure may be rendered replication-defective as a result of a homologous recombination event. Replication-defective viral vectors can be generated by mutation of an ICP4 gene and mutation of an ICP47 gene. For example, replication-defective viral vectors are generated by deletion of an ICP4 gene and deletion of an ICP47 gene.

[0167] Viral vectors of the present disclosure can be generated by deletion of loci encoding one or more ICP genes (e.g., ICP4 and ICP47) through homologous recombination. In some embodiments, generation of a viral vector of the present disclosure includes a step of homologous recombination of a first plasmid with a second plasmid. In some embodiments, the first plasmid contains nucleic acid sequences homologous to regions of an HSV genome that are adjacent to a nucleic acid region of an HSV genome that is intended to be replaced. In some embodiments, the second plasmid contains an HSV genome or fragment thereof. In some embodiments, the first plasmid contains nucleic acid sequence encoding a gene of interest between the homologous nucleic acid sequences. In some embodiments, the gene of interest may be or include a marker protein that is detectable by fluorescence, chemiluminescence, or other property, which can be used to select for vectors resulting from successful homologous recombination.

[0168] A viral vector of the present disclosure can be generated by homologous recombination of a first plasmid containing a nucleic acid sequence homologous to regions upstream of the ICP4 promoter including the viral origin contained within the short inverted repeat regions of HSV, with a second plasmid containing an HSV McKrae strain genome.

[0169] A vector can be generated by first replacing both copies of the ICP4 loci by homologous recombination using plasmid SASB3 and screening for green fluorescent protein (GFP)-expressing plaques. For example, a plasmid can be constructed by cloning the SphI to AflIII (SalI linkered) fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides124,485-126,413, KT899744, KOS strain) into SphI / SalI digested pSP72 followed by insertion of the 695 bp BglII to BamHI fragment (GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), KOS strain, nucleotides 131,931 to 132,626) containing regions upstream of the ICP4 promoter including the viral origin contained within the short inverted repeat regions into the BglII to BamHI sites of the vector plasmid. In some embodiments, a plasmid is constructed by cloning a HCMV-eGFP fragment in the BamHI site of a plasmid as described above. A plasmid as described above then can be recombined into a specific locus of a wild-type McKrae virus. The resulting viral vector can be isolated using a stable cell line that expresses one or more genes deleted or disrupted in the HSV genome that are required for replication.

[0170] In another approach, a vector can be generated by first replacing both copies of the ICP4 loci by homologous recombination using plasmid SDAXB and screening for GFP-expressing plaques. For example, a plasmid can be constructed by cloning the SphI to AflIII fragment (1,928 bp) of the HSV-1 KOS strain genome (nucleotides 124,346 to 126,273 of GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), KOS strain) into SphI / AflIII digested pSP72 to make plasmid SDA followed by changing the Afl III site to a BamHI site to make plasmid SDAB. A BamHI to BglII DNA PCR fragment containing regions upstream of the ICP4 promoter including the viral origin (nucleotides 144,933 to 145,534 of GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain) contained within the short inverted repeat regions can be cloned into the BamHI site of plasmid SDAB to make plasmid SDAXB. Next, a HCMV-eGFP fragment can be cloned into the BamHI site of a plasmid as described above. Then, the plasmid can be recombined into a specific locus of a wild-type McKrae virus. The resulting vector can be isolated using a stable cell line that expresses one or more genes deleted or disrupted in the HSV genome that are required for replication.

[0171] In another approach, a vector that includes a combined ICP4 and ICP47 deletion can be generated. For example, a plasmid sequence comprising McKrae sequence nucleotides 143,521 to 144,562 and 144,640 to 145,534 (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain) can be synthesized, and an SCMV promoter flanked by Pme I sites can be cloned between the flanking sequences. Next, a marker, for example, a red fluorescent marker, can be cloned into the plasmid so that it is operably linked to the SCMV promoter. A plasmid as described above can be recombined into a specific locus of a McKrae virus with a deletion in the ICP4 locus. Fluorescent clones can be isolated, and the insertion of the construct into the ICP47 locus can be confirmed by PCR.The resultant recombined McKrae virus has deletions of nucleotides 144,562 to 144,640 according to a reference sequence (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain). In some embodiments, the resulting viral vector is isolated using a stable cell line that expresses one or more genes deleted or disrupted in the HSV genome that are required for replication. VI. Characterization of Vectors

[0172] Viral vectors in accordance with the present disclosure can be characterized by genomic sequencing in order to determine if the expected vector was successfully created. Any method of sequencing known in the art is acceptable for this purpose. Methods of sequencing include, for example, nanopore sequencing, single molecule real time sequencing (SMRT), DNA nanoball (DNB) sequencing, pyrosequencing, and DNA arrays.

[0173] The expression of a payload from a viral vector can be detected by any method known in the art for detecting proteins or nucleic acids. Methods of detecting protein expression include immunohistochemistry, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), immune-electron microscopy, individual protein immunoprecipitation (IP), protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), immunoelectrophoresis, spectrophotometry, and bicinchoninic acid assay (BCA). Methods of detecting nucleic acid expression include Southern blotting, Northern blotting, polymerase chain reaction (PCR), quantitative PCR, and RT-PCR. VII. Formulations and Mode of Administration

[0174] Compositions comprising viral vectors as described herein may be formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions); tablets, e.g., those targeted for buccal, sublingual, and systemic absorption; boluses; powders; granules; pastes for application to the tongue; parenteral administration; for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0175] Administration can be intratumoral, parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Preferred routes of delivery include intratumoral administration. In some embodiments, pharmaceutical compositions include a viral vector in combination with a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, viral vectors are formulated in glycerol. In some embodiments, viral vectors are formulated in approximately 10% glycerol in phosphate buffered saline.

[0176] It is advantageous to formulate compositions 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 subject to be treated; each unit containing a predetermined quantity of a viral vector calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0177] As used herein, the term “administration” refers to the administration of a composition to a subject. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and vitreal. Depending upon the circumstances, administration may involve intermittent dosing and / or may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0178] The pharmaceutical composition can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks; or about 4, 5, or 6 weeks, etc. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Treatment of a subject with a viral vector can include a single treatment or, in many cases, can include a series of treatments.

[0179] In certain embodiments, the pharmaceutical composition can be administered to a patient more than one time (e.g., two, three, four, five, six, seven, eight, nine, ten, or more times), for example, as a result of the improved safety profile exhibited by the vectors described herein.

[0180] In some embodiments, the active agents, i.e., a viral vector of the disclosure and / or other agents to be administered together with a viral vector of the disclosure, are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. In some embodiments the composition is targeted to particular cell types or to cells that are infected by a virus. VIII. Methods of Treatment

[0181] Viral vectors in accordance with the present disclosure are useful for a wide variety of therapeutic applications. In one aspect, the disclosure relates to a method of treating cancer in an immune checkpoint inhibitor (ICI)-refractory subject. The method includes administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins. The vector can include a nucleic acid encoding one or more therapeutic polypeptides that function to reduce the size of a tumor. For example, the therapeutic polypeptides can include one or more therapeutic polypeptides predicted to drive therapeutic response to ICI, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0182] In addition, Applicant has surprisingly discovered that administering to a subject in need thereof a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins and expressing IL-12 and IL-15 results in a synergistic reduction of tumor size, thereby treating cancer. Thus, in certain embodiments, the disclosure relates to a method of treating cancer comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins andexpressing IL-12 and IL-15 results in a synergistic effect in reducing tumor size, thereby to treat the cancer. The reduction in tumor size is synergistic as compared to the reduction in tumor size induced by a vector (e.g., an otherwise identical vector) comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins and expresses IL-12 or IL- 15, but not both IL-12 and IL-15. In certain embodiments, the administration of the vector comprising the herpes simplex virus (HSV) genome that expresses IL-12 and IL-15 results in synergistic tumor regression as compared to administration of a herpes simplex virus. The tumor regression is synergistic as compared to the tumor regression induced by a vector (e.g., an otherwise identical vector) comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins and expresses IL-12 or IL-15, but not both IL-12 and IL-15.

[0183] In certain embodiments, the method further includes administering one or more ICI.

[0184] In certain embodiments, the cancer is an epithelial cancer. In certain embodiments, the cancer is a solid tumor, soft tissue tumor, hematopoietic tumor or metastatic lesion. In certain embodiments, the solid tumor is a sarcoma, adenocarcinoma, or carcinoma. In certain embodiments, the solid tumor is a head and neck (e.g., pharynx), thyroid, lung (e.g., small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital or genitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cell, e.g., neuroblastoma or glioma), or skin (e.g., melanoma) tumor. In certain embodiments, the hematopoietic tumor is a leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B- cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), lymphoma, Hodgkin’s disease, malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation). In certain embodiments, the cancer is selected from an endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer, fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer and liver cancer. In certain embodiments, the cancer is a breast cancer. In certainembodiments, the cancer is a lung cancer.Also provided are methods of expressing a therapeutic polypeptide in a subject, e.g., an ICI-refractory subject. The method includes administering a vector comprising a HSV genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins. The vector can include a nucleic acid encoding one or more therapeutic polypeptides that functions to reduce the size of a tumor. For example, the therapeutic polypeptides can include one or more therapeutic polypeptides predicted to drive therapeutic response to ICI, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21. In certain embodiments, the method further includes administering one or more ICI.

[0185] Also provided are methods of reducing the size of a tumor in a subject, e.g., an ICI-refractory subject. The method includes administering a vector comprising a HSV genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins. The vector can include a nucleic acid encoding a therapeutic polypeptide that functions to reduce the size of the tumor. For example, the therapeutic polypeptides can include one or more therapeutic polypeptides predicted to drive therapeutic response to ICI, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0186] In addition, Applicant has surprisingly discovered that administering to a subject in need thereof a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins and expressing IL-12 and IL-15 results in a synergistic effect in reducing tumor size. Thus, in certain embodiments, the disclosure relates to a method of treating cancer comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises an alteration that prevents the expression of one or more functional ICP4 and ICP47 proteins and expressing IL-12 and IL- 15 results in a synergistic effect in reducing tumor size.

[0187] In certain embodiments, the method further includes administering one or more ICI.

[0188] Vectors as described herein are useful to deliver one or more payloads to one or more target cells. In certain embodiments, the payload persists in target cells for up to 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, a year, or more than a year.

[0189] Target cells may reside in tissues that are poorly vascularized and difficult to reach by systemic circulation. Target cells may be susceptible to infection by HSV. In certain embodiments, target cells are particularly susceptible to infection by a McKrae strain of HSV.

[0190] Viral vectors in accordance with the present disclosure are useful for delivering one or more therapeutic polypeptides to a cell, e.g., a cell in a subject. For example, viral vectors comprising a heterologous nucleic acid segment operably linked to a promoter are useful for any disease or clinical condition associated with reduction or absence of the protein encoded by the heterologous nucleic acid segment, or any disease or clinical condition that can be effectively treated by expression of the encoded protein within the subject. Viral vectors that contain an expression cassette for synthesis of an RNAi agent (e.g., one or more siRNAs or shRNAs) are useful in treating any disease or clinical condition associated with overexpression of a transcript or its encoded protein in a subject, or any disease or clinical condition that may be treated by causing reduction of a transcript or its encoded protein in a subject. Viral vectors that comprise an expression cassette for synthesis of one or more RNAs that self-hybridize or hybridize with each other to form an RNAi agent targeted to a transcript encoding a cytokine may be used to regulate immune system responses (e.g., responses responsible for organ transplant rejection, allergy, autoimmune diseases, inflammation, etc.). Viral vectors that provide a template for synthesis of one or more RNAs that self-hybridize or hybridize with each other to form an RNAi agent targeted to a transcript of an infectious agent or targeted to a cellular transcript whose encoded product is necessary for or contributes to any aspect of the infectious process may be used in the treatment of infectious diseases.

[0191] A therapeutic polypeptide of the disclosure can include one or more of the following functional attributes: (a) targets the stroma of a tumor; (b) supports T cell (e.g., CAR T cell) and / or NK cell survival in a tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in a tumor bed; (d) promote phagocytic innate immune surveillance (e) disrupts the Sirpα / CD47 axis;(f) promotes upregulation or downregulation of one or more proteins associated with a response to ICI (e.g., IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21); and / or (g) reduces cancer cell viability.

[0192] Exemplary therapeutic polypeptides are discussed above in Section III, and are incorporated herein. IX. Combination therapy

[0193] A composition described herein can be administered in combination with one or more additional therapies, e.g., surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include chemotherapy, e.g., a cytotoxic agent. In certain embodiments the additional therapy may include a targeted therapy, e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy may include an anti- inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, e.g., a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g. methotrexate), or an NSAID. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0194] The viral vectors of the disclosure can be administered in combination with an ICI. The ICI may, for example, be selected from a PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, adenosine A2A receptor inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, BTLA inhibitor, KIR inhibitor, LAG3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, and TIGIT inhibitor.

[0195] Exemplary ICIs include a PD-1 inhibitor (e.g., an anti-PD-1 antibody e.g., pembrolizumab, nivolumab, or cemiplimab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab), a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab), and a LAG-3 inhibitor (e.g., relatlimab).

[0196] The ICI can be a PD-1 or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T-cells that serves as an immune system checkpoint that inhibits or otherwise modulates T- cell activity at the appropriate time to prevent an overactive immune response. Cancer cells, however, can take advantage of this checkpoint by expressing ligands, for example, PD-L1,that interact with PD-1 on the surface of T cells to shut down or modulate T cell activity. Exemplary PD-1 / PD-L1-based ICI include antibody-based therapeutics. Exemplary treatment methods that employ PD-1 / PD-L1-based ICI are described in U.S. Patent Nos. 8,728,474 and 9,073,994, and EP Patent No.1537878B1, and, for example, include the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos.8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos.9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0197] The viral vectors of the disclosure can be administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on a T cell with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of an antigen presenting cells (rather than cancer cells) leads to T cell inhibition. Exemplary CTLA-4 based ICI methods are described in U.S. Patent Nos.5,811,097, 5,855,887, 6,051,227. Exemplary anti- CTLA-4 antibodies are described in U.S. Patent Nos.6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International (PCT) Publication Nos. WO98 / 42752, WO00 / 37504, and WO01 / 14424, and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0198] The viral vectors of the disclosure can be administered in combination with (i) a PD-1 or PD-L1 inhibitor, e.g., a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) CTLA-4 inhibitor, e.g., a CTLA-4 inhibitor disclosed herein.

[0199] The viral vectors of the disclosure can be administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0200] Exemplary cytotoxic agents that can be administered in combination with a method or composition described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors,alkylating agents, platinating agents, inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folate analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis and radiation, or antibody molecule conjugates that bind surface proteins to deliver a toxic agent. In one embodiment, the cytotoxic agent that can be administered with a method or composition described herein is a platinum-based agent (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicin, anthracyclines (e.g., doxorubicin or epirubicin) daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.

[0201] In some embodiments, provided compositions include one or more such other active agents. In some embodiments, such other active agents are provided as part of distinct compositions. In some embodiments, combination therapy involves simultaneous administration of one or more doses or units of two or more different active agents and / or therapeutic modalities. In some embodiments, combination therapy involves simultaneous exposure to two or more different active agents and / or therapeutic modalities, for example through overlapping dosing regimens.

[0202] In some embodiments, provided compositions include or are administered in combination with one or more other active agents, e.g., an ICI, useful for the treatment of the relevant disease, disorder, and / or condition.

[0203] Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.EXAMPLES

[0204] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0205] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation and lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Example 1: IFN-γ Payload in HSV-1 mICP4 / mICP47 Vector for Oncolysis

[0206] This example describes the assessment of elicitation of an IFN-γ response gene signature and oncolysis of an HSV-1 mICP4 / mICP47 vector encoding a IFN-γ payload (HSV-1 mICP4 / mICP47 IFN-γ).

[0207] Hs578T human breast cancer cells were uninfected or infected with HSV-1 mICP4 / mICP47 IFN-γ (between 3-10 plaque forming units (PFU) / cell) and the expression profiles of genes related to the IFN-γ response signature were assessed (FIG.1). Cell viability was assessed by RealTimeGlo™ assay (RealTime-Glo™ MT Cell Viability Assay (Promega Corp., Madison, WI)) (FIG.2).

[0208] Hs578T cells were infected with 3 PFU / cell of HSV-1 mICP4 / mICP47 GFP or HSV-1 mICP4 / mICP47 IFN-γ and then cocultured with peripheral blood mononuclear cells (PBMC) for 24 hours. T-sne (t-distributed Stochastic Neighbor Embedding) analysis was used to visualize changes in PBMC populations and was assessed by flow cytometry. Dimensionality reduction (t-sne analysis) demonstrated robust phenotypic changes in several immune cell populations upon infection with HSV-1 mICP4 / mICP47 IFN-γ (data not shown).

[0209] Taken together, these results demonstrated that an HSV-1 mICP4 / mICP47 IFN-γ vector mediated a strong IFN-γ response gene signature, and that infection with HSV-1 mICP4 / mICP47 IFN-γ mediated oncolysis in a dose-dependent manner. Example 2: mCXCL13, mCCL19, and mIL-7 Payloads in HSV-1 mICP4 / mICP47 Vector for Oncolysis

[0210] This example describes the vector characterization and assessment of oncolysis of three HSV-1 mICP4 / mICP47 vectors encoding murine (m)CXCL13, mCCL19, and mIL-7, respectively (HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, and HSV-1 mICP4 / mICP47 mIL-7, respectively).

[0211] Hs578T human breast cancer cells were uninfected or infected with HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, or HSV-1 mICP4 / mICP47 mIL-7 (0, 3, or 10 PFU / cell) and payload production rates were evaluated. As shown in FIG. 3, there was a dose-dependent increase in mCXCL13, mCCL19, and mIL-7 payload production with maximal production within the first 2 days after infection in both the group dosed with 3 PFU / cell as well as the group dosed with 10 PFU / cell. Cell viability was assessed by the RealTime Glo™ MT Cell Viability Assay, in which a luciferase reaction occurs in the culture medium. Luciferase and a proprietary viability substrate were added to cell culture media, and the viability substrate then diffused into cells where it was reduced to form a luciferase substrate, which exited the cell and was used rapidly by luciferase in the media. Only metabolically active cells reduced the substrate, and light production was proportional to the number of live cells in culture. Dead cells did not reduce the substrate. As shown in FIG.4, cell viability decreased with increasing doses of vector, demonstrating that HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, and HSV-1 mICP4 / mICP47 mIL-7 mediated oncolysis in a dose-dependent manner. Example 3: Anti-CD47 Antibody Payload in HSV-1 mICP4 / mICP47 Vector for Phagocytosis and Oncolysis

[0212] The CD47 / SIRPα axis mediates a ‘don’t eat me’ signal exploited by tumor cells to escape macrophage-mediated immune surveillance (FIG.5). Anti-CD47 therapies have shown promising clinical results in solid and hematological malignancies; however, efficacy is hindered by systemic toxicity. This example describes the assessment ofincreasing phagocytosis and oncolysis of an HSV-1 mICP4 / mICP47 vector with a Sirpα-IgG fusion polypeptide (“macro1”) payload (HSV-1 mICP4 / mICP47 macro1).

[0213] M1 macrophages were matured by culturing PBMC with 50 ng / mL macrophage colony-stimulating factor (m-CSF) for 8 days with IFN-γ stimulation (50 ng / mL, 48 hours). Carboxyfluorescein succinimidyl ester (CFSE)-labelled Raji cells were incubated in conditioning media (CM) from Hs578T human breast cancer cells infected with HSV-1 mICP4 / mICP47 macro1 or control payloads (GFP and irrelevant antibody; 3 and 10 PFU / cell, 48 hours) or 40 µg / mL isotype control (IgG) or anti-CD47 antibodies prior to coculture with M1 macrophages for 3 hours. Flow cytometric analysis of cells positive for both CD14 and CSFE (FIGs.6A-6D) were used to quantify phagocytosis (FIG.7). As shown in FIG.7, CM from HSV-1 mICP4 / mICP47 macro1 infected cells resulted in a dose- dependent increase in macrophage-mediated target cell phagocytosis that was greater than the effect of an anti-CD47 antibody. Moreover, pre-treatment with CM from HSV-1 mICP4 / mICP47 vectors encoding irrelevant payloads did not significantly affect macrophage phagocytosis, demonstrating the specificity of the therapeutic payload response.

[0214] To test the effects of the anti-tumor efficacy of HSV-1 mICP4 / mICP47 macro1 in vivo, a mouse pulmonary carcinoma model was used and administered HSV-1 treatment as shown in the schematic provided in FIG.8. Briefly, 5 x 106A549 pulmonary carcinoma cells were injected into the rear flank of Balb / c nude mice. When tumors reached ~100 mm3, intratumoral (i.t.) administration of HSV-1 mICP4 / mICP47 macro1 (3 x 107PFU) or intraperitoneal (i.p.) administration of an anti-CD47 antibody or isotype control antibody (10 mg / kg) occurred (FIG.8) on Days 1, 4, 7, 10, 13, and 14, and body weight was assessed at the endpoint (Table 1), which demonstrated that HSV-1 mICP4 / mICP47 macro1 was well tolerated. Table 1

[0215] The volume of the A549 tumors was measured (FIG.9A and FIG.9B) in response to the various treatments. Treatment with HSV-1 mICP4 / mICP47 macro1 resulted in tumor growth inhibition compared to the control group. This effect was dependent on payload expression, as HSV-1 mICP4 / mICP47 GFP did not show statistically significant anti-tumor activity. There was a trend towards greater efficacy with local HSV-1 mICP4 / mICP47 macro1 delivery compared to systemic anti-CD47 antibody therapy.

[0216] A549 tumor sections were collected at Day 12 and were stained with F4 / 80 to visualize intratumoral macrophages, though no changes in macrophage infiltration in the tumors was observed (data not shown). Example 4: Assessment of Activated Pathways After Immune Checkpoint Inhibitor Therapy or Infection with HSV-1 mICP4 / mICP47 Vector

[0217] This example describes the assessment of pathways activated by infection with HSV-1 mICP4 / mICP47.

[0218] Using patient bulk RNA-seq datasets (Gide et al. (2019) CANCERCELL, 35(2): 238-255.e6; Hugo et al. (2016) CELL, 165(1): 35-44; Liu et al. (2019) NAT MED., 25(12): 1916-1927; Riaz et al. (2017) CELL, 171(4): 934-949.e16; and Van Allen et al. (2015) SCIENCE, 350(6257): 207-211), in silico analyses defined statistical associations between tumor microenvironment (TME) properties / survival and TME properties / expression of proteins, which were identified as putative potential payloads for HSV-1 mICP4 / mICP47 vectors described herein. A score was then calculated by combining the TME-to-survival and TME-to-payload p values as a -log10 meta p values (FIG.10). These analyses revealed that approximately half of the top 25 genes predicted to be drivers of the therapeutic response to immune checkpoint inhibitor (ICI) therapy were factors with reported roles in TLS formation, including CXCL13, IL-17a, LTA, LTB, CCL19, CCL21, and several TNF superfamily members.

[0219] In another study, using patient datasets from the SU2C-MARK cohort (Stand Up To Cancer – Mark Foundation non-small cell lung cancer (NSCLC) cohort) (Ravi et al. (2023) Nature Genetics 55.5: 807-819), in silico analyses identified top pathways downregulated or upregulated in response to ICI (FIG.11A) or infection with HSV-1 mICP4 / mICP47 GFP (FIG.11B), and similarities were observed, including induction of MYC and E2F targets and G2M cell cycle checkpoint responses (normalized enrichment scores of 2.4, 3.0, and 3.2, respectively), which are pathways with a known association toanti-tumor immune responses to ICI. Based upon the pathways identified, a correlation analysis was conducted and a positive correlation was observed between hallmark Gene Set Enrichment Analysis (GSEA) pathways associated with ICI response and those regulated by infection with HSV-1 mICP4 / mICP47 GFP (FIG.12), suggesting that HSV-1 mICP4 / mICP47 vectors orchestrated changes in the tumor microenvironment supportive of ICI response.

[0220] In another similar study, in silico analyses identified top GSEA pathways upregulated in response to HSV-1 mICP4 / mICP47 (FIG.20), including IFN-γ response, IL- 6 / JAK / STAT3 signaling, TNF-α signaling via NF-κB, inflammatory response, and IFN-α response.

[0221] In another study, Hs578T cells were infected with 10 PFU / cell of HSV-1 mICP4 / mICP47 or control vector (HSV-1 mICP27). Using flow cytometry, the cell-surface expression of MHC class I was measured as an indicator of antigen presentation 24 after infection (FIG.19). It was demonstrated that more MHC class I-positive cells were present in Hs578T cells infected with HSV-1 mICP4 / mICP47. Example 5: PD-1 Payload in HSV-1 mICP4 / mICP47 Vector in Combination with Immune Checkpoint Inhibitor for Oncolysis

[0222] This example describes the assessment of infection with HSV-1 mICP4 / mICP47 vectors in combination with immune checkpoint inhibitor (ICI) antibody administration for attenuating tumor size in vivo.

[0223] First, 5 x 105mammary carcinoma EMT6 cells were injected into the rear flank of Balb / c mice. After tumors reached ~100 mm3, an HSV-1 mICP4 / mICP47 GFP vector (3 x 107PFU) was administered intratumorally in combination with intraperitoneal administration of an anti-PD-1 antibody or isotype control antibody RMP1-14 (10 mg / kg, each). The HSV-1 mICP4 / mICP47 GFP had a modest anti-tumor effect, as measured by tumor volume, while the effect of the combination therapy (HSV-1 mICP4 / mICP47 GFP and anti-PD-1 antibody) was modestly greater yet (FIG.13). Example 6: Assessment of Single or Multi Gene Payload

[0224] This example describes the design and assessment of viral vectors with a single gene payload or multiple viral vectors, each encoding a single payload, thereby assessing a multi-gene payload for attenuating tumor size.

[0225] Based upon payloads identified e.g., by the methods described in Example 4, payloads were tested in vitro in multiplex assays. Briefly, EMT6 mammary tumor cells were implanted subcutaneously in the rear flank of Balb / c mice. Once tumors reached ~100 mm3, mice were randomized into groups to receive intratumoral injection of vehicle (control) or 3 x 107PFU of HSV-1 mICP4 / mICP47 GFP; or a combination of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a (“Multiplex A;” 6 x 106PFU of each vector, total dose 3 x 107PFU); or a combination of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mCCL21, HSV-1 mICP4 / mICP47 mCXCL13, and HSV-1 mICP4 / mICP47 mLTB (“Multiplex B;” 6 x 106PFU of each vector, total dose 3 x 107PFU). Treatment occurred on Days 1, 4, 7, and 10 after randomization, and tumor growth was monitored over time (FIGs.14A-14B). The HSV-1 mICP4 / mICP47 GFP viral backbone, alone, modestly decreased tumor growth, while administration of Multiplex A and Multiplex B, each expressing multiple factors with roles in TLS formation, had significant anti-tumor efficacy. Both Multiplex A and Multiplex B induced tumor growth inhibition compared to vehicle control mice (Multiplex A: 39.8 ± 10.5%, p = 0.004, Multiplex B: 38.6% ± 12.1, p = 0.011, n = 8 per group).

[0226] In another experiment, mice were randomized into groups to receive vehicle (control), 3 x 107PFU of HSV-1 mICP4 / mICP47 GFP, or a combination of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a (“Multiplex A;” 6 x 106PFU of each vector, total dose 3 x 107PFU) via cannulation of the submandibular glands. Glands were harvested and analyzed with flow cytometry 15 days later. The HSV-1 mICP4 / mICP47 GFP viral backbone, alone, enhanced immune infiltration into infected glands, as identified by a significantly increased percentage of CD45-positive cells compared to vehicle controls (FIG.15). When the glands were harvested for histology, clusters of CD11c-positive dendritic cells and CD4-positive T cells were present, demonstrating nascent TLS induction (data not shown). In the Multiplex A group, TLS were increased in number, size, and organization compared to the control HSV-1 mICP4 / mICP47 GFP vector, as demonstrated by staining with CD11c for dendritic cells, CD4 for T cells, and B220 for B cells (data not shown). Furthermore, in the Multiplex A group, the induction of TLS were accompanied by mature fibroblastic reticular cell networks and de novo expression of CCL21, as demonstrated by staining with GP38 for fibroblastic reticular cells co-localizedwith CD4 for T cells, as well as CCL21 co-localized with GP38 for fibroblastic reticular cells and B220 for B cells (data not shown).

[0227] Together, these data demonstrated that viral-mediated expression of a combination of factors induced TLS formation and anti-tumor immunity. It is contemplated that this anti-tumor immunity occured through a combination of viral vector- and payload- dependent effects.

[0228] In another set of experiments, Hs578T breast cancer cells were infected with HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 IFN-γ or other vectors encoding payloads in singleplex or multiplex (total 1 PFU / cell), then cultured with or without PBMC (“T cell activator”) for 72 hours. Tumor cell viability was assessed by flow cytometry (FIG. 16), which demonstrated that specific payload combinations, such as IL-15 (“B”), IFN-γ + IL-12 (“E”), IFN-γ + IL-15 (“F”), IL-12 + IL-15 (“G”), IL-15 + IL-21 (“H”), IFN-γ + IL-12 + IL-15 (“I”), and IFN-γ + IL-12 + IL-15 + IL-21 (“J”) enhanced PBMC-mediated cancer cell killing. A lasso regression analysis was used to identify immune cell populations positively (right of x-axis) or negatively (left of x-axis) correlated with PBMC-mediated tumor cell killing (FIG.17). Example 7: Assessment of Infection with HSV-1 mICP4 / mICP47 Vectors Expressing Tertiary Lymphoid Structure-Related Payloads

[0229] It is contemplated that driving the formation of TLS in suppressive TME will improve response to ICI, ultimately impacting patient survival. Yet, there are a limited number of therapeutic approaches that have been developed and tested to successfully form TLS in tumors. This example describes novel HSV-1 mICP4 / mICP47 vectors expressing data-driven selected combinations of factors regulating TLS formation.

[0230] Based upon payloads identified e.g., by the methods described in Example 4, gene selection payloads are selected and tested. For example, to induce TLS-mediated tumor control, two combinations of HSV-1 mICP4 / mICP47 vectors, Multiplex A (HSV-1 mICP4 / mICP47 LTA, HSV-1 mICP4 / mICP47 CCL19, HSV-1 mICP4 / mICP47 TNF, HSV-1 mICP4 / mICP47 IL-17, and HSV-1 mICP4 / mICP47 IL-7; or an HSV-1 mICP4 / mICP47 vector expressing LTA, CCL19, TNF, IL-17, and IL-7) and Multiplex B (HSV-1 mICP4 / mICP47 LTB, HSV-1 mICP4 / mICP47 CCL19, HSV-1 mICP4 / mICP47 CCL21, HSV-1 mICP4 / mICP47 CXCL13, and HSV-1 mICP4 / mICP47 IL-7; or an HSV-1 mICP4 / mICP47 vector expressing LTB CCL19 CCL21 CXCL13, or IL-7) are selected andtested in 80 subcutaneous EMT6 tumor-bearing Balb / c mice (6-9 weeks old) to define their ability to induce formation of phenotypically mature TLS. Vehicle and a GFP-expressing vector are used as controls. Additional combinations of one or more therapeutic polypeptides may be tested, including hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene, an anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, or VEGF-C, or combinations thereof. Twenty tumor-bearing mice (10 female, 10 male) are included in each experimental group. Test articles are administered (1 x 107PFU in 20 µL DPBS with 10% glycerol) by intratumoral injection on Days 1, 4, and 7 after randomization. Tumor growth is monitored using caliper measurement 2 times per week, body weight is measured, and clinical observations are annotated.

[0231] In another set of experiments, an alternative model is used, including a model of intraperitoneal tumor development (Rodriguez et al. (2021) CELL REP., 36(3): 109422) for modeling tumor lesions more permissive to TLS induction (e.g., lymph node metastases). Additionally, as yet another model, a genetically engineered mouse model with slower tumor progression is tested (Joshi et al. (2015) IMMUNITY, 43(3): 579-90).

[0232] Half of the animals in each group are culled on Day 15 (timepoint 1) and the remaining animals are culled when tumors reach 2,000 mm3(timepoint 2) to allow for assessment of time-dependent effects on TLS maturation and stability. At study endpoint, tumor, draining lymph node, and blood (for plasma and blood cell isolation) are collected and analyzed. Histologic and morphometric characterization of the TLS is completed using immunofluorescent staining for common lymphocyte markers of TLS maturation (T / B cells), follicular dendritic cell formation, secretion of survival factors and lymphoid chemokine expression, and high endothelial venules formation using fluorophore-conjugate antibodies for CD3, CD4, CD19, B220, CD11c, CD31, podoplanin, CCL21, and CXCL13 etc. Flow cytometry-based assessment of tumors and tumor-draining lymph nodes are performed to measure viral infection, tumor composition, and payload expression. A series of genes associated with a TLS signature are quantified in available samples by qPCR analysis. Flow cytometry-based immunophenotyping of blood cells including CD4- and CD8-positive T cells, T regulatory cells, B cells, NK and NKT cells, monocytes, and dendritic cells is used to define changes in peripheral immune responses. TLS formation within the TME are detected. Evidence of T / B cell zone segregation and formation of germinal centers isdetermined by histological and morphometric analyses, revealing the formation of mature TLS. Flow cytometry analyses show the maturity of the T and B cell response, and transcriptomic analyses confirm a gene signature compatible with TLS formation. Phenotypic changes in the tumor, compatible with increased tumor disruption, are revealed by smaller areas of tumor-positive cells (by histology) and reduced number of tumor cells (by flow cytometry). These changes are associated with an increased density of tumor infiltrating lymphocytes (TILs) in the TME. Together, these data characterize TLS, their evolution over time in tumors, and evaluate phenotypic and transcriptional changes within tumors, peripheral immune responses, and their relationship with tumor burden.

[0233] Therapeutic combinations with ICI are also tested (see Example 8). Example 8: Assessment of Tumor Regression with HSV-1 mICP4 / mICP47 Vectors Expressing Various Payloads

[0234] This example describes the in vivo efficacy of novel HSV-1 mICP4 / mICP47 vectors expressing data-driven select payloads.

[0235] A library of single or multi-geneencoding HSV-1 mICP4 / mICP47 vectors was constructed, each expressing one of the in silico predicted payload components (See e.g., Example 4 and FIG.10; e.g., IL-12, IL-15, IL-21, and IFN-γ).

[0236] As shown in FIG.21, there was a dose-dependent increase in IFN-γ and IL-12 payload production with maximal production within the first 2 days after infection in both the group dosed with 3 PFU / cell as well as the group dosed with 10 PFU / cell. Cell viability was assessed by the RealTime Glo™ MT Cell Viability Assay, in which a luciferase reaction occurs in the culture medium. Luciferase and a proprietary viability substrate were added to cell culture media, and the viability substrate then diffused into cells where it was reduced to form a luciferase substrate, which exited the cell and was used rapidly by luciferase in the media. Only metabolically active cells reduced the substrate, and light production was proportional to the number of live cells in culture. Dead cells did not reduce the substrate. As shown in FIG.21 (right panel), cell viability decreased with increasing doses of vector, demonstrating that HSV-1 mICP4 / mICP47 IFN-γ and HSV-1 mICP4 / mICP47 IL-12 mediated oncolysis in a dose-dependent manner.

[0237] The top scoring combinations of viral gene constructs were tested for their ability to induce immune activation and cancer cell killing activity using an ex vivo cancer / peripheral blood mononuclear cell (PBMC) co-culture system and flow cytometry-based readouts using methods described e.g., in Example 6. In vivo anti-tumor activity was assessed in Hs578T, EMT6, and LLC1 tumor-bearing mice using methods described throughout the Examples.

[0238] It was demonstrated that infection of cancer cells with single gene encoding HSV-1 mICP4 / mICP47 vectors induced PBMC-mediated cancer cell killing in an infection- and payload-dependent manner (FIGs.22A-22B). Cell killing was associated with significant changes in the composition and function of immune cells within the co-culture (FIG.23). Correlation analysis identified payload-mediated regulation of multiple immune cell populations with the expansion and activation of NK and CD8-positive T cell populations correlated with effective PBMC-mediated cancer cell killing (Lasso regression, Alpha = 0.1, R2 = 0.79) (FIG.24). Measurement of dendritic cells and Ki67-positive / Gzmb- positive CD8-positive T cells (FIG.25), as well as Ki67-positive and PD1-positive CD8- positive T cells (FIGs.27A-27B), also revealed payload-mediated regulation of multiple immune cell populations, The combination of IL-12 and IL-15 was then prioritized for in vivo testing, and it was demonstrated that intratumoral treatment with HSV-1 mICP4 / mICP47 vectors encoding IL-12 and IL-15 resulted in significant tumor growth inhibition in both tumor models tested (EMT6: 60.0% ± 12.6, p = 0.002; LLC1: 37.2% ± 9.4, p = 0.011) (FIGs. 26A-26D). Tumor regression was observed in 25% of EMT6 tumor-bearing mice. Immune activation, both within tumors and in the periphery, was associated with antitumor activity. Taken together, these results demonstrated that a multi-gene payload, specifically, IL-12 and IL-15, identified through a multiparameter decision matrix had a synergistic anti-tumor effect. Example 9: Assessment of Infection with HSV-1 mICP4 / mICP47 Vectors Alone or in Combination with Anti-PD-1 Therapy

[0239] Top-performing vectors from Example 7 are tested alone and in combination with anti-PD-1 antibody therapy (clone RMP1-14 sourced from BioXCell). Experimental groups include: 1. Vehicle + isotype control 2. HSV-1 mICP4 / mICP47 GFP + isotype control 3. Multiplex A from Example 6 or 7 or a vector from Example 7 + isotype control4. Multiplex B from Example 6 or 7 or a vector from Example 7 + isotype control 5. Vehicle + anti-PD1 6. HSV-1 mICP4 / mICP47 GFP + anti-PD1 7. Multiplex A from Example 6 or 7 or a vector from Example 7 + anti-PD1 8. Multiplex B from Example 6 or 7 or a vector from Example 7 + anti-PD1.

[0240] Eight (8) tumor-bearing mice are included in each experimental group. Viral vector treatment is 1 x 107PFU in 20 µL of DPBS with 10% glycerol by intratumoral injection on Days 1, 4, 7, and 10 with DPBS with 10% glycerol used as the vehicle control beginning when tumors reach ~100 mm3, as identified by caliper measurement of tumors 3x per week. Anti-PD-1 antibody treatment is 200 µg in PBS by intraperitoneal injection, 2 times a week for 2 weeks, with rat IgG2a in PBS used as the isotype control. The study endpoint is when tumors reach 1000 mm3(to allow time for the immunological response to evolve) or 3 weeks, whichever comes first. Endpoint analyses include histologic, morphometric, flow cytometric, and qPCR / RNASeq analyses, as are common in the art.

[0241] Similar experimental designs are used to additionally test anti-CTLA-4 and anti-PD-1 + anti-CTLA-4 antibody therapies in combination with the vector(s) from Example 7.

[0242] Additionally, experiments are conducted to evaluate the timing of vector- mediated TLS induction by sequencing treatment of the vector(s) from Example 7 and anti- PD-1, anti-CTLA-4, or anti-PD-1 + anti-CTLA-4 antibody therapies (e.g., vector then antibody vs. antibody then vector).

[0243] It is expected that additional ICI therapy will increase the antitumoral effect of the multiplex or vector(s) from Examples 6 and 7, and this will be associated with changes in the TME, indicative of immune-mediated tumor control as well as immune activation in the periphery. Example 10: Assessment of Survival After Infection with HSV-1 mICP4 / mICP47 Vectors in Combination with Anti-PD-1 Therapy

[0244] This example describes the assessment of combinations of viral vectors, each encoding a payload, thereby assessing a multi-gene payload for attenuating tumor size.

[0245] EMT6 mammary tumor cells were implanted subcutaneously in the rear flank of Balb / c mice. Once tumors reached ~100 mm3, mice were randomized into groups toreceive intratumoral injection of vehicle; or a combination of the individual vectors HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a (“Multiplex A;” 6 x 106PFU of each vector, total dose 3 x 107PFU); or a combination of the individual vectors HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mCCL21, HSV-1 mICP4 / mICP47 mCXCL13, and HSV-1 mICP4 / mICP47 mLTB (“Multiplex B;” 6 x 106PFU of each vector, total dose 3 x 107PFU). Treatment occurred on Days 1, 4, 7, and 10 after randomization. In combination with vector administration, mice were treated with an anti-PD-1 antibody (clone: RMP1-14) or isotype control antibody (IgG, 10 mg / kg) and tumor growth (FIG.18) and survival (Table 2) were monitored over time. Multiplex A and Multiplex B in combination with anti-PD-1 antibody therapy increased the number of long-term survivors (> 40 days) as compared to controls. Table 2

[0246] Taken together, these experiments demonstrated in vivo anti-tumor efficacy of the HSV-1 mICP4 / mICP47 vectors and multiplexed payloads, which regulate TLS induction, as evidenced by enhanced long-term survival. INCORPORATION BY REFERENCE

[0247] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0248] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in an immune checkpoint inhibitor (ICI)-refractory subject, the method comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein.

2. A method of expressing a therapeutic polypeptide in an ICI-refractory subject, the method comprising administering to the ICI-refractory subject a vector comprising a HSV genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein.

3. A method of reducing the size of a tumor in an ICI-refractory subject, the method comprising administering to the ICI-refractory subject a vector comprising a HSV genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein, and wherein the vector comprises a nucleic acid encoding a therapeutic polypeptide that functions to reduce the size of the tumor.

4. The method of any one of claims 1-3, wherein the HSV-1 genome comprises one or more alterations that prevent the expression of two ICP4 proteins.

5. The method of any one of claims 1-4, wherein the HSV genome comprises one or more alterations that prevent the expression of two ICP4 proteins and an ICP47 protein.

6. The method of claim 4 or 5, wherein the two ICP4 proteins are characterized as having the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 (ICP4).

7. The method of any one of claims 1-6, wherein the ICP47 protein is characterized as having the amino acid sequence of SEQ ID NO: 5 (ICP47).

8. The method of any of claims 1-7, wherein the HSV genome is an HSV-1 genome.

9. The method of any one of claims 1-7, wherein the HSV-1 genome is a McKrae genome.

10. The method of any one of claims 1-9, wherein the vector, when administered to the subject, results in one or more of:(a) delayed oncolysis; (b) increased immunogenicity; (c) increased immune activation; (d) formation of tertiary lymphoid structures (TLS) in a tumor bed; (e) upregulation or downregulation of one or more proteins associated with a response to ICI; (f) cancer cell viability reduction; and (g) a combination of any one or more of (a) – (f).

11. The method of any one of claims 1-10, wherein the vector comprises a nucleic acid encoding one or more therapeutic polypeptides, and wherein the one or more therapeutic polypeptides: (a) target the stroma of a tumor; (b) support T cell survival in a tumor microenvironment; (c) induce tertiary lymphoid structures (TLS) in a tumor bed; (d) promote phagocytic innate immune surveillance (e.g., disrupts the Sirpα / CD47 axis); (e) promote upregulation or downregulation of one or more proteins associated with a response to ICI; (f) reduce cancer cell viability; or (g) a combination of any one or more of (a) – (f).

12. The method of claim 11, wherein the one or more therapeutic polypeptides target the stroma of a tumor by degrading an extracellular matrix protein.

13. The method of claim 12, wherein the one or more therapeutic polypeptides comprises hyaluronidase, MMP-9, or an inhibitor of lysyl oxidase.

14. The method of claim 11, wherein the one or more therapeutic polypeptides target the stroma of a tumor by activating local endothelium to increase T cell infiltration.

15. The method of claim 14, wherein the one or more therapeutic polypeptides comprise a proinflammatory cytokine.

16. The method of claim 15, wherein the proinflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

17. The method of claim 11, wherein the one or more therapeutic polypeptides support T cell survival in the tumor microenvironment by enhancing recruitment of T cells to a site of a tumor.

18. The method of claim 17, wherein the one or more therapeutic polypeptides comprise CCL19 or CCL21.

19. The method of claim 11, wherein the one or more therapeutic polypeptides support T cell survival in the tumor microenvironment by supporting T cell function.

20. The method of claim 19, wherein the one or more therapeutic polypeptides comprise a T cell trophic factor.

21. The method of claim 20, wherein the T cell trophic factor is selected from IL-7, IL-12, IL-15, IL-18, and IFN-γ.

22. The method of any one of claims 11 and 17-21, wherein the T cell is a CAR T cell.

23. The method of claim 22, wherein the one or more therapeutic polypeptides comprise soluble TGFβRII.

24. The method of claim 22, wherein the one or more therapeutic polypeptides comprise an antigen recognized by the CAR T cell.

25. The method of claim 24, wherein the antigen comprises mesothelin.

26. The method of claim 11, wherein the one or more therapeutic polypeptides comprise a co- stimulatory molecule.

27. The method of claim 26, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

28. The method of claim 11, wherein the one or more therapeutic polypeptides induce tertiary lymphoid structures (TLS) in a tumor bed.

29. The method of claim 28, wherein the one or more therapeutic polypeptides comprise CCL19, lymphotoxin β, CXCL13, or TNF.

30. The method of claim 29, wherein the one or more therapeutic polypeptides promote phagocytic innate immune surveillance.

31. The method of claim 30, wherein the one or more therapeutic polypeptides disrupt the Sirpα / CD47 axis.

32. The method of claim 30, wherein the one or more therapeutic polypeptides comprise a Sirpα-IgG fusion transgene.

33. The method of claim 30, wherein the or more therapeutic polypeptides comprise an anti- CD47 antibody.

34. The method of claim 11, wherein the one or more therapeutic polypeptides support anti- tumor macrophage polarization.

35. The method of claim 34, wherein the one or more therapeutic polypeptides comprise TNF, IL-1, IL-12, IL-17, or IFN-γ.

36. The method of claim 11, wherein the one or more therapeutic polypeptides comprise hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene, an anti-CD47 antibody, IL-1, or IL-17.

37. The method of claim 11, wherein the one or more therapeutic polypeptides comprise IL- 21, LTA, LTB, CXCL12, or VEGF-C.

38. The method of claim 11, wherein the one or more therapeutic polypeptides comprise hyaluronidase, MMP-9, an inhibitor of lysyl oxidase, TNF, IL-1β, IL-6 IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, a Sirpα-IgG fusion transgene, an anti-CD47 antibody, IL-1, IL- 17, IL-21, LTA, LTB, CXCL12, or VEGF-C.

39. The method of claim 38, wherein the one or more therapeutic polypeptides comprise:a) LTA, CCL19, TNF, IL-17, and IL-7; b) LTB, CCL19, CCL21, CXCL13, and IL-7; c) IFN-γ and IL-12; d) IFN-γ and IL-15; e) IL-12 and IL-15; f) IL-15 and IL-21; g) IFN-γ, IL-12, and IL-15; h) IFN-γ, IL-12, IL-15, and IL-21; or i) CCL19, IL-7, TNF, and IL-17.

40. The method of any one of claims 1-39, wherein the method further comprises administering an immune checkpoint inhibitor (ICI).

41. The method of claim 40, wherein the ICI is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

42. The method of claim 41, wherein the ICI comprises a PD-1 inhibitor.

43. The method of claim 42, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

44. The method of claim 43, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or cemiplimab.

45. The method of claim 41, wherein the ICI comprises a PD-L1 inhibitor.

46. The method of claim 45, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

47. The method of claim 41, wherein the ICI comprises a CTLA-4 inhibitor.

48. The method of claim 47, wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

49. The method of claim 41, wherein the ICI comprises a LAG-3 inhibitor.

50. The method of claim 49, wherein the LAG-3 inhibitor comprises relatlimab.

51. The method of claim 41, wherein the method comprises administering a plurality of ICIs selected from the group consisting of a PD-1 inhibitor and a CTLA-4 inhibitor.

52. The method of claim 51, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

53. The method of claim 52, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or cemiplimab, and wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

54. The method of claim 8, wherein the one or more proteins associated with a response to ICI comprise IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

55. The method of claim 8, wherein inducing tertiary lymphoid structures (TLS) in a tumor bed comprises promoting the number, size, and / or organization of TLS structures.

56. A method of treating cancer in subject in need thereof, the method comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises one or more alterations that prevent the expression of a functional ICP4 protein and / or a functional ICP47 protein and expresses an effective amount of IL-12 and IL-15, thereby to treat the cancer.

57. The method of claim 56, wherein the method further comprises administering an immune checkpoint inhibitor (ICI).

58. The method of claim 57, wherein the ICI is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

59. The method of claim 57, wherein the ICI comprises a PD-1 inhibitor.

60. The method of claim 58, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

61. The method of claim 60, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or cemiplimab.

62. The method of claim 58, wherein the ICI comprises a PD-L1 inhibitor.

63. The method of claim 62, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

64. The method of claim 58, wherein the ICI comprises a CTLA-4 inhibitor.

65. The method of claim 64, wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

66. The method of claim 58, wherein the ICI comprises a LAG-3 inhibitor.

67. The method of claim 66, wherein the LAG-3 inhibitor comprises relatlimab.

68. The method of claim 58, wherein the method comprises administering a plurality of ICIs selected from the group consisting of a PD-1 inhibitor and a CTLA-4 inhibitor.

69. The method of claim 68, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

70. The method of claim 69, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or cemiplimab, and wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

71. The method of any one of claims 56-70, wherein the administration of the vector comprising the herpes simplex virus (HSV) genome that expresses IL-12 and IL-15 results in synergistic tumor regression as compared to administration of a vector comprising a herpes simplex virus (HSV) genome that expresses (a) IL-12 and not IL-15 or (b) only IL-15 and not IL-12.

72. The method of any one of claims 56-71, wherein the subject is ICI-refractory.

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