Compositions and methods for viral vectors

Abstract

The present invention relates generally to replication-deficient HSV-1 vectors, and more particularly, the present invention relates to replication-deficient HSV-1 vectors and their use for delivering one or more genes encoding transgenic proteins that stimulate tumor immune disruption.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 331,903, filed on April 18, 2022; the disclosure of which is incorporated herein by reference in its entirety for all purposes. Field of the Invention

[0003] The present disclosure relates generally to HSV-1 vectors, and more particularly, the present invention relates to replication-defective HSV-1 vectors and their use for delivering one or more genes encoding transgenic proteins that stimulate immune destruction of tumors. Background Art

[0004] The development of immuno-oncology has provided new therapies for treating cancers that have escaped successful treatment (because cancer has evolved mechanisms to escape the subject's immune system). However, despite significant progress in treating various cancers with immunostimulatory therapies (such as checkpoint inhibitors and CAR T cells) to date, a large number of patients are still unresponsive to immunotherapy treatment. In addition, considering the complexity of the tumor microenvironment (TME) of solid tumors, solid tumors may be more difficult to treat using immuno-oncology methods, and the tumor microenvironment contains epithelial cells, endothelial cells, mesenchymal cells, stromal cells, cancer cells, and immune cells. In some solid tumors, cancer cells affect the activity or inactivity of immune cells in TME.

[0005] Viral vectors have been developed to deliver therapeutic agents to cells. However, many such vectors exhibit insufficient persistence in target cells, preventing adequate expression of the therapeutic agent. In addition, many existing vectors cause nonspecific inflammation and tissue damage, making delivery unsafe. Therefore, despite the progress made to date, there remains a need for methods and compositions that can stimulate immune activity against tumor cells, which methods and compositions, alone or in combination with other cancer drugs, can effectively treat a tumor in a given subject. Summary of the invention

[0006] The present disclosure is based in part on the discovery that HSV-1 vectors comprising alterations (such as gene deletions) that prevent expression of one or more functional infected cell polypeptide 4 (ICP4) and infected cell polypeptide 47 (ICP47) proteins have reduced replication capacity, are oncolytic, have extended payload expression time, are cytotoxic to proliferating cells (e.g., cancer cells), and exhibit enhanced immunogenicity. Thus, it has been found that such vectors can be used to deliver therapeutic payloads (e.g., one or more therapeutic polypeptides) that can be used to reduce tumor size, such as for the treatment of cancer in a subject in need thereof. The vectors can induce delayed oncolysis, thereby allowing for sustained expression of therapeutic payloads (e.g., one or more therapeutic polypeptides). In addition, the vectors exhibit increased immunogenicity (e.g., by increasing expression of human leukocyte antigens (HLA) on the surface of proliferating cells (e.g., cancer cells), thereby increasing immune activity against tumor cells. These features are unexpected given the unpredictability of the replication capacity, oncolytic activity, and persistence in tissues of viruses, particularly different strains of viruses.

[0007] The present disclosure is also based in part on the discovery that specific combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of immune cells in a recipient to kill cancer cells, thereby reducing tumor size. For example, certain therapeutic polypeptides that target the tumor stroma, support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment, induce tertiary lymphoid structures (TLS) in the tumor bed, and / or promote innate immune surveillance of phagocytes can be expressed from viral vectors (e.g., viral vectors lacking functional ICP4 and ICP47 proteins) to increase the ability of immune cells to kill tumor cells, for example, to treat cancer in subjects in need. The vector can be designed to include a specific combination of therapeutic polypeptides that will effectively treat a given tumor type, including selecting one or more therapeutic polypeptides that reduce suppressive features in a specific tumor microenvironment that may prevent the immune system from effectively attacking the tumor.

[0008] Thus, in one aspect, the present disclosure relates to a vector comprising a herpes simplex virus (HSV) genome, wherein the vector comprises changes that prevent the expression of one or more functional ICP4 and ICP47 proteins. In certain embodiments, the functional ICP4 protein is characterized by the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 and the functional ICP47 protein is characterized by the amino acid sequence of SEQ ID NO:5. In certain embodiments, the HSV genome is an HSV-1 genome, such as a McKrae strain genome. In certain embodiments, when administered to a subject, the vector causes one or more of the following: (a) delayed oncolysis; (b) increased immunogenicity; and (c) increased immune activation.

[0009] In certain embodiments, the vector comprises a nucleic acid sequence encoding one or more therapeutic polypeptides. In certain embodiments, the therapeutic polypeptide may have one or more of the following properties:

[0010] (a) Targeting tumor stroma;

[0011] (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment;

[0012] (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0013] (d) Promotes innate immune surveillance in phagocytes.

[0014] In certain embodiments, the therapeutic polypeptide targets the tumor matrix by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, inhibitors of hyaluronidase (SEQ ID NO: 6), MMP-9 (SEQ ID NO: 7) or lysyl oxidase. In certain embodiments, the therapeutic polypeptide targets the tumor matrix by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, proinflammatory cytokines, such as TNF (SEQ ID NO: 11), IL-1β (SEQ ID NO: 8), IL-6 (SEQ ID NO: 9) or IL-18 (SEQ ID NO: 18).

[0015] In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 (SEQ ID NO: 12) or CCL21 (SEQ ID NO: 13). In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from the following: IL-7 (SEQ ID NO: 14), IL-12 (SEQ ID NO: 15 and 16), IL-15 (SEQ ID NO: 17), IL-18 (SEQ ID NO: 18) and IFNγ (SEQ ID NO: 19).

[0016] Depending on the circumstances, such as the indication to be treated, the T cell is a CAR T cell. In such embodiments, the therapeutic polypeptide may comprise soluble TGFβRII (SEQ ID NO: 20 or SEQ ID NO: 21). Alternatively, the therapeutic polypeptide comprises an antigen recognized by the CAR T cell, such as mesothelin. Alternatively or in addition, the therapeutic polypeptide may comprise a co-stimulatory molecule, such as a co-stimulatory molecule selected from CD40L (SEQ ID NO: 22) and OX40L (SEQ ID NO: 23).

[0017] In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2 (SEQ ID NO: 24), CX3CL1 (SEQ ID NO: 25), CXCL16 (SEQ ID NO: 26), CCL5 (SEQ ID NO: 27), CXCL9 (SEQ ID NO: 28), CXCL10 (SEQ ID NO: 29), CXCL11 (SEQ ID NO: 30). In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, such as NK cell trophic factors selected from the following: IL-2 (SEQ ID NO: 31), IL-15 (SEQ ID NO: 17), IL-18 (SEQ ID NO: 18) and IFNα (SEQ ID NO: 32).

[0018] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may comprise CCL19 (SEQ ID NO: 12), lymphotoxin beta (SEQ ID NO: 33), CXCL13 (SEQ ID NO: 34), or TNF.

[0019] In certain embodiments, the therapeutic polypeptide promotes phagocyte innate immune surveillance, and the therapeutic polypeptide may include, but is not limited to, a therapeutic polypeptide that disrupts the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene (SIRPα SEQ ID NO: 37).

[0020] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1 (SEQ ID NO: 35), IL-12 (SEQ ID NO: 15 and 16), IL-17 (SEQ ID NO: 36) or IFNγ (SEQ ID NO: 19).

[0021] In another aspect, the present disclosure relates to a method for expressing a polypeptide in a subject, the method comprising administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing changes that render the variant incapable of expressing functional ICP4 and ICP47 proteins. In certain embodiments, the functional ICP4 protein is characterized by the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 and the functional ICP47 protein is characterized by the amino acid sequence of SEQ ID NO:5. In certain embodiments, the HSV strain is an HSV-1 strain, such as a McKrae strain. In certain embodiments, when administered to a subject, the vector causes one or more of the following: (a) delayed oncolysis; (b) increased immunogenicity; and (c) increased immune activation.

[0022] In certain embodiments, the HSV strain comprises a nucleic acid encoding a therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide may have one or more of the following attributes:

[0023] (a) Targeting tumor stroma;

[0024] (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment;

[0025] (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0026] (d) Promotes innate immune surveillance in phagocytes.

[0027] In certain embodiments, the therapeutic polypeptide targets the tumor matrix by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, inhibitors of hyaluronidase, MMP-9, or lysyl oxidase. In certain embodiments, the therapeutic polypeptide targets the tumor matrix by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, proinflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0028] In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from the following: IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0029] Depending on the circumstances, such as the indication to be treated, the T cell is a CAR T cell. In such embodiments, the therapeutic polypeptide may comprise soluble TGFβRII. Alternatively, the therapeutic polypeptide comprises an antigen recognized by the CAR T cell, such as mesothelin. Alternatively or in addition, the therapeutic polypeptide may comprise a co-stimulatory molecule, such as a co-stimulatory molecule selected from CD40L and OX40L.

[0030] In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, such as NK cell trophic factors selected from the following: IL-2, IL-15, IL-18, and IFNα.

[0031] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may comprise CCL19, lymphotoxin beta, CXCL13, or TNF.

[0032] In certain embodiments, the therapeutic polypeptide promotes phagocyte innate immune surveillance, and the therapeutic polypeptide may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0033] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

[0034] In another aspect, the disclosure relates to a method for preparing a vector comprising a variant herpes simplex virus (HSV) genome, the vector containing changes that make the variant unable to express functional ICP4 and ICP47 proteins. The vector expresses at least one therapeutic polypeptide. The method includes incubating cells transfected with the following: (a) a first nucleic acid molecule, the first nucleic acid molecule comprising (i) a portion of the HSV genome, but not encoding functional ICP4 and ICP47 proteins, and (ii) a sequence encoding a marker element, wherein the sequence encoding the marker element is flanked by a first homology region (HR1) and a second homology region (HR2); and (b) a second nucleic acid molecule, the second nucleic acid molecule comprising a sequence encoding a therapeutic polypeptide, wherein the sequence encoding the therapeutic polypeptide is flanked by a first homology region (HR1_) and a second homology region (HR2_). HR1 is homologous to HR1_ and HR2 is homologous to HR2_, so that the sequence encoding the therapeutic polypeptide is integrated into the first nucleic acid molecule via homologous recombination. In certain embodiments, the cell is an ICP4 and / or ICP47 complementation cell. In certain embodiments, the method further comprises the step of purifying viral plaques that do not express the marker element.

[0035] In certain embodiments, the HSV genome is an HSV-1 genome, such as a McKrae strain genome.

[0036] In certain embodiments, the therapeutic polypeptide comprises one or more of the following attributes:

[0037] (a) Targeting tumor stroma;

[0038] (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment;

[0039] (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0040] (d) Promotes innate immune surveillance in phagocytes.

[0041] In certain embodiments, the therapeutic polypeptide targets the tumor matrix by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, inhibitors of hyaluronidase, MMP-9, or lysyl oxidase. In certain embodiments, the therapeutic polypeptide targets the tumor matrix by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, proinflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0042] In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from the following: IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0043] Depending on the circumstances, such as the indication to be treated, the T cell is a CAR T cell. In such embodiments, the therapeutic polypeptide may comprise soluble TGFβRII. Alternatively, the therapeutic polypeptide comprises an antigen recognized by the CAR T cell, such as mesothelin. Alternatively or in addition, the therapeutic polypeptide may comprise a co-stimulatory molecule, such as a co-stimulatory molecule selected from CD40L and OX40L.

[0044] In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. Therapeutic polypeptides can support NK cells to survive in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, such as NK cell trophic factors selected from the following: IL-2, IL-15, IL-18, and IFNα.

[0045] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may comprise CCL19, lymphotoxin beta, CXCL13, or TNF.

[0046] In certain embodiments, the therapeutic polypeptide promotes phagocyte innate immune surveillance, and the therapeutic polypeptide may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0047] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization, which may include, but is not limited to, TNF, IL-1, IL-12, IL-17, or IFNγ.

[0048] In another aspect, the present disclosure relates to a variant HSV strain comprising a vector as described herein.

[0049] In another aspect, the disclosure relates to a cell transduced with a vector as described herein.

[0050] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a carrier as described herein and a pharmaceutically acceptable carrier.

[0051] In another aspect, the present disclosure relates to a method of reducing tumor size in a subject in need thereof. The method comprises administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing an alteration that renders the variant unable to express functional ICP4 and ICP47 proteins. In addition, the vector comprises a nucleic acid encoding one or more therapeutic polypeptides, the one or more therapeutic polypeptides being used to reduce tumor size.

[0052] In certain embodiments, the variant is unable to express functional ICP4 and ICP47 proteins characterized by the amino acid sequences of SEQ ID NOs: 3 and 4 and SEQ ID NO: 5, respectively. In certain embodiments, the HSV strain is an HSV-1 strain, such as the McKrae strain.

[0053] In certain embodiments, the therapeutic polypeptide comprises one or more of the following attributes:

[0054] (a) Targeting tumor stroma;

[0055] (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment;

[0056] (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0057] (d) Promotes innate immune surveillance in phagocytes.

[0058] In certain embodiments, the therapeutic polypeptide targets the tumor matrix by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides include, but are not limited to, inhibitors of hyaluronidase, MMP-9, or lysyl oxidase. In certain embodiments, the therapeutic polypeptide targets the tumor matrix by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides include, but are not limited to, proinflammatory cytokines, such as TNF, IL-1β, IL-6, or IL-18.

[0059] In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, CCL19 or CCL21. In certain embodiments, the therapeutic polypeptide supports T cells to survive in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides include, but are not limited to, T cell trophic factors, such as T cell trophic factors selected from the following: IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0060] Depending on the circumstances, such as the indication to be treated, the T cell is a CAR T cell. In such embodiments, the therapeutic polypeptide may comprise soluble TGFβRII. Alternatively, the therapeutic polypeptide comprises an antigen recognized by the CAR T cell, such as mesothelin. Alternatively or in addition, the therapeutic polypeptide may comprise a co-stimulatory molecule, such as a co-stimulatory molecule selected from CD40L and OX40L.

[0061] In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by enhancing the recruitment of NK cells to the tumor site. Examples of such therapeutic polypeptides include, but are not limited to, chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11. In certain embodiments, the therapeutic polypeptide supports NK cells to survive in the tumor microenvironment by supporting NK cell function. Examples of such therapeutic polypeptides include, but are not limited to, NK cell trophic factors, such as NK cell trophic factors selected from the following: IL-2, IL-15, IL-18, and IFNα.

[0062] In certain embodiments, the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed. Such therapeutic polypeptides may comprise CCL19, lymphotoxin beta, CXCL13, or TNF.

[0063] In certain embodiments, the therapeutic polypeptide promotes phagocyte innate immune surveillance, and the therapeutic polypeptide may include, but is not limited to, therapeutic polypeptides that disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene.

[0064] In certain embodiments, the nucleic acid further comprises a therapeutic polypeptide that supports anti-tumor macrophage polarization. In certain embodiments, the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

[0065] In another aspect, the present disclosure relates to a vector comprising a herpes simplex virus (HSV) genome, wherein the vector comprises an alteration that prevents expression of one or more functional ICP4 and ICP47 proteins, and wherein the vector encodes one or more therapeutic polypeptides that support survival of NK cells in a tumor microenvironment (TME).

[0066] In certain embodiments, NK cells are natural NK cells or CAR NK cells. In certain embodiments, one or more therapeutic polypeptides include NK cell recruiting factors. In certain embodiments, NK cell recruiting factors are chemokine ligands. In certain embodiments, chemokine ligands are selected from CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10 and CXCL11. In certain embodiments, one or more therapeutic polypeptides include one or more NK cell trophic factors selected from the following: IL-2, IL-15, IL-18 and IFNα. In certain embodiments, one or more therapeutic polypeptides include soluble TGFβRII.

[0067] These and other aspects and features of the present invention are described in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The foregoing and other objects, features and advantages of the present disclosure will become apparent from the following description of the preferred embodiments as illustrated in the accompanying drawings. Similar reference elements identify common features in the corresponding drawings. The drawings are not necessarily drawn to scale, but emphasis is placed on illustrating the principles of the present disclosure.

[0069] Figures 1A to 1C is a schematic diagram of the McKrae strain HSV-1 genome. Figure 1A The wild-type McKrae strain HSV-1 genome is shown, showing essential genes (encoding UL27 (gB), UL48 (VP16), ICP27 and ICP4) and non-essential genes (encoding ICP0, LAT, UL37, UL38, UL41 (vhs), LAT, ICP0, ICP22 and ICP47). The genome is organized into two segments connected via a junction region: a unique long (UL) segment and a unique short (US) segment. ICP4 is encoded at two loci and ICP47 is encoded at one locus. TR represents terminal repeats; IR represents inverted repeats. Figure 1B Schematic diagram of the HSV-1 genome of the McKrae strain with a deletion in the ICP4 locus is shown. Figure 1C A schematic diagram of the HSV-1 genome of the McKrae strain in which both the ICP4 and ICP47 loci are deleted is shown.

[0070] Figure 2Exemplary vectors designed to deliver multiple payloads (e.g., genes 1-5) to cancer cells are illustrated. The upper figure shows the general design of a promoter suitable for driving the expression of a payload cassette. The lower figure shows an exemplary design of a payload cassette with an HCMV immediate early promoter (HCMV IEp), payload genes 1-5, and a poly A tail (pA). Thosea asigna virus 2A (T2) and teschovirus-1 2A (P2) display exemplary ribosomal skipping sites.

[0071] Figure 3A Graph showing the viability of cancer cells infected with the McKrae HSV-1 strain with ICP4 deletion at three MOIs (0, 3, and 10) monitored over 8 days. As shown, infection with the ICP4-deleted virus caused delayed oncolysis. Figure 3B Provided are graphs showing payload expression in cancer cells infected with the McKrae HSV-1 strain with ICP4 deletion at three MOIs (0, 3, and 10) monitored over 8 days. As shown, infection with the ICP4-deleted virus resulted in sustained payload expression. Figure 3C is a graph showing the viability of Hs578T cancer cells infected with mutICP4mutICP47 McKrae HSV-1 strain at three MOIs (0, 3, and 10) monitored over 6 days. Figure 3D is a graph showing IFNγ payload expression on days 2, 4, and 6 in Hs578T cancer cells infected at an MOI of 10 with mutICP4mutICP47 McKrae HSV-1 strain.

[0072] Figure 4A 24 hours later, the expression of payload (GFP) in cells infected with one of two exemplary oncolytic viruses, mutICP4McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, at three MOIs (0.3, 1, and 3) is shown. "mutICP4" is a McKrae HSV-1 strain in which the gene encoding ICP4 is deleted. "mutICP4mutICP47" is a McKrae HSV-1 strain in which the genes encoding ICP4 and ICP47 are deleted. These data indicate that similar mutICP4 and mutICP4mutICP47 infections were obtained. Figure 4B24 hours later, the cells were infected with one of two exemplary oncolytic viruses, mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, at three MOIs (0.3, 1, and 3). These data indicate that almost all cells are HLA positive. Figure 4C It is a flow cytometric graph of HLA expression in cells infected with one of two exemplary oncolytic viruses, mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1, according to flow cytometry after 24 hours at three MOIs (0.3, 1, and 3). These data show that in viruses in which both ICP4 and ICP47 are deleted, increased cell surface expression of HLA (a component of MHC class I molecules) is observed at all MOIs compared to ICP4 deletion alone. Figure 4D is a graph showing Tap1 expression in Hs578T cells infected with one of two exemplary oncolytic viruses, mutICP4mutICP47 McKrae HSV-1 and mutICP27 McKrae HSV-1, as measured by RNAseq. Figure 4E is a graph showing MHC class I expression in Hs578T cells infected with one of two exemplary oncolytic viruses, mutICP4mutICP47 McKrae HSV-1 and mutICP27 McKrae HSV-1, as measured by flow cytometry. Figure 4F is a diagram showing the cell signaling pathways upregulated in cancer cells following mutICP4mutICP47McKraeHSV-1 infection. Figure 4G is a diagram showing cell signaling pathways upregulated in cancer cells following mutICP27 McKrae HSV-1 infection.

[0073] Figure 4H is a graph showing the expression of TNF superfamily members (TNF, LTB, TNFSF14, PYCARD, and CASP10) in cancer cells after infection with mutICP4mutICP47 McKrae HSV-1 or mutICP27McKrae HSV-1.

[0074] FIG. 5A to FIG. 5B is a graph showing the number of CD31+ endothelial cells and PDGFRa+PDPN+ stromal cells in mouse salivary glands following infection with mutICP4mutICP47 McKrae HSV-1 virus carrying a payload of murine LTB, CXCL13, CCL19, CCL21, and IL-7. Figure 5Cis a graph showing the expression of TGFβ1 in Hs578T cells after infection with mutICP4mutICP47 (with encoded GFP as a marker) as measured by RNAseq.

[0075] Fig. 6A is a graph showing the viability of Hs578T cells after infection with the viral backbone alone (mutICP4mutICP47-GFP) or mutICP4mutICP47 vector encoding human IFNγ, human IL-12, or human IFNγ and human IL-12 or recombinant rhIL-15 added after co-culture with PBMCs. Figure 6B and Figure 6C is a graph showing the number of Ki67+ granzyme B+CD8+ T cells after 24h and 72h of co-culture of PBMCs with Hs578T cells after infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (skeleton). Fig.6D is a graph showing the number of granzyme B+Ki67-NK cells after 24 h of co-culture of PBMCs with Hs578T cells following infection with MutI CP4mutICP47-IFNγ or MutICP4mutICP47-GFP (backbone). Fig. 6E is a graph showing the number of antigen presenting cells (CD11c+CD16+CD14-Ki67+MHCII++) after 24 h of co-culture of PBMCs with Hs578T cells after infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (scaffold). Fig. 6F is a graph showing the viability of Hs578T cells after infection with MutICP4mutICP47-IFNγ or MutICP4mutICP47-GFP (scaffold) with or without PBMC co-culture. Figure 6G is a graph showing the expression of various T and NK cell chemokines (CCL 2, CXCL9, CXCL10, CXCL11, CCL5, CX3CL1 and CXCL16) by Hs578T cells 24 h after infection with mutICP4mutICP47-GFP or mutICP4mut ICP47-IFNγ as measured by RNAseq.

[0076] Fig. 7A is a graph showing the percentage (%) of CD45+ cells in mouse salivary glands harvested 15 days after delivery of mutICP4mutICP-GFP HSV-1 vector or vehicle to mice.

[0077] Figure 7BShown are images of cells isolated from mouse salivary glands harvested 15 days after delivery of the mutICP4mutICP-GFPHSV-1 vector to mice, collected by microscopy at 40x resolution. Cells were stained for CD4+ (T cell marker), B220 (B cell marker), CD11c (dendritic cell marker), and DAPI (nuclear marker). Figure 7C Shown are pictures of salivary glands from mice harvested 15 days after delivery of a combination of mutICP4mutICP47 HSV-1 vectors expressing murine TNF, CCL19, IL-17a, and IL-7 to mice collected by microscopy at 10x and 40x resolution. Cells were stained for CD4+ (T cell marker), B220 (B cell marker), CD11c (dendritic cell marker), and DAPI (nuclear marker).

[0078] Figure 8 Graph showing the percentage (%) of phagocytosis of labeled Raji cells by macrophages. Raji cells were pretreated with conditioned medium collected from Hs578T cells infected with mutICP4mutICP47-SIRPα-IgG (MOI of 3 or 10), conditioned medium collected from mutICP4mutICP47-GFP cells, IgG, or anti-CD47 antibody. DETAILED DESCRIPTION

[0079] The present invention is based in part on the discovery that HSV-1 vectors comprising alterations that prevent expression of one or more functional ICP4 and ICP47 proteins (such as gene deletions or inactivating mutations) have reduced replication capacity, are oncolytic, have extended payload expression time, are cytotoxic to proliferating cells (e.g., cancer cells), and exhibit enhanced immunogenicity. Thus, it has been found that the vectors can be used to deliver therapeutic payloads (e.g., therapeutic polypeptides) that can be used to reduce tumor size, e.g., for treating cancer in a subject in need thereof. The vectors can induce delayed oncolysis, thereby allowing sustained expression of therapeutic payloads (e.g., therapeutic polypeptides). In addition, the vectors exhibit increased immunogenicity, thereby increasing immune activity against tumor cells.

[0080] The present invention is also based in part on the discovery that specific 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 reducing tumor size. For example, therapeutic polypeptides that target tumor stroma, support T cells (e.g., CAR T cells) and / or NK cells to survive in the tumor microenvironment, induce tertiary lymphoid structures (TLS) in the tumor bed, and / or promote innate immune surveillance by phagocytes can be expressed from viral vectors (e.g., viral vectors lacking functional ICP4 and ICP47 proteins) to increase the ability of immune cells to kill tumor cells, for example, to treat cancer in subjects in need. The vector can be designed to include a specific combination of therapeutic polypeptides that will effectively treat a given tumor type, including selecting one or more therapeutic polypeptides that reduce suppressive features in a specific tumor microenvironment that may prevent the immune system from effectively attacking the tumor.

[0081] I. Definitions

[0082] In this application, unless otherwise clear from the context, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to cover the components or steps listed itemized, whether presented alone or together with one or more other components or steps; and (iv) where a range is provided, the endpoints are included.

[0083] As used herein, the term "administering" refers to administering a composition to a subject or system. Administration to an animal subject (e.g., to a human) can be performed by any appropriate route. For example, in some embodiments, administration can be transbronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, surface, transtracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous. Depending on the circumstances, administration may involve intermittent administration and / or may involve continuous administration (e.g., perfusion) for at least one selected period of time.

[0084] As used herein, the term "agent" refers to a compound or entity of any chemical class, including, for example, polypeptide, nucleic acid, sugar, lipid, small molecule or a combination thereof. In some embodiments, the agent is a natural product or comprises a natural product, because it is found in nature and / or obtained from nature. In some embodiments, the agent is one or more artificial entities or comprises one or more artificial entities, because it is designed, engineered and / or produced by artificial action and / or is not found in nature. Some specific embodiments of the agent that can be used according to the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (such as siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc.

[0085] As used herein, the term "ameliorate" refers to preventing, alleviating or alleviating a condition, or improving a subject's condition. Amelioration includes but does not require complete restoration or complete arrest of a disease, disorder or condition.

[0086] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to people of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates and / or pigs). In some embodiments, animals include but are not limited to mammals, birds, reptiles, amphibians, fish, insects and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals and / or clones. Depending on the context, the terms "people," "patient" and "subject" are used interchangeably herein.

[0087] The terms "about" and "approximately" may be understood to allow for standard variations as would be understood by one skilled in the art. For example, unless otherwise indicated or inferred, as used herein, the term "about" refers to a variation of ±10% from the nominal value.

[0088] As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered with overlapping dosing regimens.

[0089] As used herein, the term "engineered" refers to aspects that have been artificially manipulated. For example, when two or more sequences are artificially manipulated to be directly connected to each other in an engineered polynucleotide (the two or more sequences are not connected together in this order in nature), the polynucleotide is considered to be "engineered". For example, in some embodiments of the present disclosure, the engineered polynucleotide comprises a regulatory sequence, which is found in nature to be operably associated with a first coding sequence but not operably associated with a second coding sequence, and is artificially connected so that it is operably associated with a second coding sequence. Similarly, if a cell or organism has been manipulated so that its genetic information is changed (for example, a new genetic material that did not exist previously has been introduced, such as by transformation, mating, somatic cell hybridization, transfection, transduction or other mechanisms, or, for example, by substitution or deletion mutation or by mating schemes. Preexisting genetic material is changed or removed), then the cell or organism is considered to be "engineered". As is understood by those skilled in the art as a convention, the offspring of engineered polynucleotides or cells are still generally referred to as "engineered", although the actual manipulation is performed on existing entities.

[0090] 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 the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0091] As used herein, the term "homology" refers to the overall correlation between polymer molecules, such as between nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, if the sequence of a polymer molecule is at least 75%, 80%, 85%, 90%, 95% or 99% identical, then they are considered to be "homologous" to each other. In some embodiments, if the sequence of a polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% similar, then they are considered to be "homologous" to each other.

[0092] As used herein, the term "isolated" means that a substance and / or entity has been (1) separated from at least some of the components with which it was originally associated when it was originally produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which it was originally associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the separation percentage or purity of the substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biopolymer (such as a naturally occurring polypeptide or polynucleotide) is considered "isolated" when it a) is not associated with some or all of the components that accompany it in its native state in nature, due to its origin or source of derivation; b) is substantially free of other polypeptides or nucleic acids of the same species as the species in which it is produced in nature; c) is expressed by a cell or other expression system that is not of the species in which it is produced in nature or is otherwise associated with components from a cell or other expression system that is not of the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system that is different from the cell system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide if it has been separated from other components with which it is associated a) in nature; and / or b) when originally produced.

[0093] As used herein, the term "marker element" refers to a detectable or selectable agent. In some embodiments, a "marker element" is a detectable or selectable nucleic acid sequence. In some embodiments, a "marker element" is an expression product (e.g., RNA or protein) whose presence or absence is detectable and / or selectable in a cell. In some embodiments, the expression product is an enzyme or comprises an enzyme. In some embodiments, the expression product is a fluorophore.

[0094] As used herein, the term "nucleic acid" refers to any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are incorporated into or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is RNA or comprises RNA; in some embodiments, "nucleic acid" is DNA or comprises DNA. In some embodiments, nucleic acid is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues, or consists of them. In some embodiments, nucleic acid is one or more nucleic acid analogs, comprises one or more nucleic acid analogs, or consists of them. In some embodiments, nucleic acid analogs are different from nucleic acids because they do not use a phosphodiester backbone. For example, in some embodiments, nucleic acid is one or more "peptide nucleic acids", comprises one or more "peptide nucleic acids", or consists of them, and the one or more peptide nucleic acids are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, which are considered to be within the scope of the present disclosure. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate bonds and / or 5'-N-phosphoramidite bonds, rather than phosphodiester bonds. In some embodiments, the nucleic acid is, comprises or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine). In some embodiments, the nucleic acid is, comprises or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiolcytidine, methylated bases, intercalated bases, and combinations thereof. In some embodiments, the nucleic acid comprises one or more sugars that are modified compared to those in natural nucleic acids (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or protein). In some embodiments, nucleic acid includes one or more introns. In some embodiments, nucleic acid is prepared by one or more of the following: separation from natural sources, synthesis (in vivo or in vitro) by polymerase based on complementary templates, replication and chemical synthesis in recombinant cells or systems.In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, or more residues in length. In some embodiments, the nucleic acid is single-stranded; in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element encoding a polypeptide or is the complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0095] As used herein, the terms "patient" or "subject" are used interchangeably and refer to any organism to which the provided compositions are or can be applied, for example, for experimental, diagnostic, preventive, cosmetic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates and / or humans). In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or diseases. In some embodiments, the subject exhibits one or more symptoms of a disorder or disease. In some embodiments, the subject has been diagnosed with one or more disorders or diseases. In some embodiments, the subject is receiving or has received certain therapies for diagnosing and / or treating a disease, disorder or disease.

[0096] As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a carrier) formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration with a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as a drench (aqueous or non-aqueous solution or suspension), tablet (such as those targeted for buccal, sublingual, and systemic absorption), bolus, powder, granules, paste for application to the tongue; parenteral administration, such as in the form of a sterile solution or suspension or a sustained release formulation, such as by subcutaneous, intramuscular, intravenous, or epidural injection; topical application, such as in the form of a cream, ointment, or controlled release patch or spray suitable for use on the skin, lungs, or oral cavity; intravaginal or rectal use, such as in the form of a pessary, cream, or foam; sublingual; ocular; transdermal; or nasal, pulmonary, and to other mucosal surfaces.

[0097] As used herein, the term "pharmaceutically acceptable" as applied to a 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.

[0098] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle that participates in the transport or transportation of the compounds of the invention from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that 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 carboxymethylcellulose, ethylcellulose, 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 glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0099] As used herein, the terms "prevent" and "prevention" when used in conjunction with the occurrence of a disease, disorder, and / or condition refer to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention is considered complete when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0100] As used herein, the term "treatment (treatment / treat / treating)" refers to any administration of a substance that partially or completely alleviates, improves, alleviates, inhibits, delays the onset of one or more symptoms, characteristics and / or causes of a specific disease, disorder and / or illness (e.g., cancer), reduces its severity and / or reduces its incidence. Such treatment can be the treatment of subjects who do not show signs of the relevant disease, disorder and / or illness and / or the treatment of subjects who only show early signs of the disease, disorder and / or illness. Alternatively or in addition, such treatment can be the treatment of subjects who show one or more determined signs of the relevant disease, disorder and / or illness. In some embodiments, treatment can be the treatment of subjects who have been diagnosed with the relevant disease, disorder and / or illness. In some embodiments, treatment can be the treatment of subjects who are known to have one or more sensitive factors that are statistically associated with an increased risk of developing the relevant disease, disorder and / or illness.

[0101] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be linked. Another type of vector is a viral vector, in which other DNA segments can be linked to a viral genome or part thereof. Some vectors can replicate autonomously in a host cell introduced therein (e.g., a bacterial vector, an additional mammalian vector, a herpes simplex virus (HSV) vector with a bacterial origin of replication). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of a gene operably connected thereto. Such vectors are referred to herein as "expression vectors".

[0102] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally implemented in the art or as described herein. The aforementioned techniques and procedures can be performed generally according to conventional methods well known in the art and as described in various general and more specific references cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[0103] The use of sections is not meant to limit the present invention. Each section may apply to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated or clearly separated from the context.

[0104] Throughout this specification, 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 there are additional compositions of the invention consisting essentially of, or consisting of, the recited components, and that there are processes and methods according to the invention consisting essentially of, or consisting of, the recited processing steps.

[0105] In the present application, where an element or component is said to be included in and / or selected from a series of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group consisting of two or more of the listed elements or components.

[0106] In addition, it should be understood that, without departing from the spirit and scope of the present invention, the elements and / or features of the compositions or methods described herein can be combined in various ways, whether or not explicitly or implicitly herein. For example, unless otherwise understood from the context, when referring to a particular compound, the compound can be used in various embodiments of the compositions of the present invention and / or the methods of the present invention. In other words, within the application, embodiments have been described and depicted in a manner that enables writing and drawing clear and concise applications, but it is expected and will be understood that, without departing from the teachings of the present invention and the invention, embodiments can be combined or separated differently. For example, it should be understood that all features described and displayed herein can be applied to all aspects of the present invention described and displayed herein.

[0107] It should be understood that, unless otherwise understood from the context and usage, the expression "at least one of" includes each of the objects listed after the expression individually and various combinations of two or more of the objects listed. Unless otherwise understood from the context, the expression "and / or" in combination with three or more of the objects listed should be understood to have the same meaning.

[0108] Unless specifically stated or understood otherwise from the context, the terms “include / includes / including”, “have has having” or “contain / contai ns / containing” (including grammatical equivalents thereof) should generally be understood as open and non-restrictive, for example, not excluding other unlisted elements or steps.

[0109] It should be understood that the order of steps or the order in which certain actions are performed is not important, as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0110] The use of any and all examples or exemplary language (e.g., "such as" or "including") herein is intended only to better illustrate the present invention and does not limit the scope of the present invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the present invention.

[0111] II. Viral vectors and HSV-1 (McKrae)

[0112] Viral vectors can be used to facilitate nucleic acid transfer into cells. HSV-1 vectors can usually accommodate up to 25 kb of foreign DNA sequences. HSV-1 has an approximately 152-kb double-stranded linear DNA genome that can be additionally maintained in the nucleus. HSV-1 virions are enveloped and approximately 110 nm in diameter.

[0113] At least 17 HSV-1 strains 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).

[0114] A.McKrae

[0115] The McKrae strain was isolated from a patient with herpes simplex keratitis and subsequently passaged in tissue culture. The partial genomic sequence of McKrae is provided in SEQ ID NO: 1 (GenBank Accession No.: JQ730035.1).

[0116] Interstrain differences in HSV-1 peripheral replication and virulence were observed after injection into animals. McKrae undergoes spontaneous or induced reactivation at a higher frequency than other known strains and is among the most virulent HSV-1 strains.

[0117] HSV genes affect viral characteristics and phenotype. There are at least 9 genes and several non-coding sequences that are unique to the McKrae strain. In addition to those genes related to pathogenesis and latent reactivation (such as RL1, RSI and RL2), three UL genes (UL36, UL49A, UL56) and three US genes (US7, US10 and US11) are unique to the McKrae strain. In addition to genetic variation, non-coding sequences (such as LAT, 'a' sequences and miRNA) also contain variations unique to McKrae.

[0118] One or more of the following genes and non-coding sequences may be considered characteristic of the McKrae strain. In McKrae, RL1 (ICP34.5) has an extended PAT repeat between residues 159 and 160, which results in 8 iterations, while other strains contain only 3-5 iterations. The PAT repeat is thought to affect the cellular localization of the ICP34.5 protein. (Mao et al. (2012) JB IOL .C HEM .277(13):11423-31). ICP34.5 is considered to be a neurovirulence factor involved in viral replication and anti-host response.

[0119] The McKrae strain also contains an extended repeat element of six iterations of the internal tandem repeat STPSTTT (SEQID NO: 38) located within the coding sequence of US07 (gl). In addition, in McKrae, UL 36 contains an early stop codon introduced by the deletion of a G nucleotide in a single nucleotide string encoding amino acid residue 2453 (nt 72,535), and UL 56 (180aa) contains a single base pair insertion at nucleotide 115,992 (amino acid 97). The McKrae strain also contains an extended ORF in US10 generated by a single bp insertion at nucleotide 143,416, and the frameshift causes the loss of the stop codon in McKrae and a unique C-terminal protein sequence. Compared with other strains, McKrae has amino acid differences at UL49A residues 28 and 51. 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. In addition, the McKrae strain contains a reduced number of tandem repeats found at the UL-RL junction (49 bp in McKrae, compared to 181 bp in strains 17 and KOS), and is missing approximately 330 nucleotides immediately following the UL-RL junction repeat. McKrae also contains a unique variation within the 'a' sequence direct repeat 2 (DR2) array. The McKrae DR2 repeat is interrupted twice by the same guanine-rich sequence, rather than a series of uninterrupted tandem repeats.

[0120] The major variation within the LAT intron between strains is due to differences in the repeat element (GCACCCC CACTCCCAC) (SEQ ID NO: 39), which has different numbers of iterations starting at nucleotide 119,482 in the McKrae strain, with McKrae containing 13 repeats, while strains F, H129 and 17 contain 9 repeats, and KOS contains 15 repeats. In addition, tandem repeat variation was found between strains starting at base 125,520 in McKrae. The McKrae repeat element includes twelve iterations of CCCCAGCCCTCCCCAG (SEQ ID NO: 40) and eight iterations of CCCCTCGCCCCCTCCCG (SEQ ID NO: 41). The first repeat unit is unique from the other strains in that it contains a GA transition, and strain McKrae contains three more iterations than any other strain. The second repeat element of the McKrae strain was collapsed, losing 188 nucleotides relative to all other strains, and was separated from the upstream repeat by a 105 bp 100% conserved sequence containing miR-H5.

[0121] McKrae also contains a unique ICP4 coding sequence not found in other known strains (Watson et al., (2012) supra). ICP4 is an immediate early transcriptional regulator and is involved in reactivation. While 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 substitution block present in McKrae is close to the nuclear localization signal (NLS) (amino acids 728-734). Changes in the conformation of this region can alter the NLS and, in turn, affect not only the localization of ICP4, but also the localization of other viral proteins (e.g., ICP0, ICP8) that are affected by ICP4 localization (Knipe and Smith, Molecular and Cellular Biology, (1986), 6 (7), 2371-2381). Therefore, this region may affect the viral phenotype in part by altering the localization of the protein in the nucleus.

[0122] B. Modification of HSV-1 vector

[0123] A vector comprising an HSV-1 genome (e.g., from a McKrae strain) may have one or more HSV genes necessary for the replication of a loss of function. For example, a vector comprising an HSV genome may contain changes that prevent the 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 that is expressed shortly after infection and maintains the HSV-1 viral cycle. The ICP47 of HSV-1 is an 87 amino acid cytoplasmic polypeptide, 88 residues if the start methionine is included. It binds to the TAP1-TAP2 heterodimer in human cells, but does not bind in mouse cells, and blocks the transport of peptides by blocking the peptide binding site of TAP. Therefore, MHC class I molecules cannot load peptides. The resulting empty class I molecules are retained in the ER, and the presentation of epitopes to CTLs is eliminated in HSV infected human cells. Changes that prevent expression of one or more functional HSV genes (e.g., ICP4 and ICP47) may include mutations (e.g., missense mutations, nonsense mutations, insertions, deletions, etc.) in the coding sequence of the gene or in a regulatory sequence (e.g., promoter) that affects gene expression. ICP47 mutations include, but are not limited to, mutations in A4, D27, K31, R32, R34, or R41 relative to SEQ ID NO: 5 and combinations thereof (as described in Mozzie et al. (2022) MOL BIOL EVOL. 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 and 210 at 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 present disclosure provides replication-deficient HSV vectors having alterations in one or more of ICP0, ICP4, ICP22, ICP27, and ICP47.

[0124] The HSV-1IE promoter contains one or more copies of an IE-specific regulatory sequence with a consensus of TAATGARAT (SEQ ID NO: 44) (wherein R is a purine). These motifs are typically located within a few hundred base pairs of the proximal IE promoter sequence, but in conjunction with their flanking sequences, they are discrete functional entities that can confer IE-specific regulation on other proximal promoter elements of different temporal categories. In some embodiments, a replication-deficient virus is formed by deleting nucleotides in an IE-specific regulatory sequence (e.g., an IE-specific regulatory sequence that affects the expression of one or more of ICP0, ICP4, ICP22, ICP27, and ICP47). In some embodiments, the IE-specific regulatory sequence contains an internal deletion. In some embodiments, the IE-specific regulatory sequence contains a terminal deletion. In some embodiments, the IE-specific regulatory sequence is completely deleted.

[0125] In some embodiments, the present disclosure provides HSV vectors with non-functional ICP4 and ICP47 genes. In certain embodiments, the ICP4 gene or a regulatory sequence affecting the expression of ICP4 comprises a mutation, and the ICP47 gene or a regulatory sequence affecting the expression of ICP47 comprises a mutation. For example, the ICP4 gene may comprise a mutation and the ICP47 gene may comprise a mutation. In another example, the regulatory sequence affecting the expression of ICP4 comprises a mutation and the regulatory sequence affecting the expression of ICP47 comprises a mutation. In a specific example, the ICP4 gene or a regulatory sequence affecting the expression of ICP4 comprises a deletion (e.g., a complete deletion or a partial deletion sufficient to eliminate activity), and the ICP47 gene or a regulatory sequence affecting the expression of ICP47 comprises a deletion (e.g., a complete deletion or a partial deletion sufficient to eliminate activity). The ICP4 gene may include a deletion and the ICP47 gene may include a deletion. In addition, combinations of the foregoing situations are contemplated, such as a deletion of ICP4 and a mutation in the regulatory sequence of ICP47.

[0126] In some embodiments, the present disclosure provides an HSV vector or HSV strain having a non-functional ICP47 gene. In some embodiments, the present disclosure provides an HSV vector or HSV strain having non-functional ICP4 and ICP47 genes. In some embodiments, the present disclosure provides an HSV vector or HSV strain with ICP4 and ICP47 deletions. In some embodiments, the gene encoding ICP4 and the gene encoding ICP47 are completely or partially deleted without disrupting the expression of any other immediate early genes.

[0127] HSV-1 vectors with altered (e.g., mutated) HSV genes can be produced in cell lines that express defective proteins in trans. In some embodiments, HSV-1 vectors are produced in mammalian cell lines (e.g., mammalian cell lines of the Vero pedigree). 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 (SV40pA). Depending on the circumstances, viral vectors can be produced in Vero 6-5C cells or Vero D cells.

[0128] In certain embodiments, the viral vector of the present disclosure is a McKrae HSV-1 viral vector. The wild-type McKrae strain HSV-1 comprises two copies of a gene encoding ICP4 and one copy of a gene encoding ICP47. In some embodiments, the present disclosure provides an HSV vector having non-functional ICP4 and ICP47 genes. In certain embodiments, at least one ICP4 gene (i.e., one or two copies of a gene) or a regulatory sequence affecting the expression of ICP4 comprises a mutation and the ICP47 gene or a regulatory sequence affecting the expression of ICP47 comprises a mutation. For example, at least one copy of the ICP4 gene (i.e., one or two copies of the ICP4 gene) may comprise a mutation and the ICP47 gene may comprise a mutation. In another example, a regulatory sequence affecting the expression of at least one ICP4 gene (i.e., one or two copies of the ICP4 gene) comprises a mutation and a regulatory sequence affecting the expression of ICP47 comprises a mutation. In a specific example, at least one ICP4 gene (i.e., one or two copies of the ICP4 gene) or a regulatory sequence affecting the expression of at least one ICP4 gene (i.e., one or two copies of the ICP4 gene) comprises a deletion and the ICP47 gene or a regulatory sequence affecting the expression of ICP47 comprises a deletion. At least one ICP4 gene (i.e., one or two copies of the ICP4 gene) may comprise a deletion and the ICP47 gene may comprise a deletion.

[0129] In some embodiments, the present disclosure provides an HSV vector or HSV strain having a non-functional ICP47 gene. In some embodiments, the present disclosure provides an HSV vector or HSV strain having non-functional ICP4 and ICP47 genes. In some embodiments, the present disclosure provides an HSV vector or HSV strain having an ICP4 deletion and one or two copies of ICP47 deletion. In some embodiments, one or two copies of the gene encoding ICP4 are completely or partially deleted and the gene encoding ICP47 is completely or partially deleted without disrupting the expression of any other immediate early genes.

[0130] C. Application of HSV-1 vector

[0131] The viral vectors described herein (eg, HSV-1 vectors) can exhibit characteristics that are advantageous for use in treating disease (eg, for treating cancer).

[0132] In certain embodiments, the viral vectors as described herein exhibit delayed oncolysis (e.g., when administered to a subject). The delayed oncolysis allows the persistence of the virus in target (e.g., cancer) cells, sustained payload expression, exposure of tumor-associated antigens in the tumor microenvironment caused by the payload, and limited nonspecific inflammation and tissue damage. The delayed oncolysis allows the persistence of the virus in target cells, sustained payload expression, exposure of tumor-associated antigens in the tumor microenvironment caused by the payload, and limited nonspecific inflammation and tissue damage.

[0133] 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 herein in the Examples, 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.

[0134] 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 an increase or decrease in RNA expression in a pathway associated with immune cell activation, including but not limited to an IFNα response pathway, an inflammatory response pathway, a fungal signaling pathway, and / or an MtorC1 signaling pathway. The genes involved in such pathways are described on the GSEA website. See, for example, gsea-msigdb.org / gse a / msigdb / collections.jsp. Immune activation can also be detected by detecting increased expression of TNF superfamily members (including tumor necrosis factor-α (TNF), lymphocyte toxin β (LTB), APRIL (TN FSF13), and LIGHT (TNFSF14)) and / or detecting expression of immunogenic cell death regulators indicating changes in pyroptosis and / or increased apoptosis (such as detecting an increase in expression of PYCARD and caspase 10 (CASP10)).

[0135] III. Payload

[0136] Viral vectors according to the present disclosure include nucleic acid molecules containing the payload of the vector. Payload includes nucleic acid molecules encoding one or more polypeptides. It is expected that the payload may include nucleic acid molecules containing sequences complementary to the nucleic acid sequences encoding the polypeptides. Payload may encode nucleic acid molecules with regulatory functions, such as small interfering RNA (siRNA) polynucleotides or micro RNA (miRNA) polynucleotides.

[0137] The payload can be a nucleic acid molecule encoding a protein that is exogenous to the target tissue or the subject to which the vector is administered. For example, the payload can be a nucleic acid molecule encoding a protein that is endogenous to the target tissue or the subject to which the vector is administered. In some embodiments, the nucleic acid molecule is codon optimized.

[0138] 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.

[0139] The therapeutic polypeptide encoded on the vector may have one or more of the following functional attributes:

[0140] (1) Targeting tumor stroma;

[0141] (2) Supporting T cell (e.g., CAR T cell) survival in the tumor microenvironment;

[0142] (3) induction of tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0143] (4) Promote innate immune surveillance of phagocytes.

[0144] One or more therapeutic polypeptides from one or more classes (1-4 above) can be encoded on the same vector to target multiple pathways for increasing immune activity against tumors. For example, the vector can encode 1, 2 or 3 therapeutic polypeptides from class 1 and 1, 2 or 3 therapeutic polypeptides from class 2, 3 or 4. Alternatively, the vector can encode 1, 2 or 3 therapeutic polypeptides from class 2 and 1, 2 or 3 therapeutic polypeptides from class 3 or 4. Alternatively, the vector can encode 1, 2 or 3 therapeutic polypeptides from class 3 and 1, 2 or 3 therapeutic polypeptides from class 4. Alternatively, the vector can encode 1 or 2 therapeutic polypeptides from class 1 and 1 or 2 therapeutic polypeptides from class 2, 3 and / or 4. Alternatively, the vector can encode 1 or 2 therapeutic polypeptides from class 2 and 1 or 2 therapeutic polypeptides from class 3 and / or 4. Similarly, the vector can encode 1 or 2 therapeutic polypeptides from class 3 and 1 or 2 therapeutic polypeptides from class 4.

[0145] The therapeutic polypeptide in the payload can be cloned in series and driven by the same promoter, and the joint sequence separates the individual polypeptides. In certain embodiments, the joint sequence comprises a ribosome jump site (sequence), such as a 2A peptide. In an expression construct, when such a ribosome jump site is included in the joint between the two polypeptide sequences, the translation of the transcribed mRNA causes two polypeptides to be produced separately, rather than in the form of a fusion protein. The 2A peptide sequence shares the core sequence motif of DXEXNPGP, where X is any amino acid (SEQ ID NO: 45). Non-limiting examples of suitable 2A peptide sequences include T2A (EGRGSLLTC GDVEENPGP; SEQ ID NO: 46), P2A (ATNFSLLKQAGDVEENPG P; SEQ ID NO: 181), 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-terminus of the 2A peptide to improve efficiency.

[0146] A. Targeting the Tumor Matrix

[0147] The viral vector may 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 reducing tumor size.

[0148] Tumor stroma can be targeted by different mechanisms. For example, the extracellular matrix can be dissolved by proteins involved in the degradation of the extracellular matrix (ECM), including but not limited to hyaluronidase, MMP-9 and inhibitors of lysyl oxidase. Therefore, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides involved in the degradation of the extracellular matrix (ECM), including but not limited to inhibitors of hyaluronidase, MMP-9 and lysyl oxidase.

[0149] In addition, cancer-associated fibroblasts can be targeted by proteins that interfere with the TGFβ pathway. Therefore, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides that interfere with the TGFβ pathway, including, for example, polypeptides that include an external domain (e.g., a non-transmembrane domain) of TGFβ. In certain embodiments, the therapeutic polypeptide includes a fusion protein comprising an external domain of TGFβ, an IgG domain (e.g., an Fc domain), and an external domain of TGFβRII.

[0150] The local endothelium can be activated to facilitate T cell infiltration by expressing proinflammatory cytokines, including but not limited to TNF, IL-1β, IL-6 and IL-18. The expression of enzymes that degrade the extracellular matrix of tumor cells will simultaneously destroy the physical barriers within the tumor to allow immune cell infiltration. Therefore, in certain embodiments, the viral vector encodes one or more proinflammatory cytokines, including, for example, TNF, IL-1β, IL-6 and IL-18.

[0151] Tumor stroma targeting and local endothelial activation can be regulated together or separately by localized expression of these therapeutic polypeptides.

[0152] In certain embodiments, one or more genes encoding hyaluronidase, MMP-9, inhibitors of lysyl oxidase, therapeutic polypeptides that interfere 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, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0153] In certain embodiments, the effects of cancer cells that secrete factors that lyse the tumor stroma or activate local endothelium for T cell infiltration on the tumor stroma or local endothelium can be assessed in vitro or in vivo using methods known in the art.

[0154] B. Support T cells (such as CAR T cells) to survive in the tumor microenvironment

[0155] In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support T cells to survive in a tumor microenvironment (TME). In the embodiments described herein, the T cells can be natural T cells (e.g., T cells present in a subject to which a viral vector is administered) or CAR T cells (e.g., CAR T cells administered to a subject). In certain embodiments, the viral vector is administered together with a T cell (e.g., CAR T cell), for example, to treat a tumor.

[0156] T cells in the tumor microenvironment can be supported by different mechanisms. For example, the recruitment of T cells (such as natural or CAR T cells) to the tumor site can be improved by the presence of T cell recruitment factors (such as chemokine ligands CCL19 or CCL21) in the tumor microenvironment.

[0157] Local T cells can be supported to induce strong and lasting anti-tumor responses. For example, local expression of T cell trophic factors such as IL-7, IL-12, IL-15, IL-18, and IFNγ can induce strong and lasting anti-tumor responses from local or recruited T cells. Therefore, in certain embodiments, the viral vector encodes one or more T cell trophic factors, including, for example, IL-7, IL-12, IL-15, IL-18, and IFNγ.

[0158] Additionally, TGFβ signaling in cells within a tumor may be inhibited due to expression of soluble TGFβRII. Thus, in certain embodiments, the therapeutic polypeptide comprises soluble TGFβRII.

[0159] Depending on the target TME, local expression of a gene encoding a co-stimulatory molecule such as CD40L or OX40L can stimulate local or recruited T cells. Thus, in certain embodiments, the therapeutic polypeptide comprises CD40L or OX40L.

[0160] T cell support or T cell recruitment can be modulated by these therapeutic polypeptides together or individually.

[0161] In certain embodiments, one or more genes encoding CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFNγ, 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, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0162] The effects of cancer cells secreting these factors on T cell recruitment or T cell function can be assessed in vitro or in vivo using methods known in the art.

[0163] C. Supporting NK cells (e.g., CAR NK cells) to survive in the tumor microenvironment

[0164] In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support NK cells to survive in a tumor microenvironment (TME). In the embodiments described herein, NK cells can be natural NK cells (e.g., NK cells present in a subject to which a viral vector is administered) or CAR NK cells (e.g., CAR NK cells administered to a subject). In certain embodiments, a viral vector is administered together with a NK cell (e.g., CAR NK cell), for example, to treat a tumor.

[0165] NK cells in the tumor microenvironment can be supported by different mechanisms. For example, the recruitment of NK cells (such as natural or CAR NK cells) to the tumor site can be improved by the presence of NK cell recruitment factors (such as chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11) in the tumor microenvironment.

[0166] Local NK cells can be supported to induce strong and lasting anti-tumor responses. For example, local expression of NK cell trophic factors (such as IL-2, IL-15, IL-18 and IFNα) can induce strong and lasting anti-tumor responses from local or recruited NK cells. Therefore, in certain embodiments, the viral vector encodes one or more NK cell trophic factors, including, for example, IL-2, IL-15, IL-18 and IFNα.

[0167] Additionally, TGFβ signaling in cells within a tumor may be inhibited due to expression of soluble TGFβRII. Thus, in certain embodiments, the therapeutic polypeptide comprises soluble TGFβRII.

[0168] NK cell support or NK cell recruitment can be modulated by these therapeutic polypeptides together or individually.

[0169] In certain embodiments, one or more genes encoding CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11 IL-2, IL-15, IL-18 and IFNα 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, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0170] The effects of cancer cells secreting these factors on NK cell recruitment or NK cell function can be assessed in vitro or in vivo using methods known in the art.

[0171] D. Induction of tertiary lymphoid structures in the tumor bed

[0172] In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that induce tertiary lymphoid structures (TLS) within the tumor bed.

[0173] The formation of TLS in tumors can play a role in anti-tumor immunity and is associated with the response to immune checkpoint inhibition. Thus, viral vectors encoding therapeutic polypeptides that induce TLS formation can be used according to the methods described herein. In certain embodiments, viral vectors are used in combination with other solid tumor therapeutic agents, including but not limited to CAR T cell therapy and immune checkpoint inhibitors.

[0174] In certain embodiments, one or more genes encoding therapeutic polypeptides involved in the establishment and maintenance of TLS (including but not limited to CCL19, lymphotoxin beta, CXCL13, and TNF) 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, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae strain HSV-1 virus.

[0175] The effects of cancer cells secreting these factors on tertiary lymphoid structures (TLS) within the tumor bed can be assessed in vitro or in vivo using methods known in the art.

[0176] E. Promotes innate immune surveillance by phagocytes

[0177] In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that promote innate immune surveillance by phagocytes and elimination of tumor cells.

[0178] CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal regulatory protein alpha (SIRPα) on macrophages, enabling these cancer cells to escape macrophage-mediated phagocytosis. The Sirpα / CD47 axis can be disrupted, for example, by the presence of a Sirpα-IgG fusion transgene to induce the promotion of phagocyte innate immune surveillance and tumor cell elimination. Disruption of the Sirpα / CD47 axis can promote phagocyte innate immune surveillance and tumor cell elimination by exposing the tumor's 'eat me' signal. Therefore, in certain embodiments, the viral vector encodes a therapeutic polypeptide that disrupts the Sirpα / CD47 axis, thereby promoting phagocyte innate immune surveillance and tumor cell elimination. In certain embodiments, the therapeutic polypeptide is an anti-CD47 antibody. In certain embodiments, the viral vector encodes a Sirpα-IgG fusion polypeptide.

[0179] Additionally, the vector may encode therapeutic polypeptides for supporting anti-tumor macrophage polarization, including but not limited to TNF, IL-1, IL-12, IL-17, and IFNγ.

[0180] In certain embodiments, one or more therapeutic polypeptides involved in the establishment and maintenance of TLS in tissues (including but not limited to Sirpα-IgG fusion transgenes, TNF, IL-1, IL-12, IL-17 and IFNγ) can be cloned into a non-replicating McKrae strain HSV-1 vector.

[0181] The effects of cancer cells secreting these factors on innate immune surveillance by phagocytes can be assessed in vitro or in vivo using methods known in the art.

[0182] IV. Regulatory Elements

[0183] Contemplated herein are the inclusion of non-native regulatory sequences, gene control sequences, promoters, non-coding sequences, introns or coding sequences in the nucleic acids disclosed herein. Also contemplated herein are nucleic acids that include nucleic acid tags or signal sequences or encoded protein tags or protein signal sequences. Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0184] The promoters included in the nucleic acids of the present disclosure may be tissue-specific or cell-type-specific promoters, promoters specific to multiple tissues or cell types, organ-specific promoters, promoters specific to multiple organs, systemic or ubiquitous promoters, or nearly systemic or ubiquitous promoters. Promoters with stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, inconstant, or unpredictable expression are also included in the present disclosure. Promoters of the present disclosure may include any of the above features or other promoter features known in the art.

[0185] Examples of known promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CMV / human β3 globulin promoter, the GFAP promoter, the chicken β-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.

[0186] In some embodiments, the promoter is a neuron-specific promoter because it is a promoter that has specific expression in neurons, preferential expression in neurons, or generally drives the expression of related coding sequences in neurons or neuronal subsets 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 αI, neuron-specific enolase, microtubule-associated protein IB (MAP1B), and platelet-derived growth factor β chain promoters and their derivatives. In some embodiments, the promoter is a calcitonin gene-related peptide (CGRP) promoter or a derivative thereof.

[0187] Other regulatory elements may additionally be operably linked to the payload, such as an enhancer and a polyadenylation site. In some embodiments, the enhancer comprises a human cytomegalovirus (HCMV) sequence. In some embodiments, the polyadenylation site comprises a bovine growth hormone (BGH) polyadenylation signal.

[0188] In some embodiments, the promoter is a chimera of one or more promoters or regulatory elements found in nature. In some embodiments, the viral vector comprises a payload, the expression of which is driven by a CGRP promoter with an HCMV enhancer sequence.

[0189] V. Preparation of vector

[0190] The present disclosure is particularly related to replication-deficient McKrae strain viral vectors. Viral vectors can be produced by mutations (e.g., deletions) of one or more immediate early genes or regulatory sequences that affect their expression. Viral genes can be mutated using methods of recombinant technology known in the art. The viral vectors of the present disclosure may become replication-deficient due to homologous recombination events. Replication-deficient viral vectors can be produced by mutations of the ICP4 gene and mutations of the ICP47 gene. For example, replication-deficient viral vectors are produced by deletions of the ICP4 gene and deletions of the ICP47 gene.

[0191] In some embodiments, the viral vector of the present invention is produced by homologous recombination through the deletion of the locus encoding one or more ICPs (e.g., ICP4 and ICP47). In some embodiments, the viral vector of the present invention is produced including the step of homologous recombination of a first plasmid and a second plasmid. In some embodiments, the first plasmid contains a nucleic acid sequence homologous to an HSV genomic region close to the HSV genomic nucleic acid region that is desired to be replaced. In some embodiments, the second plasmid contains the HSV genome or a fragment thereof. In some embodiments, the first plasmid contains a nucleic acid sequence encoding a gene of interest between homologous nucleic acid sequences. In some embodiments, the gene of interest may be or include a marker protein, which may be detected by fluorescence, chemiluminescence, or other properties that can be used to select a vector produced by successful homologous recombination.

[0192] In some embodiments, the viral vector of the present disclosure is produced by homologous recombination of a first plasmid containing a nucleic acid sequence homologous to a region upstream of the ICP4 promoter including the viral origin contained in a short inverted repeat region of HSV, and a second plasmid containing the HSV McKrae strain genome.

[0193] In some embodiments, the vector is prepared by first replacing two copies of the ICP4 locus by homologous recombination using plasmid SASB3 and screening for plaques expressing green fluorescent protein (GFP). In some embodiments, the plasmid is constructed as follows: the Sph I to AflIII (Sal I linker) fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides 124485-126413, KT899744, KOS strain) is cloned into Sph I / Sal I digested pSP72, and then a 695 bp Bgl II to BamH I fragment (GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), KOS strain, nucleotides 131931 to 132626) containing the region upstream of the ICP4 promoter including the viral origin contained in the short inverted repeat region is inserted into the BglII to BamH I site of the vector plasmid. In some embodiments, a plasmid is constructed by cloning a HCMV-eGFP fragment at the BamHI site of a plasmid as described above. In some embodiments, a plasmid as described above is then recombined into a specific locus of a wild-type McKrae virus. In some embodiments, a stable cell line is used to isolate the resulting viral vector, which expresses one or more genes deleted or disrupted in the HSV genome required for replication.

[0194] In some embodiments, the vector is prepared by first replacing two copies of the ICP4 locus by homologous recombination using plasmid SDAXB and screening for plaques expressing green fluorescent protein (GFP). In some embodiments, the plasmid is constructed as follows: the Sph I to Afl III fragment (1928 bp) of the HSV-1 KOS strain genome (GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), nucleotides 124346 to 126273 of the KOS strain) is cloned into Sph I / Afl III digested pSP72 to prepare plasmid SDA, and then the Afl III site is changed to a BamHI site (plasmid SDAB). A BamHI to Bgl II DNA PCR fragment containing a region upstream of the ICP4 promoter including the viral origin contained in the short inverted repeat region (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), nucleotides 144933 to 145534 of the McKrae strain) is cloned into the BamHI site of plasmid SDAB to prepare plasmid SDAXB. In some embodiments, the plasmid is constructed by cloning the HCMV-eGFP fragment at the BamHI site of a plasmid as described above. In some embodiments, the plasmid as described above is then recombined into a specific locus of the wild-type McKrae virus. In some embodiments, the resulting vector is isolated using a stable cell line that expresses one or more genes deleted or disrupted in the HSV genome required for replication.

[0195] In some embodiments, a vector comprising a combined ICP4 and ICP47 deletion is prepared. In some embodiments, a plasmid sequence comprising McKrae sequence base pairs 143521 to 144562 and 144640 to 145534 (GenBank Accession Number: JQ730035.1 (SEQ ID NO: 1), McKrae strain) can be synthesized. The SCMV promoter flanking the Pme I site can be cloned between these flanking sequences. In some embodiments, a marker (e.g., a red fluorescent marker) can be placed in an appropriate orientation for the SCMV promoter. In some embodiments, the plasmid as described above is then recombined into a specific locus of the McKrae virus with a deletion in the ICP4 locus. In some embodiments, fluorescent clones can be isolated, and the insertion of the construct into the ICP47 locus can be confirmed by PCR. According to the reference sequence (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain), the resulting recombinant McKrae virus has a deletion of base pairs 144562 to 144640. In some embodiments, a stable cell line is used to isolate the resulting viral vector, which expresses one or more genes deleted or disrupted in the HSV genome required for replication.

[0196] VI. Characterization of Vectors

[0197] The viral vector according to the present disclosure can be characterized by genome sequencing to determine whether the expected vector has been successfully formed. Any sequencing method known in the art is acceptable for this purpose. Sequencing methods include, for example, nanopore sequencing, single molecule real-time sequencing (SMRT), DNA nanoball (DNB) sequencing, pyrophosphate sequencing, and use of DNA arrays.

[0198] Payload expression from viral vectors can be detected by any method known in the art for detecting proteins or nucleic acids. Methods for detecting protein expression include immunohistochemistry, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy, individual protein immunoprecipitation (IP), protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), immunoelectrophoresis, spectrophotometry, and bicinchoninic acid assay (BCA). Methods for detecting nucleic acid expression include Southern blotting, Northern blotting, polymerase chain reaction (PCR), quantitative PCR, and RT-PCR.

[0199] VII. Application / Use

[0200] Viral vectors according to the present disclosure can be used for multiple therapeutic applications. Carriers as described herein can be used for delivering one or more payloads to one or more target cells. In certain embodiments, the payload is maintained in the target cell 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, 3 weeks, 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, one year or more than one year.

[0201] In some embodiments, the target cell is present in a tissue that is poorly vascularized and difficult to reach through the systemic circulation. In some embodiments, the target cell is a cell susceptible to infection by HSV. In some embodiments, the target cell is particularly susceptible to infection by the McKrae strain of HSV.

[0202] Viral vectors according to the present disclosure can be used to deliver one or more therapeutic polypeptides to cells, such as cells in a subject. For example, a viral vector comprising a heterologous nucleic acid segment operably connected to a promoter can be used for any disease or clinical disorder associated with a reduction or absence of a protein encoded by the heterologous nucleic acid segment, or any disease or clinical disorder that can be effectively treated by expressing the encoded protein in a subject. A viral vector containing an expression cassette for synthesizing an RNAi agent (e.g., one or more siRNAs or shRNAs) can be used to treat any disease or clinical disorder associated with overexpression of a transcript or its encoded protein in a subject, or any disease or clinical disorder that can be treated by reducing a transcript or its encoded protein in a subject. A viral vector comprising an expression cassette for synthesizing one or more RNAs can be used to regulate immune system responses (e.g., responses responsible for organ transplant rejection, allergic reactions, autoimmune diseases, inflammation, etc.), wherein the one or more RNAs are self-hybridized or hybridized with each other to form an RNAi agent targeting a transcript encoding a cytokine. Viral vectors that provide a template for the synthesis of one or more RNAs that self-hybridize or hybridize with each other to form RNAi agents that target transcripts of an infectious agent or target cellular transcripts whose encoded products are necessary for or contribute to any aspect of the infectious process can be used to treat infectious diseases.

[0203] In one aspect, the present disclosure relates to a method for reducing tumor size in a subject in need thereof. The method comprises administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing changes that render the variant unable to express functional ICP4 and ICP47 proteins. In addition, the vector comprises a nucleic acid encoding a therapeutic polypeptide for reducing tumor size. In certain embodiments, the variant is unable to express functional ICP4 and ICP47 proteins characterized by the amino acid sequences of SEQ ID NOs: 3 and 4 and SEQ ID NO: 5, respectively. In certain embodiments, the HSV strain is an HSV-1 strain, such as a McKrae strain.

[0204] In certain embodiments, the therapeutic polypeptide comprises one or more of the following functional attributes:

[0205] (a) Targeting tumor stroma;

[0206] (b) supporting the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment;

[0207] (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or

[0208] (d) Promotes innate immune surveillance in phagocytes.

[0209] Exemplary therapeutic polypeptides are discussed in Section III above and are incorporated herein.

[0210] VIII. Administration

[0211] Compositions comprising viral vectors as described herein can be formulated for delivery by any available route, including but not limited to intratumoral, parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (surface), transmucosal, rectal, and vaginal. Preferred delivery routes include intratumoral delivery. In some embodiments, the pharmaceutical composition includes a combination of a viral vector and 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, etc. that are compatible with drug administration. Supplementary active compounds can also be incorporated into the composition. In some embodiments, the viral vector is formulated in glycerol. In some embodiments, the viral vector is formulated in phosphate buffered saline containing about 10% glycerol.

[0212] It is advantageous to formulate the composition 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 contains a predetermined quantity of viral vector calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.

[0213] The pharmaceutical composition can be administered at various intervals and for different time periods as desired, e.g., once a week for about 1 to 10 weeks, 2 to 8 weeks, about 3 to 7 weeks, about 4, 5 or 6 weeks, etc. The skilled artisan will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the overall health and / or age of the subject, and the presence of other diseases. Treatment of a subject with a viral vector may include a single treatment, or in many cases, may include a series of treatments.

[0214] In certain embodiments, a pharmaceutical composition may be administered to a patient more than once (e.g., two, three, four, five, six, seven, eight, nine, ten or more times), for example due to the improved safety profile exhibited by the vectors described herein.

[0215] In some embodiments, the active agent (i.e., the viral vector of the present invention and / or other agents to be administered with the viral vector of the present invention) is prepared in the presence of a carrier (such as a controlled release formulation, including implants and microencapsulation delivery systems) that will prevent the compound from being rapidly eliminated from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. In some embodiments, the compositions target specific cell types or cells infected by the virus.

[0216] IX. Combination therapy

[0217] According to the present disclosure, provided compositions can be administered in combination with one or more other active agents and / or treatment modes (such as known therapeutic agents and / or independently active biologically active agents). 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 a unique composition. In some embodiments, combination therapy involves simultaneous administration of one or more doses or units of two or more different active agents and / or treatment modes; in some embodiments, combination therapy involves simultaneous exposure to two or more different active agents and / or treatment modes, for example, by overlapping dosing regimens.

[0218] In some embodiments, provided compositions include or are administered in combination with one or more additional active agents useful for treating related diseases, disorders, and / or conditions.

[0219] Throughout this specification, where devices, apparatuses, and systems 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 there are additional devices, apparatuses, and systems of the present invention that consist essentially of, or consist of, the enumerated components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the enumerated processing steps.

[0220] The practice of the present 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 present invention in any way.

[0221] Example

[0222] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not meant to limit the present invention in any way. The present examples, together with the methods described herein, currently represent preferred embodiments, are exemplary, and are not intended to be limiting of the scope of the present invention. Those skilled in the art will appreciate variations therein and other uses encompassed within the spirit of the present invention as defined by the scope of the claims.

[0223] Example 1 : Generation of ICP4- and ICP47-modified McKrae strain HSV-1 vectors

[0224] This example describes the cloning and preparation of a non-replicating McKrae strain HSV-1 vector containing alterations in the ICP4 and ICP47 regions.

[0225] Wild-type McKrae strain HSV-1 contains two copies of the gene encoding ICP4 ( Figure 1A ). First, a plasmid containing the full-length viral genome is modified using homologous recombination by replacing two copies of the two ICP4 loci with modified loci, for example as described in WO 2017 / 165813, and screening for plaques expressing green fluorescent protein (GFP). Briefly, the plasmid was constructed as follows: the Sph I to Afl III fragment (1928 bp) of the HSV-1 KOS strain genome (GenBank Accession No.: KT899744.1 (SEQ ID NO: 2), nucleotides 124346 to 126273 of the KOS strain) was cloned into Sph I / AflIII-digested pSP72 to prepare plasmid SDA, and then the III site was changed to a BamHI site (plasmid SDAB). A BamHI to Bgl II DNA PCR fragment containing a region upstream of the ICP4 promoter including the viral origin contained in the short inverted repeat region (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), nucleotides 144933 to 145534 of the McKrae strain) was cloned into the BamHI site of plasmid SDAB to prepare plasmid SDAXB. In addition, the HCMV-eGFP fragment was cloned into the BamHI site of plasmid SDAXB. The SDAXB plasmid was then recombined into the ICP4 locus of the wild-type McKrae viral genome to generate mutICP4 McKrae HSV-1. Figure 1B The resulting viral vector structure is shown in .

[0226] Secondly, the ICP47 locus of mutICP4 McKrae HSV-1 was modified. Briefly, a plasmid sequence containing McKrae sequence base pairs 143521 to 144562 and 144640 to 145534 (GenBank Accession Number: JQ730035.1 (SEQ ID NO: 1), McKrae strain) was synthesized. The SCMV promoter flanking the Pme I site was cloned between these flanking sequences. In addition, a red fluorescent marker was placed in an appropriate orientation for the SCMV promoter and the resulting plasmid was used to recombinant into mutICP4 McKrae HSV-1, thereby forming mutICP4mutICP47 McKrae HSV-1. Fluorescent clones were isolated, and PCR was performed to confirm that the construct was inserted into the ICP47 locus. According to the reference sequence (GenBank Accession No.: JQ730035.1 (SEQ ID NO: 1), McKrae strain), the resulting virus has a deletion of base pairs 144562 to 144640. Figure 1C The resulting viral vector structure is shown in .

[0227] After the mutICP4mutICP47 vectors were generated, vector stocks were prepared by infecting complementing cells and purifying the vector from the supernatant.

[0228] Example 2 : Cloning, preparation and evaluation of single or multi-gene payloads

[0229] This example describes the cloning, preparation, and evaluation of a multigene payload viral vector.

[0230] Briefly, mutICP4 McKrae HSV-1 or mutICP4mutICP47McKrae HSV-1 vectors containing marker elements (e.g., GFP) in place of genes encoding ICP4 and / or ICP47 were further modified by replacing the marker elements with one or more genes of interest (GOIs) (e.g., therapeutic polypeptides) to prepare constructs for the following examples. In order to replace the marker elements with genes of interest (GOIs) in mutICP4 McKrae HSV-1, mutICP27, or mutICP4mutICP47McKrae HSV-1, the GOI flanked by the HCMV immediate early protein gene (IEp) and the polyadenylation signal (HCMV-GOI-pA) was cloned into a plasmid and transduced into a stable Vero cell line expressing ICP4. After cell transduction, plaques that did not express the marker elements were isolated and tested for GOI expression by ELISA. In the examples that follow, the GOIs IFNγ, LTB, CXCL13, CCL19, CCL21 and IL-7 were cloned into vectors and expressed. Figure 2 An exemplary multi-gene payload viral vector is shown in FIG.

[0231] Example 3 : Evaluation of oncolysis and payload expression

[0232] This example describes the evaluation of oncolysis onset and payload expression of mutICP4 McKrae HSV-1 expressing murine IL-17a and mutICP4mutICP47-IFNγMcKrae HSV-1 expressing IFNγ in human breast cancer cells.

[0233] mutICP4 McKrae HSV-1

[0234] Hs578T human breast cancer cells were infected with increasing amounts of mutICP4McKrae HSV-1 expressing murine IL-17a (multiplicity of infection (MOI) of 0, 3, and 10) and then assayed by CellTiter-Glo ( Cell viability was assessed by luminescent cell viability assay (Promega Corp., Madison, WI). Figure 3A ) and expression of the murine IL-17a payload was assessed by ELISA for 8 days ( Figure 3B ).

[0235] These results demonstrate that mutICP4 McKrae HSV-1-mediated changes in cell viability were not observed until several days after infection and that robust expression of the payload was maintained for up to 8 days.

[0236] mutICP4mutICP47 McKrae HSV-1

[0237] Hs578T human breast cancer cells were infected with increasing amounts of IFNγ-expressing mutICP4mutICP47-IFNγMcKrae HSV-1 (multiplicity of infection (MOI) of 0, 3, and 10) and then analyzed by real-time Glo TM Cell viability was assessed by ELISA (Promega Corp., Madison, WI). Figure 3C ) and expression of IFNγ payload was assessed by ELISA for 6 days ( Figure 3D ).

[0238] These results confirmed that mutICP4mutICP47-IFNγ infection inhibited cell growth in a dose-dependent manner ( Figure 3C IFNγ production was highest in the first 2 days after infection and was above baseline by 6 days after infection. #p<0.0001( Figure 3D). The results showed that no changes in mutICP4mutICP47-IFNγ-mediated activity were observed until several days after infection, and robust expression of the payload was maintained for up to 6 days ( Figure 3C and Figure 3D ). Delayed oncolysis allows for persistence of the virus in target cells, sustained payload expression, exposure of tumor-associated antigens within the payload-induced tumor microenvironment, and limited nonspecific inflammation and tissue damage. It is believed that delayed oncolysis will result in an improved safety profile and the vector will be suitable for repeated dosing.

[0239] Example 4 : Assessment of immunogenicity and immune activation

[0240] Immunogenicity

[0241] This embodiment describes the assessment of the impact of mutICP4 McKrae HSV-1 and mutICP4mutICP47McKrae HSV-1 on immunogenicity in human breast cancer cells. This embodiment also describes the assessment of the impact of mutICP4mutICP47McKrae HSV-1 on immunogenicity compared with another replication-defective McKrae HSV-1 with ICP27 (mutICP27) with mutation in human breast cancer cells. ICP27 is an immediate early gene (like ICP4), which makes the virus replication-defective when destroyed. The mutICP27 vector allows the mutICP4mutICP47 replication-defective vector to be compared with another replication-defective vector with a completely complete ICP47 locus in the same strain. Because ICP4 and ICP47 loci are very close, it is difficult to destroy the ICP4 locus without affecting the ICP47 locus, and the ICP4 control used in the embodiment may also destroy the ICP47 function. Therefore, the mutICP27 vector provides a good alternative for comparison of control vectors.

[0242] Hs578T human breast cancer cells were infected with increasing amounts of mutICP4McKrae HSV-1-GFP or mutICP4mutICP47 McKrae HSV-1-GFP at multiplicity of infection (MOI) of 0.3, 1 and 3. After 24 h, infection (GFP+ cells) and cell surface MHC class I expression (HLA) were assessed by flow cytometry.

[0243] The mutICP4 McKrae HSV-1 and mutICP4mutICP47 McKrae HSV-1 strains showed similar infection rates at all three MOIs tested ( Figure 4AThe results also showed that almost all cells were HLA positive ( Figure 4B ) and that cell surface expression of HLA was increased for mutICP4mutICP47 McKrae HSV-1 compared to mutICP4 McKrae HSV-1 ( Figure 4C ).

[0244] These results indicate that mutICP4mutI CP47 McKrae HSV-1 induces increased immunogenicity in breast cancer cells compared with mutICP4 McKrae HSV-1.

[0245] Next, Hs578T cells were infected with 10 PFU per cell of mutICP4mutI CP47 or mutICP27, each HSV-1 strain encoding GFP as a marker. After 24 hours of infection, cells were harvested for (1) RNAseq analysis of Tap 1 (a protein that mediates unidirectional translocation of peptide antigens from the cytosol to the endoplasmic reticulum (ER) for loading onto MHC class I) and (2) flow cytometric analysis of cell surface MHC class I (MHC-I) expression (HLA).

[0246] like Figure 4D As shown in , mutICP4mutICP47 infection resulted in increased antigen presentation as measured by increased Tap1 gene expression ( Figure 4D ) and cell surface MHC-I was upregulated compared with mutICP27 infection ( Figure 4E The results showed that the cell surface expression of MHC-I was not down-regulated in the case of mutICP4mutICP47 vector infection as observed in the case of mutICP27 vector infection, but it was increased by about 50% over the level of uninfected cells. ( Figure 4E ; **p<0.01, #p<0.0001). Therefore, infection with mutICP4mutICP47 vectors in breast cancer cells resulted in increased immunogenicity compared to infection with mutICP27 vectors.

[0247] Immune activation-gene set enrichment analysis

[0248] The response of cells to the viral backbone can be measured by gene expression analysis as determined by RNAseq. Gene set enrichment analysis (GSEA) is a bioinformatics method for identifying gene-related pathways that are altered by treatment using this RNAseq data (see, for example, the GSEA website at gsea-msigdb.org / gsea / msigdb / collections.jsp). By identifying pathways that are significantly altered in response to treatment, GSEA provides insight into the cellular mechanisms that underlie the response to the viral backbone.

[0249] To determine genes that were up-regulated or down-regulated by mutICP4mutICP47 or mutICP27 infection, Hs578T cells were infected with 10 PFU of mutICP4mutICP47 (using encoded GFP as a marker) or mutICP27 per cell for 24 hours, and RNA was extracted from the cells for RNAseq analysis. RNAseq data were analyzed using GSEA software and reported as standardized enrichment scores and p values.

[0250] The results showed that the main pathways upregulated by mutICP4mutICP47 compared with mutICP27 were immune activation pathways, including the marker interferon α response (HALLMARK_INTE RFERON_ALPHA_RESPONSE) and the marker inflammatory response (HALLMARK_INFL AMMATORY_RESPONSE) ( Figure 4F The major pathways upregulated by mutICP27 infection were those involved in cellular responses, including HALLMARK_MYC_TARGETS and MTORC1 signaling (MTORC1_SIGNALING) ( Figure 4G ). The marker gene sets are carefully curated and concisely summarize unique well-defined biological states or processes, showing consistent expression patterns (see, e.g., Liberzon et al. (2015) CELL SYST.; 1(6):417-425). For example, the marker interferon alpha response pathway includes genes that are upregulated in response to the alpha interferon protein, such as Tap1. The mutICP4mutICP47 vector induced greater immune activation compared to the mutI CP27 vector.

[0251] Immune activation-TNFSF expression

[0252] TNF superfamily members (TNFSF) are important factors in the induction of innate and adaptive immune responses. These molecules act by directly inducing immunogenic forms of cell death (including apoptosis and pyroptosis) and by directly stimulating immune cells.

[0253] To determine whether TNFSF is involved in mutICP4mutICP47 or mutICP27 cellular responses, Hs578T cells were infected with 10 PFU per cell of mutICP4mutICP47 or mutICP27, each HSV-1 strain encoding GFP as a marker, for 24 h, and RNA was extracted from the cells for RNAseq analysis.

[0254] The results showed that mutICP4mutICP47 induced higher expression of TNF superfamily members including tumor necrosis factor-α (TNF), lymphotoxin β (LTB), APRIL (TNFSF13), and LIGHT (TNFSF14) compared with mutICP27 ( Figure 4H ). Each of these genes can act as both an inducer of immunogenic cell death and a stimulator of immune response. In addition, mutICP4mutICP47 upregulated the expression of PYCARD and caspase 10 (10), regulators of pyroptosis and apoptosis, to a greater extent than mutICP27 infection. Compared with mutICP27 virus infection, mutICP4mutICP47 infection preferentially activated immune response and immunogenic cell death pathways.

[0255] Example 5: Multigene Payloads for Targeting Tumor Stroma

[0256] This example describes the design and evaluation of viral vectors with multigene payloads for targeting the tumor stroma and activating the local endothelium to facilitate T cell infiltration of the tumor, thereby reducing tumor size.

[0257] Tumor stroma can be targeted by different mechanisms. For example, the extracellular matrix can be solubilized by proteins involved in extracellular matrix (ECM) degradation, including but not limited to hyaluronidase, MMP-9, and inhibitors of lysyl oxidase. In addition, cancer-associated fibroblasts can be targeted by proteins that interfere with the TGFβ pathway.

[0258] The local endothelium can be activated to favor T cell infiltration by proinflammatory cytokines, including but not limited to TNF, IL-1β, IL-6, and IL- 18. It is expected that the inclusion of enzymes that degrade the tumor extracellular matrix will simultaneously destroy the physical barriers within the tumor to allow immune cell infiltration.

[0259] Tumor stroma targeting and local endothelial activation can be regulated by the localized expression of these factors together or individually.

[0260] Thus, one or more of hyaluronidase, MMP-9, inhibitors of lysyl oxidase, genes that interfere with the TGFβ pathway, TNF, IL-1β, IL-6, or IL-18 can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2 and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more McKrae strain HSV-1 viruses containing the GOI at different MOIs as described in Example 3, and oncolysis, payload expression and / or secretion, and immunogenicity can be assessed as described in Examples 3 and 4.

[0261] The effects of cancer cells that secrete factors that dissolve tumor stroma or activate local endothelium for T cell infiltration on tumor stroma or local endothelium can be assessed in vitro or in vivo. It is expected that expression of genes that target tumor stroma and / or local endothelium will increase T cell infiltration and reduce tumor size.

[0262] Example 6: Multigene payloads for targeting tumor stroma in mutICP4mutICP47

[0263] This example describes the design and evaluation of a mutICP4mutICP47 HSV-1 vector with a multigene payload for targeting the tumor stroma and activating the local endothelium to favor T cell infiltration of the tumor, thereby reducing tumor size, using a salivary gland model (see, e.g. (Barone et al. PNAS (2015) 112(35) 11024-11029).

[0264] In simple terms, in the presence of 3×10 7 PFU of a combination of mutICP4mutICP47 HSV-1 vector expressing GFP or mutICP4mutICP47 HSV-1 vector expressing each of murine LTB, CXCL13, CCL19, CCL21, or IL-7 (6×10 6 PFU of each vector, total dose 3x 10 7 PFU, "payload" prepared as described in Example 2), both submandibular salivary glands of wild-type BALB / c mice (8-10 weeks old) were cannulated via excretory cannula under anesthesia and then harvested 15 days later for flow cytometric analysis using antibodies against CD31, PDGFRa and PDPN (a marker of stromal cell activation). The number of positive cells was quantified.

[0265] The results showed that the mutICP4mutICP47 virus encoding the payload increased the number of CD31+ endothelial cells and PDGFRa+PDPN+ stromal cells in the salivary glands ( FIG. 5A to FIG. 5B). These data demonstrate that stromal and endothelial activation can be achieved using a combination of mutICP4mutICP47 viruses, each carrying a payload of one of the murine LTB, CXCL13, CCL19, CCL21, and IL-7 genes.

[0266] Example 7 : Evaluation of the expression of TGFβ pathway components after mutICP4mutICP47 infection

[0267] This example describes the evaluation of the expression of TGFβ pathway components following infection with mutICP4mutICP47-GFP HSV-1 vectors.

[0268] The TGFβ pathway is a multifunctional cytokine that regulates stromal, endothelial, and immune cells, and therapeutic targeting of this pathway has been described in cancer.

[0269] To determine the expression of TGFβ pathway components in the context of mutICP4mutICP47 viruses after infection of cancer cells, Hs578T cells were infected with 10 PFU of mutICP4mutICP47-GFP (using the encoded GFP as a marker, without payload) per cell (uninfected cells served as controls) for 24 hours, and RNA was extracted from the cells for RNAseq analysis. Quantification of TGFβ1 mRNA expression.

[0270] The results showed that after infection with the mutICP4mutICP47 vector, TGFβ1 expression was reduced by about 50% at 24 h post infection ( Figure 5C ). Therefore, infection with the mutICP4mutICP47 viral backbone itself reduced the expression of TGFβ1.

[0271] Example 8 : Multigene payload for T cell survival in the tumor microenvironment

[0272] This example describes the design and evaluation of viral vectors with multigene payloads for supporting T cell survival in the tumor microenvironment (TME).

[0273] T cells in the tumor microenvironment can be supported by different mechanisms. For example, the recruitment of T cells to tumor sites can be improved by the presence of T cell recruitment factors (such as chemokine ligands CCL19 or CCL21) in the tumor microenvironment.

[0274] Local T cells can be supported to induce strong and lasting anti-tumor responses. For example, local expression of T cell trophic factors (such as IL-7, IL-12, IL-15, IL-18 and IFNγ) can induce strong and lasting anti-tumor responses from local or recruited T cells. In addition, TGFβ signaling in cells within the tumor can be inhibited by soluble TGFβRII expression. In addition, local expression of transgenics encoding costimulatory molecules (such as CD40L or OX40L) according to the target TME and in combination with other vectors or therapeutic agents can stimulate local or recruited T cells.

[0275] T cell support or T cell recruitment can be regulated by these factors together or individually.

[0276] One or more of CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFNγ, soluble TGFβRII, CD40L, or OX40L can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2, and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more McKrae strain HSV-1 viruses containing the GOI at different MOIs as described in Example 3, and oncolysis, payload expression and / or secretion, and immunogenicity can be assessed as described in Examples 3 and 4.

[0277] The effects of cancer cells secreting these factors on T cell recruitment or T cell function can be assessed in vitro or in vivo. It is expected that the expression of genes for T cell recruitment or T cell function will result in the recruitment of T cells to the tumor site and support the function of local T cells, thereby reducing tumor size.

[0278] Example 9 : Multiple genes in mutICP4mutICP47 that enable T cells and NK cells to survive in the tumor microenvironment Due to the effective load

[0279] This example describes the design and evaluation of mutICP4mutICP47 HSV-1 vectors with a multigene payload for supporting T cell and NK cell survival in the tumor microenvironment (TME).

[0280] Immune-mediated tumor cell killing can be assessed in ex vivo co-culture models that include peripheral blood mononuclear cells (PBMCs) and target tumor cells. Cytotoxic cells in these models include CD8+ T and NK cells present in PBMCs.

[0281] To determine the cytotoxic activity of mutICP4mutICP47 HSV-1 vectors with or without multigene payloads in an in vitro breast cancer model, CellTracker Red-labeled Hs578T cells (a human breast cancer cell line) were infected with mutICP4mutICP47 vectors encoding GFP, human IFNγ, human IL-12, or a combination thereof at a total viral dose of 0.5 PFU per cell. After 2 h, CellTracker Purple-labeled human PBMCs were added to the cultures at a ratio of approximately 10 PBMCs per 1 Hs578T cell and incubated for 72 h. Recombinant human IL-15 (rh IL-15) was added to mutICP4mutICP47-GFP infected cells to simulate viral-mediated delivery of human IL-15. PBMC activator cocktail (ImmunoCult TM Human CD3 / CD28 / CD2 T cell activator + rh IL-2) served as a positive control for maximal tumor cell killing under these conditions. After 72 hours, samples were collected and live Hs578 T cells were quantified by flow cytometry.

[0282] The results showed that virus-mediated expression of IFNγ or IL-12 in Hs578T:PBMC co-cultures reduced the number of viable Hs578T cells to a greater extent than viral backbone treatment alone (mutICP4mutICP47-GFP). Fig. 6A ). Similarly, the addition of rh IL-15 to mutICP4mutICP47-GFP infected cells also increased Hs578T tumor cell killing. The combination of IFNγ-expressing and IL-12-expressing mutICP4mutICP47 vectors further enhanced immune-mediated tumor cell killing in this assay. These data support the use of human IFNγ, IL-12, IL-15 and their combinations encoded in the designed vectors to support T or NK cell function and immune-mediated tumor cell killing in the tumor microenvironment.

[0283] Example 10 : IFNs in mutICP4mutICP47 for T and NK cell survival in the tumor microenvironment Gamma Payload

[0284] This example describes the evaluation of mutICP4mutICP47 HSV-1 vectors with an IFNγ payload for supporting T cell and NK cell survival in the tumor microenvironment (TME).

[0285] To determine T cell and NK cell activity after treatment with mutICP4mutICP47HSV-1 vectors with or without IFNγ payload, Hs578T cells were infected with 1 or 3 PFU of mutICP4mutICP47 encoding GFP or mutICP4mutICP47 encoding human IFNγ per cell in OPTI-MEM+5% FBS for 2 hours and then co-cultured with PBMCs for 24 or 72 hours. Cells were harvested after 24 or 72 hours, stained with fluorescent conjugated antibodies, and analyzed by flow cytometry.

[0286] The results showed that after 24 hours, mutICP4mutICP47 infection increased Ki67 + Granzyme B + CD8 + The number of T cells increased, and this effect was further increased after 72 hours of co-culture ( Figure 6B and Figure 6C ). Compared with mutICP4mutICP47-GFP, mutICP4mutICP47-IFNγ has a stronger effect in T cell activation.

[0287] The results showed that after 24 h, mutICP4mutICP47-IFNγ induced a greater number of granzyme B compared with uninfected controls or mutICP4mutICP47-GFP. + Ki67-NK cells and antigen presenting cells (CD11c+CD16+CD14-Ki67+MHCII++)( Fig.6D and Fig. 6E ).

[0288] Infection with mutICP4mutICP47-GFP or mutICP4mutICP47-IFNγ reduced the number of viable tumor cells and impaired PBMC-mediated target cell killing ( Fig. 6F The effect of the mutICP4mutICP47 virus on T cell activation was dose-dependent. These data confirm that cancer cells infected with the mutICP4mutICP47 vector encoding human IFNγ modulate T and NK cell function, increase antigen presentation capacity, and activate immune-mediated tumor cell killing.

[0289] Embodiment 11 : Evaluation of chemokine expression in cancer cells after infection with mutICP4mutICP47-IFNγ

[0290] This example describes the evaluation of the effects of mutICP4mutICP47 HSV-1 vectors with an IFNγ payload on chemokine expression in cancer cells.

[0291] Chemokines in the tumor microenvironment are important factors regulating the recruitment of T and NK cells into tumors.

[0292] To determine the expression of chemokines after cancer cell infection with mutICP4mutICP47-GFP or mutICP4mutICP47-IFNγ, Hs578T cells were infected with 10 PFU of mutICP4mutICP47 (with encoded GFP as a marker) or mutICP4mutICP47 vector encoding human IFNγ per cell for 24 h, and RNA was extracted from cells for RNAseq analysis. The number of gene counts for each gene of interest is reported (****p<0.0001).

[0293] The results showed that infection with the mutICP4mutICP47 vector upregulated the expression of multiple T and NK cell chemokines, including CCL2, CXCL9, CXCL10, CXCL11, and CX3CL1, and this effect was further enhanced by the human IFNγ encoded in the vector ( Figure 6G ). The data suggest that responses to the mutICP4mutICP47 viral backbone itself support T and NK cell recruitment to tumors.

[0294] Example 12 : Multigene payloads for CAR T cell survival in the TME

[0295] This example describes the design and evaluation of viral vectors with multigene payloads for supporting CAR T cell survival in the TME.

[0296] T cells in the tumor microenvironment can be supported by different mechanisms. For example, the recruitment of CAR T cells to tumor sites can be improved by the presence of T cell recruitment factors (such as chemokine ligands CCL19 or CCL21) in the tumor microenvironment.

[0297] Local CAR T cells can be supported to induce strong and lasting anti-tumor responses. For example, local expression of CAR T cell trophic factors (such as IL-7, IL-15 and IL-18) can induce strong and lasting anti-tumor responses from local or recruited CAR T cells. In addition, TGFβ signaling in cells within the tumor can be inhibited by soluble TGFβRII expression. In addition, local expression of transgenics encoding costimulatory molecules (such as CD40L or OX40L) according to the target TME and combinations with other vectors or therapeutic agents can stimulate local or recruited CAR T cells.

[0298] Ectopic or overexpression of a cognate CAR antigen (such as mesothelin) in tumor cells can redirect CAR T cells to target tumor cells expressing the antigen.

[0299] CAR T cell support or CAR T cell recruitment can be regulated by these factors together or individually.

[0300] One or more of CCL19, CCL21, IL-7, IL-15, IL-18, soluble TGFβRII, CD40L, or OX40L can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2, and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more McKrae strain HSV-1 viruses containing the GOI at different MOIs as described in Example 3, and oncolysis, payload expression and / or secretion, and immunogenicity can be assessed as described in Examples 3 and 4.

[0301] The effects of cancer cells secreting these factors on CAR T cell recruitment or CAR T cell function can be evaluated in vitro or in vivo. It is expected that the expression of genes for CAR T cell recruitment or CAR T cell function will recruit CAR T cells to the tumor site and support the function of local CAR T cells and reduce tumor size. In addition, it is expected that the expression of ectopic tumor antigens in the tumor will recruit CAR T cells with homologous CAR to the tumor and reduce tumor size.

[0302] Example 13 : mutICP4mutICP47 HSV-1 vectors for induction of tertiary lymphoid structures in the tumor bed Multigene payload

[0303] This example describes the design and evaluation of mutICP4mutICP47 HSV-1 vectors with a multigene payload for inducing tertiary lymphoid structures (TLS) within the tumor bed.

[0304] Tertiary lymphoid structures (TLS) serve as functional niches that mature T and B cell responses against locally displayed tumor antigens and, although originally described in the context of chronic inflammation, have recently been identified as elements of tissues capable of supporting robust immune responses against solid tumors. TLS formation appears to be associated with improved immunotherapy efficacy in mice and humans, providing ICI-activated TILs with recruitment and survival signals within the tumor itself.

[0305] To determine the induction of TLS in the tumor bed by the mutICP4mutICP47 HSV-1 vector, a model of intraglandular delivery of vectors in salivary glands was used. This model is an environment that has been shown to be highly permissive for TLS formation. Briefly, 3x10 7PFU of mutICP4mutICP HSV-1 vector expressing GFP or a combination of mutICP4mutICP47 HSV-1 vector expressing murine TNF, CCL19, IL-17a, and IL-7 (6x10 6 PFU of each vector including the vector encoding GFP, total dose 3x10 7 PFU), both submandibular glands of wild-type BALB / c mice (8-10 weeks old) were cannulated via excretory cannulas under anesthesia, and salivary glands were harvested 15 days later for flow cytometry and immunofluorescence staining ( Fig. 7A Dendritic cells (CD11c), CD4+ T cells (CD4), B cells (B220) and nuclei (DAPI) were visually observed ( Figure 7B and Figure 7C ). Two regions of interest (A1, A2) are shown at low magnification (10X) and high magnification (40X) ( Figure 7C ).

[0306] The results showed that the mutICP4mutICP47 viral backbone alone enhanced immune infiltration into the salivary glands (measured as CD45+ cells by flow cytometry) ( Fig. 7A ) and form small immune cell aggregates characterized by dendritic cells and CD4+ T cells ( Figure 7B and Figure 7C Administration of a combination of mutICP4mutICP47 vectors expressing TNF, CCL19, IL-17a, and IL-7, reported to play a role in TLS formation, increased the number, size, and organization of observed TLS structures, as demonstrated by spatially defined B and T cell zones of infiltrating dendritic cells in immunofluorescence images. The data suggest that responses to the mutICP4mutICP47 viral backbone itself support potential lymphoid neogenesis processes, and that this may be enhanced by virally mediated expression of a combination of factors known to regulate TLS formation, such as TNF, CCL19, IL-17a, and IL-7. Lymphotoxin beta and CXCL13, which are believed to play a role in TLS establishment and maintenance, may also be used to support TLS formation.

[0307] Embodiment 14 : Multigene payloads for promoting innate immune surveillance in phagocytes

[0308] This example describes the design and evaluation of viral vectors with multigene payloads for promoting phagocyte innate immune surveillance and tumor cell elimination.

[0309] SIRPα of tumor cells binds to CD47 of macrophages, leading to inhibition of phagocytosis. This "don't eat me" signal allows tumor cells to evade immune destruction by macrophages. To block this pathway, a mutICP4mutICP47 vector encoding a SIRPα-IgG fusion protein was constructed. To determine the activity of this vector in an ex vivo phagocytosis model, macrophages were co-cultured with mutICP4mutICP47 SIRPα-IgG medium. Briefly, the STEMCELL EasySep TM Human Monocyte Enrichment Kit enriches CD14+ cells from human PBMCs. The enriched cells are plated in 24-well plates and cultured for 8 days. STEMCELL ImmunoCult TM -SF macrophage differentiation medium and 50ng / ml m-CsF. At the 4th day, fresh medium was added, and at the 6th day, 50ng / ml IFNγ was added to stimulate cell polarization to M1 macrophages. At the 8th day, CFSE-labeled Raji cells were pretreated (30min) with IgG control or anti-CD47 antibody (positive control) or with the conditioned medium of Hs578T cells from mutICP4mutICP47-GFP infected with 10PFU per cell or 3 or 10PFU per cell of mutICP4mutICP47-SIRPα-IgG. Then macrophages were added to CFSE-labeled Raji cells and co-cultured for 3 hours. Samples were collected, and phagocytosis was measured using flow cytometry. Percent phagocytosis was defined as the percentage of CFSE-positive macrophages (CD14+).

[0310] The results showed that conditioned medium from Hs578T cells infected with mutICP4mutICP47-SIRPα-IgG induced a higher percentage of phagocytosis compared with IgG control, anti-CD47 antibody, or mutICP4mutICP47-GFP, and this effect was dose-dependent ( Figure 8 ). These data confirm that infection of cells with mutICP4mutICP47 viral vectors encoding SIRPα-IgG fusion proteins increases macrophage phagocytosis.

[0311] Embodiment 15 : Multigene payloads for NK cell or CAR NK cell survival in the TME

[0312] This example describes the design and evaluation of viral vectors with multigene payloads for promoting survival of NK cells and CAR NK cells in the TME.

[0313] NK cells and CAR NK cells in the tumor microenvironment can be supported by different mechanisms. For example, the recruitment of NK cells and CAR NK cells to the tumor site can be improved by the presence of NK cells and CAR NK cell recruitment factors (such as chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11 or CAR antigens) in the tumor microenvironment.

[0314] Local NK cells and CAR NK cells can be supported to induce strong and lasting anti-tumor responses. For example, local expression of NK cell and CAR NK cell trophic factors (such as IL-2, IL-15, IL-18 and IFNα) can induce strong and lasting anti-tumor responses from local or recruited NK cells and CAR NK cells. In addition, TGFβ signaling in cells within the tumor can be inhibited by soluble TGFβRII expression.

[0315] NK cell and CAR NK cell support or NK cell and CAR NK cell recruitment can be regulated by these factors together or individually.

[0316] One or more of CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11, IL-2, IL-15, IL-18, IFNα, or soluble TGFβRII can be cloned into a non-replicating McKrae strain HSV-1 vector as described in Example 2, and expressed and purified as described in Example 1. Cancer cells can be transduced with one or more McKrae strain HSV-1 viruses containing the GOI at different MOIs as described in Example 3, and oncolysis, payload expression and / or secretion, and immunogenicity can be assessed as described in Examples 3 and 4.

[0317] The effects of cancer cells secreting these factors on NK cell and CAR NK cell recruitment or NK cell and CAR NK cell function can be evaluated in vitro or in vivo. It is expected that the expression of genes for NK cell and CAR NK cell recruitment or NK cell and CAR NK cell function will recruit NK cells and CAR NK cells to the tumor site and support the function of local NK cells and CAR NK cells, thereby reducing tumor size.

[0318] Incorporated by Reference

[0319] The entire disclosure of each of the patent and scientific documents mentioned herein is incorporated herein by reference for all purposes.

[0320] Equivalent

[0321] The present invention may be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments should be considered to be illustrative in all aspects, rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and scope of the equivalents of the claims are intended to be included therein.

[0322] Sequence Listing

[0323] SEQ ID NO: 1 - GenBank Accession No.: JQ730035.1

[0324] starting point

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] SEQ ID NO:2—GenBank Accession No.: KT899744.1

[0371] starting point

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] SEQ ID NO:3—ICP4 protein (McKrae strain, AFK50409.1)

[0418]

[0419]

[0420] SEQ ID NO:4—ICP4 protein (McKrae strain, AFK50423.1)

[0421]

[0422] SEQ ID NO:5—ICP47 protein (McKrae strain)

[0423]

[0424] SEQ ID NO:6—Cell surface hyaluronidase

[0425]

[0426] SEQ ID NO:7—human matrix metalloproteinase-9 (MMP-9)

[0427]

[0428] SEQ ID NO:8—Human interleukin-1β

[0429]

[0430] SEQ ID NO:9—Human interleukin-6

[0431]

[0432] SEQ ID NO:10—Human interleukin-18

[0433]

[0434] SEQ ID NO:11-TNFA_Human Tumor Necrosis Factor

[0435] SEQ ID NO:12-CCL19_Human CC motif chemokine 19

[0436]

[0437] SEQ ID NO:13-CCL21_Human CC motif chemokine 21

[0438]

[0439] SEQ ID NO: 14-IL7_human interleukin-7

[0440]

[0441] SEQ ID NO:15-IL12A_Human interleukin-12 subunit alpha (p35)

[0442]

[0443] SEQ ID NO:16-IL12B_Human interleukin-12 subunit beta (p40)

[0444]

[0445] SEQ ID NO:17-IL15_Human interleukin-15

[0446]

[0447] SEQ ID NO:18-IL18_human interleukin-18

[0448]

[0449] SEQ ID NO:19 -IFNG_human interferon gamma

[0450]

[0451] SEQ ID NO:20-TGFR2_human TGF-β receptor type 2

[0452] SEQ ID NO:21 - TGFR2 - Human isoform 3 of TGF-beta receptor type 2

[0453]

[0454] SEQ ID NO:22—CD40L_human CD40 ligand

[0455] SEQ ID NO:23-TNFL4_Human tumor necrosis factor ligand superfamily member 4

[0456]

[0457] SEQ ID NO:24-CCL2_Human CC motif chemokine 2

[0458]

[0459] SEQ ID NO:25-CX3CL1_human chemokine fractalin

[0460] SEQ ID NO:26-CXL16_Human CXC motif chemokine 16

[0461]

[0462] SEQ ID NO:27-CCL5_Human CC motif chemokine 5

[0463]

[0464] SEQ ID NO:28-CXCL9_human CXC motif chemokine 9

[0465]

[0466] SEQ ID NO:29-CXL10_Human CXC motif chemokine 10

[0467]

[0468] SEQ ID NO:30-CXL11_Human CXC motif chemokine 11

[0469]

[0470] SEQ ID NO:31-IL2_human interleukin-2

[0471]

[0472] SEQ ID NO:32-IFNA1_human interferon alpha-1 / 13

[0473]

[0474] SEQ ID NO:33-TNFC_human lymphocyte toxin-β

[0475]

[0476] SEQ ID NO:34-CXL13_Human CXC motif chemokine 13

[0477]

[0478] SEQ ID NO:35-IL1A_human interleukin-1α

[0479]

[0480] SEQ ID NO:36-IL17_Human interleukin-17

[0481]

[0482] SEQ ID NO:37-SHPS1_Human tyrosine protein phosphatase non-receptor type substrate 1 (SIRPα)

[0483]

Claims

1. A vector comprising a herpes simplex virus (HSV) genome, wherein the vector comprises an alteration that prevents expression of one or more functional ICP4 and ICP47 proteins.

2. The vector of claim 1, wherein the functional ICP4 and ICP47 proteins are characterized by the amino acid sequences of SEQ ID NOs: 3 and 4 (ICP4) and SEQ ID NO: 5 (ICP47).

3. A vector as described in claim 1 or claim 2, wherein the HSV genome is an HSV-1 genome.

4. The vector of claim 3, wherein the HSV genome is a McKrae strain genome.

5. The vector of claim 3, wherein when administered to a subject, the vector causes one or more of the following: (a) Delayed oncolysis; (b) increased immunogenicity; and (c) Increased immune activation.

6. The vector of any one of claims 1-5, wherein the vector comprises a nucleic acid sequence encoding one or more therapeutic polypeptides.

7. The vector of claim 6, wherein the therapeutic polypeptide: (a) Targeting tumor stroma; (b) Supporting T cell survival in the tumor microenvironment; (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promotes innate immune surveillance in phagocytes.

8. The vector of claim 7, wherein the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins.

9. The vector of claim 8, wherein the therapeutic polypeptide comprises an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

10. The vector of claim 7, wherein the therapeutic polypeptide targets the tumor stroma by activating local endothelium to increase T cell infiltration.

11. The vector of claim 10, wherein the therapeutic polypeptide comprises a pro-inflammatory cytokine.

12. The vector of claim 11, wherein the proinflammatory cytokine comprises TNF, IL-1β, IL-6 or IL-18.

13. The vector of claim 7, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by enhancing T cell recruitment to tumor sites.

14. The vector of claim 13, wherein the therapeutic polypeptide comprises CCL19 or CCL21.

15. The vector of claim 7, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by supporting T cell function.

16. The vector of claim 15, wherein the therapeutic polypeptide comprises a T cell trophic factor.

17. The vector of claim 16, wherein the T cell trophic factor is selected from IL-7, IL-12, IL-15, IL-18 and IFNγ.

18. The vector of claim 7, wherein the T cell is a CAR T cell.

19. The vector of claim 18, wherein the therapeutic polypeptide comprises soluble TGFβRII.

20. The vector of claim 18, wherein the therapeutic polypeptide comprises an antigen recognized by the CAR T cell.

21. The vector of claim 20, wherein the antigen recognized by the CAR T cells comprises mesothelin.

22. The vector of claim 21, wherein the therapeutic polypeptide comprises a co-stimulatory molecule.

23. The vector of claim 22, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

24. The vector of claim 7, wherein the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed.

25. The vector of claim 24, wherein the therapeutic polypeptide comprises CCL19, lymphotoxin beta, CXCL13, or TNF.

26. The vector of claim 7, wherein the therapeutic polypeptide promotes phagocyte innate immune surveillance.

27. The vector of claim 26, wherein the therapeutic polypeptide disrupts the Sirpα / CD47 axis.

28. The vector of claim 27, wherein the therapeutic polypeptide comprises a Sirpα-IgG fusion transgene.

29. The vector of claim 7, wherein the therapeutic polypeptide supports anti-tumor macrophage polarization.

30. The vector of claim 29, wherein the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

31. A method of expressing a polypeptide in a subject, the method comprising administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing an alteration that renders the variant incapable of expressing functional ICP4 and ICP47 proteins.

32. The method of claim 31, wherein the variant is unable to express functional ICP4 and ICP47 proteins characterized by the amino acid sequences of SEQ ID NOs: 3 and 4 (ICP4) and SEQ ID NO: 5 (ICP47).

33. The method of claim 31 or 32, wherein the HSV strain is an HSV-1 strain.

34. The method of claim 33, wherein the HSV strain is a McKrae strain.

35. The method of any one of claims 31-34, wherein when administered to the subject, the vector causes one or more of the following: (a) Delayed oncolysis; (b) increased immunogenicity; and (c) Increased immune activation.

36. The method of any one of claims 31-35, wherein the HSV strain comprises a nucleic acid encoding a therapeutic polypeptide.

37. The method of claim 36, wherein the therapeutic polypeptide: (a) Targeting tumor stroma; (b) Supporting T cell survival in the tumor microenvironment; (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promotes innate immune surveillance in phagocytes.

38. The method of claim 37, wherein the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins.

39. The method of claim 38, wherein the therapeutic polypeptide comprises an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

40. The method of claim 37, wherein the therapeutic polypeptide targets the tumor stroma by activating local endothelium to increase T cell infiltration.

41. The method of claim 40, wherein the therapeutic polypeptide comprises a pro-inflammatory cytokine.

42. The method of claim 41, wherein the proinflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

43. The method of claim 37, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by enhancing T cell recruitment to tumor sites.

44. The method of claim 43, wherein the therapeutic polypeptide comprises CCL19 or CCL21.

45. The method of claim 37, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by supporting T cell function.

46. ​​The method of claim 45, wherein the therapeutic polypeptide comprises a T cell trophic factor.

47. The method of claim 46, wherein the T cell trophic factor is selected from the group consisting of IL-7, IL-12, IL-15, IL-18 and IFNγ.

48. The method of claim 37, wherein the T cells are CAR T cells.

49. The method of claim 48, wherein the therapeutic polypeptide comprises soluble TGFβRII.

50. The method of claim 48, wherein the therapeutic polypeptide comprises an antigen recognized by the CAR T cell.

51. The method of claim 50, wherein the antigen recognized by the CAR T cells comprises mesothelin.

52. The method of claim 48, wherein the therapeutic polypeptide comprises a co-stimulatory molecule.

53. The method of claim 52, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

54. The method of claim 37, wherein the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed.

55. The method of claim 54, wherein the therapeutic polypeptide comprises CCL19, lymphotoxin beta, CXCL13, or TNF.

56. The method of claim 37, wherein the therapeutic polypeptide promotes phagocyte innate immune surveillance.

57. The method of claim 56, wherein the therapeutic polypeptide disrupts the Sirpα / CD47 axis.

58. The method of claim 57, wherein the therapeutic polypeptide comprises a Sirpα-IgG fusion transgene.

59. The method of claim 36, wherein the therapeutic polypeptide supports anti-tumor macrophage polarization.

60. The method of claim 59, wherein the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

61. A method of preparing a vector comprising a variant herpes simplex virus (HSV) genome, the vector containing changes that render the variant incapable of expressing functional ICP4 and ICP47 proteins, and wherein the vector expresses a therapeutic polypeptide, the method comprising incubating cells transfected with: (a) First Nucleic Acid Molecule: (i) contains a portion of the HSV genome but does not encode functional ICP4 and ICP47 proteins; and (ii) comprising a sequence encoding a marker element, wherein said sequence encoding said marker element is flanked by a first region of homology (HR1) and a second region of homology (HR2); and (b) a second nucleic acid molecule comprising a sequence encoding a therapeutic polypeptide, wherein the sequence encoding the therapeutic polypeptide is flanked by a first homologous region (HR1_) and a second homologous region (HR2_), wherein HR1 is homologous to HR1_ and HR2 is homologous to HR2_, such that the sequence encoding the therapeutic polypeptide is integrated into the first nucleic acid molecule via homologous recombination.

62. The method of claim 61, wherein the cell is an ICP4 and / or ICP47 complementing cell.

63. The method of claim 61, further comprising the step of purifying viral plaques that do not express the marker element.

64. A method as described in any of claims 61-64, wherein the HSV genome is an HSV-1 genome.

65. The method of claim 64, wherein the HSV genome is a McKrae strain genome.

66. The method of any one of claims 61-65, wherein the therapeutic polypeptide: (a) Targeting tumor stroma; (b) Supporting T cell survival in the tumor microenvironment; (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promotes innate immune surveillance in phagocytes.

67. The method of claim 66, wherein the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins.

68. The method of claim 67, wherein the therapeutic polypeptide comprises an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

69. The method of claim 66, wherein the therapeutic polypeptide targets the tumor stroma by activating local endothelium to increase T cell infiltration.

70. The method of claim 69, wherein the therapeutic polypeptide comprises a pro-inflammatory cytokine.

71. The method of claim 70, wherein the proinflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

72. The method of claim 66, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by enhancing T cell recruitment to tumor sites.

73. The method of claim 72, wherein the therapeutic polypeptide comprises CCL19 or CCL21.

74. The method of claim 73, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by supporting T cell function.

75. The method of claim 74, wherein the therapeutic polypeptide comprises a T cell trophic factor.

76. The method of claim 75, wherein the T cell trophic factor is selected from IL-7, IL-12, IL-15, IL-18 and IFNγ.

77. The method of claim 66, wherein the T cells are CAR T cells.

78. The method of claim 77, wherein the therapeutic polypeptide comprises soluble TGFβRII.

79. The method of claim 77, wherein the therapeutic polypeptide comprises an antigen recognized by the CAR T cell.

80. The method of claim 79, wherein the antigen recognized by the CAR T cells comprises mesothelin.

81. The method of claim 77, wherein the therapeutic polypeptide comprises a co-stimulatory molecule.

82. The method of claim 81, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

83. The method of claim 66, wherein the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed.

84. The method of claim 83, wherein the therapeutic polypeptide comprises CCL19, lymphotoxin beta, CXCL13, or TNF.

85. The method of claim 66, wherein the therapeutic polypeptide promotes phagocyte innate immune surveillance.

86. The method of claim 85, wherein the therapeutic polypeptide disrupts the Sirpα / CD47 axis.

87. The method of claim 86, wherein the therapeutic polypeptide comprises a Sirpα-IgG fusion transgene.

88. The method of claim 66, wherein the therapeutic polypeptide supports anti-tumor macrophage polarization.

89. The method of claim 88, wherein the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

90. A variant HSV strain comprising a vector as described in any one of claims 1-30.

91. A cell transduced with the vector of any one of claims 1-30.

92. A pharmaceutical composition comprising a carrier as described in any one of claims 1-30 and a pharmaceutically acceptable carrier.

93. A method of reducing the size of a tumor in a subject in need thereof, the method comprising administering to the subject a vector comprising a variant of a herpes simplex virus (HSV) strain, the genome of the variant containing changes that render the variant unable to express functional ICP4 and ICP47 proteins, and wherein the vector comprises a nucleic acid encoding a therapeutic polypeptide for reducing the size of the tumor.

94. The method of claim 93, wherein the variant is unable to express functional ICP4 and ICP47 proteins characterized by the amino acid sequences of SEQ ID NOs: 3 and 4 (ICP4) and SEQ ID NO: 5 (ICP47), respectively.

95. The method of claim 93 or 94, wherein the HSV strain is an HSV-1 strain.

96. The method of claim 95, wherein the HSV strain is a McKrae strain.

97. The method of any one of claims 93-96, wherein when administered to the subject, the vector causes one or more of: (a) Delayed oncolysis; (b) increased immunogenicity; and (c) Increased immune activation.

98. The method of any one of claims 93-97, wherein the therapeutic polypeptide: (a) Targeting tumor stroma; (b) Supporting T cell survival in the tumor microenvironment; (c) inducing tertiary lymphoid structures (TLS) in the tumor bed; and / or (d) Promotes innate immune surveillance in phagocytes.

99. The method of claim 98, wherein the therapeutic polypeptide targets the tumor stroma by degrading extracellular matrix proteins.

100. The method of claim 99, wherein the therapeutic polypeptide comprises an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

101. The method of claim 98, wherein the therapeutic polypeptide targets the tumor stroma by activating local endothelium to increase T cell infiltration.

102. The method of claim 101, wherein the therapeutic polypeptide comprises a pro-inflammatory cytokine.

103. The method of claim 102, wherein the proinflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

104. The method of claim 98, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by enhancing T cell recruitment to tumor sites.

105. The method of claim 100, wherein the therapeutic polypeptide comprises CCL19 or CCL21.

106. The method of claim 98, wherein the therapeutic polypeptide supports T cell survival in the tumor microenvironment by supporting T cell function.

107. The method of claim 102, wherein the therapeutic polypeptide comprises a T cell trophic factor.

108. The method of claim 103, wherein the T cell trophic factor is selected from IL-7, IL-12, IL-15, IL-18 and IFNγ.

109. The method of claim 98, wherein the T cells are CAR T cells.

110. The method of claim 105, wherein the therapeutic polypeptide comprises soluble TGFβRII.

111. The method of claim 105, wherein the therapeutic polypeptide comprises an antigen recognized by the CAR T cell.

112. The method of claim 107, wherein the antigen recognized by the CAR T cells comprises mesothelin.

113. The method of claim 105, wherein the therapeutic polypeptide comprises a co-stimulatory molecule.

114. The method of claim 109, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

115. The method of claim 98, wherein the therapeutic polypeptide induces tertiary lymphoid structures (TLS) in the tumor bed.

116. The method of claim 111, wherein the therapeutic polypeptide comprises CCL19, lymphotoxin beta, CXCL13, or TNF.

117. The method of claim 98, wherein the therapeutic polypeptide promotes phagocyte innate immune surveillance.

118. The method of claim 113, wherein the therapeutic polypeptide disrupts the Sirpα / CD47 axis.

119. The method of claim 114, wherein the therapeutic polypeptide comprises a Sirpα-IgG fusion transgene.

120. The method of claim 98, wherein the therapeutic polypeptide supports anti-tumor macrophage polarization.

121. The method of claim 120, wherein the therapeutic polypeptide that supports anti-tumor macrophage polarization comprises TNF, IL-1, IL-12, IL-17, or IFNγ.

122. A vector comprising a herpes simplex virus (HSV) genome, wherein the vector comprises alterations that prevent expression of one or more functional ICP4 and ICP47 proteins, and wherein the vector encodes one or more therapeutic polypeptides that support survival of NK cells in a tumor microenvironment (TME).

123. A vector as described in claim 122, wherein the NK cell is a natural NK cell or a CAR NK cell.

124. The vector of claim 122 or 123, wherein the one or more therapeutic polypeptides comprise an NK cell recruiting factor.

125. The vector of claim 124, wherein the NK cell recruiting factor is a chemokine ligand.

126. The vector of claim 125, wherein the chemokine ligand is selected from the group consisting of CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, and CXCL11.

127. The vector of claim 122 or 123, wherein the one or more therapeutic polypeptides comprise an NK cell trophic factor.

128. The vector of claim 127, wherein the NK cell trophic factor is selected from IL-2, IL-15, IL-18 and IFNα.

129. The vector of claim 122 or 123, wherein the one or more therapeutic polypeptides comprises soluble TGFβRII.

Images