WO2025186778A1

WO2025186778A1 - Combinations of oncolytic viruses and immunomodulators

Info

Publication number: WO2025186778A1
Application number: PCT/IB2025/052480
Authority: WO
Inventors: Charles Drake; Steven R. Fuhrmann; Sylvie Laquerre; Matthew Mulvey; Manuel Sepulveda; Kathryne TAYLOR; Douglas Yamada
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2024-03-08
Filing date: 2025-03-07
Publication date: 2025-09-12
Anticipated expiration: 2026-09-08
Also published as: WO2025186778A8

Abstract

Provided herein are methods of treatment comprising administering oncolytic viruses comprising payload genes, such as genes encoding IL-12, FLT3L, CD40 agonists, and/or CTLA- 4 antibodies and antibodies to immunomodulatory agents.

Description

COMBINATIONS OF ONCOLYTIC VIRUSES AND IMMUNOMODULATORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Serial Numbers 63/562,782 filed on March 8, 2024. The entire contents of this application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 5, 2025, is named JBI6842WOPCTl_SL.xml and is 273,133 bytes in size.

TECHNICAL FIELD

[0003] Provided herein are methods of treating solid tumors comprising administration of oncolytic viruses derived from herpes simplex virus 1 (HSV-1) in combination with an immunomodulator.

BACKGROUND

[0004] Oncolytic viruses, specifically designed to replicate within and eliminate cancerous cells, exert their anti-cancer effects through various mechanisms (Bartlett DL et al., 2013, Molecular Cancer 12: 103-120). Primarily, these viruses initiate cell lysis, achieved via apoptosis, necrosis, pyroptosis, autophagy, or a combination thereof (Guo ZS et al., 2014, Front Oncol 4:74). Additionally, they can target a cancer's blood supply, inducing apoptosis and necrosis not only in infected cells but also in non-infected ones.

[0005] Furthermore, oncolytic viruses stimulate immunogenic cell death (ICD) in cancer cells. This prompts the release of danger signal molecules (signal 0) and inflammatory cytokines, while also presenting tumor-associated antigens (TAAs) to naive T cells. Consequently, this process triggers an anti -tumor immune response (Guo ZS et al., 2014, Front Oncol 4:74). These potent viruses not only evoke robust and systemic adaptive antitumor immunity but also facilitate the migration of tumor-specific CD8+ T cells into tumor tissues (Bartlett DL et al., 2013, Mol Cancer 12: 103; Guo ZS et al., 2017, 8:555). [0006] This immune reaction against cancer is not confined to infected cancer cells but extends to metastatic lesions. The approval of the pioneering drug, T-VEC, by the FDA in 2015 to treat advanced melanoma highlighted the promising potential of this innovative cancer treatment (Andtbacka RH et al., 2015, J Clin Oncol 33 :2780-8). However, despite their advantages, oncolytic viruses have faced challenges, notably the highly immunosuppressive tumor environment (Zou W., 2005, Nat Rev Cancer 5:263- 74).

[0007] To address these challenges effectively, combining oncolytic viruses with agents that modulate the immune system is desirable and could significantly improve outcomes.

SUMMARY OF THE INVENTION

[0008] Provided herein are methods of treating a solid tumor in an individual comprising administering an oncolytic virus provided herein in combination with an immunomodulator to the individual. In some embodiments, the oncolytic virus provided herein in combination with an immunomodulator triggers an abscopal response to a distant tumor and/or immunological memory to a tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0010] FIGs.lA-lB are diagrams of the genome structure and transgene cassettes of the mouse surrogate virus, mJP-OV-2 and the human virus JP-OV-2. As shown in FIG. 1 A, the mouse surrogate virus mJP-OV-2 was engineered to express the anti-CTLA-4 antagonist (maCTLA-4), CD40 agonist (mCD40ag), and IL- 12 (mscIL-12) pay loads from the US 10- 12 locus, and hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon-optimized US11 (hCoUSl 1) using the US 12 immediate early promoter and endogenous US11 using late US11 promoter. As shown in FIG. IB, the human virus JP-OV-2 was engineered to express the anti- CTLA-4 antagonist (haCTLA-4), CD40 agonist (hCD40ag), and IL- 12 (hscIL-12) payloads from the US10-12 locus, and hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon- optimized US11 (hCoUSl 1) using the US 12 immediate early promoter and endogenous US11 using late US11 promoter. “S” shown in the hexagon represents a stop codon between hCoUSl 1 and US 12, which inhibits US 12 expression. US 12 in grey indicates that the gene does not express. IRL, internal repeat long; IRS, internal repeat short; TRL, terminal repeat long; TRS, terminal repeat short; UL, unique long; US, unique short.

[0011] FIGS. 2A-2K show the effect of treatment with an oncolytic HSV-1 virus in combination with immunomodulatory agents. FIG. 2A shows a schematic for the experimental design. On Day 0, MC-38 5AG tumor cells were implanted bilaterally on each flank of mice (n=10/group). Mice were treated intratumoral on Days 8, 11, and 14 with vehicle or mJP-OV-2 (arrows). Some groups were treated intraperitoneal (IP) on Days 8, 12, 15, and 19 with anti-PDl (FIG. 2B), anti- CTLA4 (FIG. 2D), anti-OX40 (FIG. 2F), anti-4-lBB (FIG. 2G) or anti-GITR (FIG. 2H) (arrowheads). Tumor volume for the treated and untreated tumors are graphed as the mean ± SEM. Tumor growth of the treatment groups was compared statistically with growth of the vehicle control group over time to Day 29. Survival of the treatment groups was compared with that of the vehicle control group for oncolytic HSV-1 virus treatment in combination with anti- PDl (FIG. 2C), anti-CTLA4 (FIG. 2E), anti-OX40 (FIG. 2G), anti-4-lBB (FIG. 21), or anti- GITR (FIG. 2K). * p<0.05, ** p<0.01, *** p<0.001. IT, intratumoral; SEM, standard error of the mean.

[0012] FIG. 3 shows results from a tumor rechallenge of mice cured of bilateral MC-38 5 AG tumors by intratumoral treatment with oncolytic HSV-1 virus and/or Immunomodulatory agents. Mice that exhibited CRs after the initial MC-38 5 AG tumor challenge and treatment with the indicated agents were rechallenged SC with MC-38 5AG tumor cells on Day 61 (graphed as Day 0). Naive mice were challenged with MC-38 5 AG tumor cells as a positive control for tumor growth. The number of mice in each group is in parentheses. Survival over 52 days is depicted. * p<0.05 for each treatment group compared with the naive control group. CR, complete response; CTLA4, cytotoxic T lymphocyte-associated protein 4; IT, intratumoral; PD1, programmed cell death protein 1.

[0013] FIGs. 4A-4D shows the effect of different dose levels of the oncolytic HSV-1 virus alone or in combination with anti-PDl on MC-38 5 AG tumor growth and mouse survival. On Day 0, MC-38 5 AG mouse tumor cells were implanted bilaterally on each flank of mice (n=10/group). Mice were treated intratumoral in the right tumor on Days 8, 11, and 14 (downward arrows) with 50 pL of vehicle or mJP-OV-2 alone or in combination with anti-PDl. As shown in FIG. 4A and FIG. 4C, group tumor volumes are graphed for the left untreated tumors and the right treated tumors as the mean ± SEM. Treatment group tumor volumes were compared statistically with the vehicle control group over time to Day 30. As shown in FIG. 4B and FIG. 4D, survival is depicted to Day 64. * p<0.05, ** p<0.01, *** p<0.001 in comparison with the vehicle control group. Note that the doses of mJP-OV-2 in the figure are the stock concentrations of the virus, and the tumors were injected with 50 pL of each stock, or l/20th of the stock concentration. IT, intratumoral; ns, not significant; pfu, plaque-forming units; SEM, standard error of the mean.

DETAILED DESCRIPTION

DEFINITIONS

[0014] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0015] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to any particular aspect of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed.

[0016] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0017] Reference to “about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. [0018] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0019] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0020] As used herein, the terms “including,” “containing,” and “comprising” are used in their open, non-limiting sense.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0022] Units, prefixes, and symbols are denoted in their Systeme International d’Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0023] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results. For purposes of this disclosure, beneficial or desired results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, reducing recurrence rate of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of cancer. The methods described herein contemplate any one or more of these aspects of treatment.

[0024] An “effective amount” or “therapeutically effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce recurrence rate in the individual. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent occurrence and/or recurrence of tumor; (vii) delay occurrence and/or recurrence of tumor; (viii) reduce recurrence rate of tumor, and/or (ix) relieve to some extent one or more of the symptoms associated with the cancer. As is understood in the art, an “effective amount” may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint.

[0025] "In conjunction with" or “in combination with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of an oncolytic virus described herein in addition to administration of the other agent (such as immunomodulator(s)) to the same individual under the same treatment plan. As such, "in conjunction with" or “in combination with” refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the individual.

[0026] The term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered at the same time. [0027] As used herein, the term “sequential administration” or “in sequence” means that the first therapy and second therapy in a combination therapy are administered with a time separation, for example, minutes, days, or weeks.

[0028] The term “administered immediately prior to” means that the first therapy is administered no more than about 15 minutes, such as no more than about any of 10, 5 or 1 minutes before administration of the second therapy. The term “administered immediately after” means that the first therapy is administered no more than about 15 minutes, such as no more than about any of 15, 10 or 1 minutes after administration of the second therapy.

[0029] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0030] A "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. A "cancer" or "cancer tissue" can include a tumor. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. Following metastasis, the distal tumors can be said to be "derived from" the pre-metastasis tumor.

[0031] “Enhance” or “enhanced” refers to enhancement in one or more functions of a test molecule when compared to a control molecule or a combination of test molecules when compared to one or more control molecules. Exemplary functions that can be measured are tumor cell killing, T cell activation, relative or absolute T cell number, Fc-mediated effector function (e.g. ADCC, CDC and/or ADCP) or binding to an Fey receptor (FcyR) or FcRn. “Enhanced” may be an enhancement of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant enhancement.

[0032] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith,

[0033] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures (immunoglobulin molecules, fragments of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions), including but not limited to monoclonal antibodies, 4-chain antibodies (such as IgG antibodies), heavy chain antibodies, and antibody fragments thereof so long as they exhibit the desired antigen-binding activity. The term “4-chain antibody” is used herein to refer to an antibody or antigen-binding fragment having two heavy chains and two light chains. The term “heavy chain antibody,” also known as “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises two heavy chains, but lacks two light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0034] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that binds specifically to an antigen can, however, have cross-reactivity to other antigens, such as homologous antigens from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.

[0035] “Antibody fragments” comprise a portion of an antibody, preferably the antigen binding or variable region of the antibody. Examples of antibody fragments include VHHs, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see US Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0036] As used herein, the terms “binding”, "binds" or "specifically binds" in the context of the binding of an antibody to a pre-determined antigen typically is a binding with an affinity corresponding to a KD of about 10⁶ M or less, e.g. 10⁷ M or less, such as about 10⁸ M or less, such as about 10⁹ M or less, about 10¹⁰ M or less, or about 10¹¹ M or even less when determined by for instance BioLayer Interferometry (BLI) technology in a Octet HTX instrument using the antibody as the ligand and the antigen as the analyte, and wherein the antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD of binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount with which the KD of binding is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low, then the amount with which the KD of binding to the antigen is lower than the KD of binding to a non-specific antigen may be at least 10,000-fold (that is, the antibody is highly specific).

[0037] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity, as used herein, and KD are inversely related, that is that higher affinity is intended to refer to lower KD, and lower affinity is intended to refer to higher KD.

[0038] A “CDR” refers to one of three hypervariable regions (H1, H2, or H3) within the nonframework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL P-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains. Kabat et al., J. Biol. Chem. 1977, 252, 6609-6616; Kabat, Adv. Protein Chem. 1978, 32, 1-75. CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adapt different conformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures. Al- Lazikani et al., J. Mol. Biol. 1997, 273, 927-948; Morea et al., Methods. 2000, 20, 267-279. Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme. ALLazikani et al., supra (1997). Such nomenclature is similarly well known to those skilled in the art.

[0039] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxylterminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0040] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol.

150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0041] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, 4-chain antibodies and antigen-binding antibody fragments thereof comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Generally, heavy-chain antibodies comprise three HVRs (HVR1, HVR2, HVR3).

[0042] A number of HVR delineations are in use and are encompassed herein. Exemplary HVRs for 4-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31 -35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0043] The amino acid residues of a single-domain antibody (such as VHH) can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that — as is well known in the art for VH domains and for VHH domains — the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).

[0044] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., framework, “FR,” residues) are numbered herein according to Kabat et al.

[0045] The term “cassette”, “expression cassette,” or “gene cassette” refers to a sequence of DNA carrying, and capable of directing the expression of, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually a vector) to another by “cutting” the fragment out using restriction enzymes and “pasting” it back into the new context (such as a viral genome). Typically, the DNA fragment (nucleic acid sequence) is operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) (genes). Such sequence elements may include a promoter and a polyadenylation signal. [0046] A sequence “encoding” an expression product, such as a polypeptide, is a minimum nucleotide sequence that, when expressed, results in the production of that polypeptide.

[0047] The term “exogenous” refers to a combination of elements not naturally occurring. For example, an “exogenous gene” refers to a gene to be introduced to the genome of a virus, wherein that gene is not normally found in the genome of the virus or is a homolog of a gene expressed in the virus from a different species (e.g., the bovine herpes virus UL49.5 gene, which encodes for a TAP inhibitor, is exogenous when inserted into a viral genome that does not natively encode UL49.5).

[0048] As used herein, the term “herpes simplex virus” or “HSV” refers to members of the Herpesviridae family. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known by their taxonomical names Human alphaherpesvirus I and Human alphaherpesvirus 2, are two members of the human Herpesviridae family, a set of viruses that produce viral infections in the majority of humans.

[0049] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. [0050] A coding sequence is “under the control of " or “operatively associated with” a promoter in a virus or cell when RNA polymerase transcribes the coding sequence into RNA, particularly mRNA, which is then spliced (if it contains introns) and translated into the polypeptide encoded by the coding sequence.

[0051] The term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10 ^-4 M, alternatively at least about 10 ^-5 , alternatively at least about 10 ^-6 M, alternatively at least about 10 ^-7 M, alternatively at least about 10 ^-8 M, alternatively at least about 10 ^-9 M, alternatively at least about 10 ^{-1 0} M, alternatively at least about 10 ^{-1 1} M, alternatively at least about 10 ^{-1 2} M, or greater. In some embodiments, the term “specific binding” refers to binding where a molecule binds a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. KD can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.

IMMUNOMODULATORS

[0052] The methods in some embodiments comprise administration of an oncolytic virus with an immunomodulator.

[0053] “Immunomodulator” refers to an agent that when present, alters, suppresses or stimulates the body's immune system. Immunomodulators can target specific molecules, such as the checkpoint molecules, or non-specifically modulate the immune response.

[0054] Immunomodulators in particular embodiments of the present disclosure include immune checkpoint inhibitors and immune-stimulating agents. As used herein, the term "immune checkpoint inhibitors," "checkpoint inhibitors," and the like refers to compounds that inhibit the activity of control mechanisms of the immune system. Immune system checkpoints, or immune checkpoints, are inhibitory pathways in the immune system that generally act to maintain self- tolerance or modulate the duration and amplitude of physiological immune responses to minimize collateral tissue damage. Immune checkpoint inhibitors can inhibit an immune system checkpoint by stimulating the activity of a stimulatory checkpoint molecule, or inhibiting the activity of an inhibitory checkpoint molecule in the pathway. Stimulatory immune checkpoint molecules are molecules, such as proteins, that stimulate or positively regulate the immune system. Inhibitory checkpoint molecules are molecules, such as proteins, that inhibit or negatively regulate the immune system. Immune system checkpoint molecules include, but are not limited to, programmed cell death 1 protein (PD-1), programmed cell death 1 ligand 1 (PD- Ll), programmed cell death 1 ligand 2 (PD-L2), cytotoxic T-lymphocyte antigen 4 (CTLA-4), , lymphocyte activation gene 3 (LAG3), B7-1, B7-H3, B7-H4, T cell membrane protein 3 (TIM3), B- and T-lymphocyte attenuator (BTLA), V- domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), Killer-cell immunoglobulin-like receptor (KIR), and A2A adenosine receptor (A2aR). As such, checkpoint inhibitors include antagonists of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM3. For example, antibodies that bind to PD-1, PD-L1, PD-L2, CTLA-4, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM3 and antagonize their function are checkpoint inhibitors. Moreover, any molecule (e.g., peptide, nucleic acid, small molecule, etc.) that inhibits the inhibitory function of an immune system checkpoint is a checkpoint inhibitor.

[0055] In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an inhibitory immune checkpoint protein. In some embodiments, the immunomodulator is an antibody. In some embodiments, the antibody is an antagonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigenbinding subsequences of the full length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof.

[0056] In some embodiments, the immunomodulator is an immune-stimulating agent. In some embodiments, the immune-stimulating agent is a natural or engineered ligand of an immune stimulatory molecule. In some embodiments, the immune-stimulating agent is an antibody. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof.

[0057] Exemplary immune checkpoint molecules and immunomodulators thereof are discussed below. It is understood that other suitable immune checkpoint molecules and immunomodulators known in the art are also within the scope of the present disclosure.

PD-1

[0058] PD-1 is a part of the B7/CD28 family of co-stimulatory molecules that regulate T-cell activation and tolerance, and thus antagonistic anti-PD-1 antibodies can be useful for overcoming tolerance. PD-1 has been defined as a receptor for B7-4. B7-4 can inhibit immune cell activation upon binding to an inhibitory receptor on an immune cell. Engagement of the PD- 1/PD-L1 pathway results in inhibition of T-cell effector function, cytokine secretion and proliferation. (Turnis et al., Oncolmmunology 1(7): 1172-1174, 2012). High levels of PD-1 are associated with exhausted or chronically stimulated T cells. Moreover, increased PD-1 expression correlates with reduced survival in cancer patients.

[0059] Agents for down modulating PD-1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signal in an immune cell resulting in enhancement of the immune response known in the art may be used in the present disclosure. For example, Cetrelimab is a human antibody to PD-1 comprising the following CDR sequences, VH/VL sequences, and heavy and light chain sequences:

SEQ ID NO: 500 HCDR1

SYAIS

SEQ ID NO: 501

HCDR2

GIIPIFDTANYAQKFQG

SEQ ID NO: 502

HCDR3

PGLAAAYDTGSLDY

SEQ ID NO: 503

LCDR1

RASQSVRSYLA

SEQ ID NO: 504

LCDR2

DASNRAT

SEQ ID NO: 505

LCDR3

QQRNYWPLT

SEQ ID NO: 506

VH

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTAN YAQKFQGRVTIT ADES TS TAYMELS SLRSEDTAVYYC ARPGLAAAYDTGSLD YWGQGT LVTVSS

SEQ ID NO: 507

VL EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIK

SEQ ID NO: 508

HEAVY CHAIN

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTAN YAQKFQGRVTIT ADES TS TAYMELS SLRSEDTAVYYC ARPGLAAAYDTGSLD YWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAI<TI<PRE EQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 509

LIGHT CHAIN

EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKAD YEKHKVYACEVTHQGLS SPVTKSFNRGEC

CTLA-4

[0060] CTLA-4 is an immune checkpoint molecule, which is up-regulated on activated T-cells. An anti-CTLA-4 mAh can block the interaction of CTLA-4 with CD80/86 and switch off the mechanism of immune suppression and enable continuous stimulation of T-cells by DCs.

4- IBB

[0061] 4-1BB, also known as CD137 is a member of the TNFR superfamily. One characterized activity of 4- IBB is its costimulatory activity for activated T cells. 0X40

[0062] 0X40, also known as CD134 and TNFRSF4, is a member of the TNFR-superfamily of receptors. 0X40 is a co-stimulatory immune checkpoint molecule, expressed after 24 to 72 hours following activation of the T cells. The interaction of OX40L and 0X40 will sustain T cell proliferation and immune response and memory beyond the first two days. Methods for enhancing the immune response to a tumor antigen by engaging the 0X40 receptor on the surface of T-cells by an 0X40 receptor binding agent, OX40L or an 0X40 agonist during or shortly after priming of the T-cells by the antigen can be used in CLIVS as an immune checkpoint inhibitor.

GITR

[0063] GITR is a member of the TNFR superfamily. GITR is expressed in many components of the innate and adaptive immune system and stimulates both acquired and innate immunity. Agents that stimulate the activity of GITR is useful as an immune-stimulating agent.

CD40

[0064] CD40 (Cluster of differentiation 40) is a co-stimulatory protein found on antigen presenting cells and is required for their activation. Binding of CD40L (CD154) on TH cells to CD40 activates antigen presenting cells and incudes a variety of downstream effects to stimulate immune response. Agents that stimulate the activity of CD40 is useful as an immune-stimulating agent.

PD-L1/PD-L2

[0065] PD-L1 (Programmed cell death-ligand 1) is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 serves as a ligand for PD-1 to play a major role in suppressing the immune system during particular events such as pregnancy, tissue allographs, autoimmune disease and other disease states such as hepatitis and cancer. The formation of PD-1 receptor/PD-L1 ligand complex transmits an inhibitory signal which reduces the proliferation of CD8+ T cells at the lymph nodes. ONCOLYTIC VIRUSES

[0066] Immunotherapy of cancer with oncolytic viruses is an emerging and maturing treatment modality which uses replication-competent viruses that selectively infect and damage tumor cells and may also, preferably, induce an immunological response which can control both the target tumor and distal tumors. Each species of oncolytic virus has a different cellular tropism, which helps determine which tissues are preferentially infected. Engineering of the virus can expand, restrict, or modulate this host range. A variety of species of virus have been investigated for use in oncolytic therapies, including those derived from HSV, vaccinia, and reovirus.

[0067] Thus, the present application provides oncolytic viruses that are effective for treating cancer. Non-limiting examples of oncolytic viruses include those derived from a herpes simplex virus, a vaccinia virus, an adenovirus, a reovirus, or a vesicular stomatitis virus. In some embodiments, the oncolytic virus (such as an oncolytic HSV) preferentially triggers an immune response that results in killing of tumor cells. As used herein, the virus “preferentially kills” tumor cells when certain infectious doses of the virus are more likely to kill tumor cells than neighboring healthy cells (such as at least two times more likely to kill tumor cells than neighboring healthy cells at a given dose). In some embodiments, the oncolytic virus expresses one or more payload proteins described below. In some embodiments, the oncolytic virus induces an immune response to the tumor, which, in some embodiments, causes tumor cells at sites distal to the site of infection to be killed. In some embodiments, the oncolytic virus is capable of evading an individual’s immune system after administration to the individual. As used herein, evading the individual’s immune system means that the oncolytic virus is able to preferentially replicate in tumor cells. In some embodiments, the oncolytic viruses provided herein are more sensitive to an innate antiviral response than a wild-type virus, enabling preferential replication in tumor cells. In some embodiments, the oncolytic viruses provided herein have an intermediate resistance to interferon.

Oncolytic herpes simplex virus

[0068] In one aspect, the present disclosure pertains to oncolytic herpes simplex virus (HSV). In some embodiments, the oncolytic HSV is derived from HSV-1. In some embodiments, the oncolytic HSV comprises one or more expression cassettes described herein. In some embodiments, the oncolytic HSV expresses one or more payload proteins described herein. In some embodiments, the oncolytic HSV lacks one or more native HSV genes. In some embodiments, the oncolytic HSV lacks one or both copies of γ34.5. In some embodiments, the oncolytic HSV does not express one or more native HSV proteins, such as US 12. In some embodiments, the oncolytic HSV expresses one or more additional copies of a native HSV protein, such as US11. In some embodiments, the oncolytic HSV expresses a native HSV protein in a different temporal order, such as expressing immediate-early US11. The oncolytic HSV may be a component of a pharmaceutical composition described herein. The oncolytic HSV, or a pharmaceutical composition comprising the oncolytic HSV, may be administered to individual according to the methods described herein (such as the methods of treatment described herein). In some embodiments, the oncolytic HSV preferentially triggers an immune response that results in killing of tumor cells compared to the wild-type HSV from which it is derived. In some embodiments, the oncolytic HSV is capable of triggering an immune response that triggers killing tumor cells at one or more sites distal to a target site.

Viral payloads

[0069] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the gene cassette otherwise described herein, comprises one or more genes encoding one or more payload molecules. The payload molecules are generally intended to enhance the therapeutic effect of the oncolytic virus (such as an oncolytic HSV). For example, a payload molecule may promote an immune response (e.g., against the tumor target) or may enhance the cytotoxicity of the oncolytic virus.

IL-12

[0070] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding interleukin 12 (IL- 12). [0071] IL- 12 is a heterodimeric protein comprising two subunits: p35 and p40. The native p35 subunit is linked to the p40 subunit by a disulfide bond. The human and mouse p40 subunits are 70% identical, while the p35 subunits share 60% amino acid sequence homology. The p35 and p40 subunits may function in receptor binding and signal transduction, respectively (Zou, J. J., et al. (1995). Structure-function analysis of the p35 subunit of mouse interleukin 12. The Journal of biological chemistry, 270(11), 5864-5871). IL-12 is normally secreted by antigen-presenting cells, such as macrophages and dendritic cells. Biologically active IL- 12 (comprising both subunits in a heterodimer) functions to differentiate naive T cells into Thl cells, promote cytotoxic activity of NK cells and T cells, and block angiogenesis.

[0072] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding the p35 subunit of IL-12 and/or a polynucleotide encoding the p40 subunit of IL-12. In some embodiments, the p35 subunit and/or p40 subunit of IL-12 is human. In some embodiments, the p35 subunit and/or p40 subunit of IL- 12 is murine. In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a p35 subunit and a p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a p35 subunit of IL- 12 and a p40 subunit of IL- 12 connected by a peptide linker.

[0073] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human p35 subunit of IL- 12 and/or a polynucleotide encoding a human p40 subunit of IL- 12. In some embodiments, the human p35 subunit comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the human p40 subunit comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the human p40 subunit comprises the amino acid sequence of SEQ ID NOV, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the oncolytic virus (such as an oncolytic HS V), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a human p35 subunit and a human p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a human p35 subunit of IL- 12 and a human p40 subunit of IL- 12 connected by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO:4In some embodiments, the IL- 12 heterodimer comprises the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 10.

[0074] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a murine p35 subunit of IL- 12 and/or a polynucleotide encoding a murine p40 subunit of IL- 12. In some embodiments, the murine p35 subunit comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the murine p40 subunit comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the murine p40 subunit comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding an IL- 12 heterodimer comprising a murine p35 subunit and a murine p40 subunit. In some embodiments, the IL- 12 heterodimer comprises a polypeptide comprising a murine p35 subunit of IL- 12 and a murine p40 subunit of IL- 12 connected by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:7In some embodiments, the IL- 12 heterodimer comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 12. CD40 agonist

[0075] Cluster of differentiation 40 (CD40) is a costimulatory polypeptide expressed on numerous cell types, from antigen presenting cells (APCs) to epithelial cells. It is additionally present on various cancer cells. CD40 agonist, also known as cluster of differentiation 154 (CD154), comprises 261 amino acids and is a type II membrane glycopolypeptide that is expressed on the surface of activated T cells. Native CD40 agonist promotes B cell maturation. It is additionally essential for immunoglobulin class switching, as lack of CD40 agonist is associated with hyper IgM syndrome. CD40 agonist exists as a membrane-bound form, in which the extracellular domain forms a homotrimer, and a proteolytically-cleaved, soluble form, which has been shown to be biologically active.

[0076] In some embodiments, provided herein is an oncolytic virus comprising a polynucleotide encoding a CD40 agonist. In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding CD40 agonist. In some embodiments, the CD40 agonist is a CD40 ligand. In some embodiments, the CD40 agonist comprises a CD40 ligand ectodomain. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is fused to a trimerization motif, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, each of the three singlechain trimeric CD40 ligand ectodomains is fused to an Fc region, e.g., to direct formation of the trimer of three single-chain trimeric CD40 ligand ectodomains. In some embodiments, said Fc region is an IgGFc region e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region. In some embodiments, said Fc region comprises one or more amino acid substitutions, insertions, or deletions that disfavor binding of said Fc region to another Fc region, such as an IgG Fc region, e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region. In some embodiments, said Fc region comprises a substitution of the IgG interaction domain with an IgA interaction domain. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is bivalent. In some embodiments, the CD40 agonist is an agonist antibody.

[0077] In some embodiments, the CD40 agonist comprises a human CD40 ligand ectodomain. In some embodiments, the human CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric human CD40 ligand ectodomains. In some embodiments, the single-chain trimeric human CD40 ligand ectodomains comprise a polypeptide comprising three human CD40 ligand ectodomains connected by peptide linkers. In some embodiments, the single-chain trimeric human CD40 ligand ectodomain polypeptide comprises a first human CD40 ligand ectodomain connected by a peptide linker to a second human CD40 ligand ectodomain which is connected by a peptide linker to a third human CD40 ligand ectodomain. In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of glycine and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:24.

[0078] In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:30.

[0079] In some embodiments, the CD40 agonist comprises a murine CD40 ligand ectodomain. In some embodiments, the murine CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the CD40 agonist is a trimer of three single-chain trimeric murine CD40 ligand ectodomains. In some embodiments, the single-chain trimeric murine CD40 ligand ectodomains comprise a polypeptide comprising three murine CD40 ligand ectodomains connected by peptide linkers. In some embodiments, the single-chain trimeric murine CD40 ligand ectodomain polypeptide comprises a first murine CD40 ligand ectodomain connected by a peptide linker to a second murine CD40 ligand ectodomain which is connected by a peptide linker to a third murine CD40 ligand ectodomain. In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the trimerization motif is a T4 fibritin trimerization motif. In some embodiments, the T4 fibritin trimerization motif comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to each of the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to each of the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence comprising of leucine, glycine, and/or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 28 In some embodiments, the CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the CD40 agonist forms a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29. CTLA-4 binding protein

[0080] Cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4), also known as cluster of differentiation 152 (CD 152), is a polypeptide receptor that functions as an immune checkpoint and downregulates immune responses. The polypeptide contains an extracellular V- like domain, a transmembrane domain, and a cytoplasmic tail. Alternate isoforms have been characterized. CTLA-4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation, and contributes to the inhibitory function of regulatory T cells. CTLA-4 binds to CD80 and CD86, also known as B7-1 and B7-2 respectively, on APCs in order to induce its inhibitory function to T cells.

[0081] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a CTLA-4 binding protein. In some embodiments, the CTLA-4 binding protein is a CTLA-4 antagonist. For example, in some instances, the CTLA-4 binding protein inhibits the interaction between CTLA- 4 and one or more CTLA-4 ligands, such as CD80 and/or CD86. In some embodiments, the CTLA-4 binding protein specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA-4.

[0082] In some embodiments, the CTLA-4 binding protein is an anti-CTLA-4 antibody or antigen binding fragment thereof. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof specifically binds to human CTLA-4, murine CTLA-4, or both human and murine CTLA-4. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment is bivalent. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment comprises an Fc region, such as an active Fc region. In some embodiments, the anti- CTLA-4 antibody or antigen binding fragment comprises an IgGl, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgGl, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof comprises a singlechain variable fragment (scFv). In some embodiments, the anti-CTLA-4 scFv is fused to the N- terminus of an IgGl, IgG2, IgG3, or IgG4 constant domain, e.g., a human or mouse IgGl, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof comprises an anti-CTLA-4 VHH, e.g., a camelid antibody comprising an anti-CTLA-4 VHH. In some embodiments, the anti-CTLA-4 VHH is fused to the heavy chain of an IgGl, IgG2, IgG3, or IgG4 Fc, e.g., a human or mouse IgGl, IgG2, IgG3, or IgG4 Fc.

[0083] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof, such as the anti-CTLA-4 scFv, specifically binds to human CTLA-4. In some embodiments, the anti-CTLA-4 scFv is fused to the N-terminus of a IgGl constant domain, e.g., a human IgGl constant domain. In some embodiments, the human IgGl is a variant human IgGl comprising a C220S substitution, wherein the numbering of the residues is according to EU numbering. In some embodiments, the human IgGl is a Glm(17) IgGl. In some embodiments, the anti-CTLA- 4 antibody causes depletion of regulatory T (Treg) cells.

[0084] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:40; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:41; and (c) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:42; and/or wherein the VL comprises one or more of: (a) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:44; and (c) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0085] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:46; and/or a VL comprising the amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the variable heavy chain and variable light chain are connected via a linker sequence. In some embodiments, the linker sequence comprises an amino acid sequence set forth in SEQ ID NO:61. [0086] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises an IgGl constant domain comprising the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the heavy chain of the CTLA-4 antibody comprises the amino acid sequence set forth in SEQ ID NO48, with or without the C terminal lysine.

[0087] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises the amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 60.

[0088] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 scFv) comprises the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 50. [0089] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof, such as the anti-CTLA-4 VHH, specifically binds to murine CTLA-4. In some embodiments, the anti-CTLA-4 VHH is fused to the heavy chain of a murine IgG2a Fc.

[0090] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a variable heavy chain (VH), wherein the VH comprises one or more of: (a) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51; (b) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52; and (c) a CDR- H3 comprising the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a VH, wherein the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti- CTLA-4 VHH) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 58, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:58. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 59, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:59.

[0091] In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises a signal peptide sequence comprising the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the anti-CTLA-4 antibody or antigen binding fragment thereof (e.g., the anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 57.

[0092] In some embodiments, the oncolytic herpes simplex type 1 (HSV-1) virus comprising a polynucleotide encoding an antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) is administered to an individual, and an antibody to human cytotoxic T lymphocyte- associated protein 4 (αCTLA4) can further be administered to the individual. In some embodiments, the antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) administered systemically to the individual are the same antibody. In some embodiments, the antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) encoded within the oncolytic virus and antibody to human cytotoxic T lymphocyte-associated protein 4 (αCTLA4) administered systemically to the individual are the different antibodies.

FLT3 ligand

[0093] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a fms-like tyrosine kinase 3 ( FLT3) ligand (FLT3L). FLT3L is a growth and differentiation factor that enhances and expands dendritic cells (DCs) as well as recruits DCs to the tumor microenvironment.

[0094] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a human

FLT3L. In some embodiments, the human FLT3L comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:72.

[0095] In some embodiments, the human FLT3L comprises a signal peptide directing secretion to the plasma membrane. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, the human FLT3L comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:71.

[0096] In some embodiments, the FLT3L, e.g., the human FLT3L, is a homodimer. In some embodiments, the human FLT3L is proteolytically processed into soluble FLT3L. In some embodiments, the soluble FLT3L forms a homodimer.

Other payload molecules

[0097] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises one or more polynucleotides encoding a US11 protein, such as a US 11 protein from an HSV, e.g., an HSV-1 or HSV-2.

[0098] The protein kinase R (PKR) pathway is a component of the host cellular innate antiviral response. PKR becomes activated in response to binding double-stranded RNA (dsRNA), a byproduct of viral replication, leading to phosphorylation and inactivation of eukaryotic translation initiation Factor 2 Subunit 1 ( eIF2α), a translation initiation factor. Phosphorylated eIF2α prevents translation initiation, a cellular defense mechanism aimed at blocking the production of viral proteins. The US11 protein is believed to bind and sequester dsRNA, preventing the activation of the PKR pathway in host cells, and enabling enhanced viral replication. [0099] In some embodiments, the US11 protein comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 80.

[0100] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide encoding a US11 protein, wherein the polynucleotide comprises a native US11 gene nucleotide sequence, e.g., from an HSV, such as an HSV-1 or an HSV-2. In some embodiments, the native US11 gene is a native US11 late gene, wherein the US11 protein is expressed in the late stage of viral replication. In some embodiments, the native US11 late gene is under the control of the endogenous US11 promoter, e.g., from an HSV, such as an HSV-1 or an HSV-2.

[0101] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises a polynucleotide comprising a variant US11 gene. In some embodiments, the variant US11 gene is codon optimized for expression of the US11 protein in human cells. In some embodiments, the variant US11 gene encodes a wild type US11 protein, e.g., from an HSV, such as an HSV-1 or an HSV-2. In some embodiments, the variant US11 gene comprises the nucleotide sequence of SEQ ID NO:204, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:204. In some embodiments, the variant US11 gene is operably linked to a promoter. In some embodiments, the promoter directs immediate early expression of the US11 protein during viral replication. In some embodiments, the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof. [0102] In some embodiments, the oncolytic virus (such as an oncolytic HSV), or the expression cassette otherwise described herein, comprises both a polynucleotide encoding a US11 protein and comprising a native US11 gene nucleotide sequence, e.g., as described above; and a polynucleotide comprising a variant US11 gene, e.g., as described above.

[0103] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette otherwise described herein, comprises a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, such as a viral TAP inhibitor. In general, viral TAP inhibitors prevent TAP from transporting peptides into the lumen of the endoplasmic reticulum, thus impairing peptide loading onto major histocompatibility complex (MHC) Class I molecules for display at the cell surface (Verweij et al. Viral inhibition of the transporter associated with antigen processing (TAP): A striking example of functional convergent evolution. PLoS Pathog. 2015; 11(4): el 004743). Although TAP inhibition disrupts the transport of newly-expressed MHC molecules to the cell surface, this does not block preexisting antigen display. Thus, TAP inhibition by a TAP inhibitor can prevent the display of viral antigens on the cell surface, preventing premature clearance of infected cells and enabling virus persistence throughout multiple rounds of virus replication.

[0104] In some embodiments, the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5. In some embodiments, the TAP inhibitor comprises the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO:83. In some embodiments, the TAP inhibitor further comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the TAP inhibitor comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having any of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the TAP inhibitor is expressed during the immediate early phase of viral replication, i.e., it is expressed as an immediate early gene. In some embodiments, the polynucleotide encoding the TAP inhibitor is expressed under the control of an immediate early promoter, such as a CMV promoter, e.g., an hCMV promoter.

Expression cassettes

[0105] Provided herein are one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CTLA- 4 binding protein, a polynucleotide encoding an FLT3 ligand (FLT3L), or any combination thereof.

Expression cassettes encoding IL- 12, a CD40 agonist, and/or a CTLA-4 binding protein [0106] Provided herein are expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein.

[0107] In some embodiments, the expression cassettes of the disclosure comprise a promoter operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary and non-limiting promoters thatmay be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EFla promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HSV promoter sequence, such as the HSV-1 or HSV-2 US 12 promoter, or the HSV-1 or HSV-2 US11 promoter.

[0108] In some embodiments, the expression cassettes of the disclosure comprise a polyadenylation signal operably linked to each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable polyadenylation signal may be used in the cassettes of the disclosure. Exemplary and non-limiting polyadenylation signals (polyA or pA) that may be used include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA(hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence, such as the US10-12 polyA or the US9-10 polyA from HSV-1 or HSV-2.

[0109] In some embodiments, the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein.

[0110] In some embodiments, the expression cassettes of the disclosure comprise an RNA Polymerase II transcriptional pause signal positioned after each of the polynucleotide encoding IL- 12, the polynucleotide encoding the CD40 agonist, and/or the polynucleotide encoding the CTLA-4 binding protein. Any suitable RNA Polymerase II transcriptional pause signal may be used in the cassettes of the disclosure. Exemplary and non-limiting RNA polymerase II transcriptional pause signals include the human complement C2 protein terminator (C2) and the human Gastrin terminator (hGT).

[0111] In some embodiments, an expression cassette of the disclosure comprises, in order, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide encoding the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12 are in the same orientation in the expression cassette, and the polynucleotide encoding the CD40 agonist is in the reverse orientation relative to the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding the IL- 12.

[0112] In some embodiments, the polynucleotide encoding the CTLA-4 binding protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an mCMV promoter. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a polyA derived from the human GAPDH gene.. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CTLA- 4 binding protein In some embodiments, the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein. In some embodiments, the RNA polymerase II transcriptional pause signal is a C2 RNA polymerase II transcriptional pause signal. In some embodiments, the encoded CTLA-4 binding protein is any of the CTLA-4 binding proteins described herein, e.g., in Section III-C, above. [0113] In some embodiments, the polynucleotide encoding the CD40 agonist is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is the AOHV1 promoter. In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the CD40 agonist, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hBGpA. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding the CD40 agonist. In some embodiments, the expression cassette further comprises an RNA polymerase II transcriptional pause signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcriptional pause signal known in the art or described herein. In some embodiments, the RNA polymerase II transcriptional pause signal is the hGT RNA polymerase II transcriptional pause signal. In some embodiments, the encoded CD40 agonist is any of the CD40 agonists described herein, e.g., in Sections III-B or V, herein. In some embodiments, the polynucleotide encoding the CD40 agonist is in the reverse orientation within the expression cassette relative to the polynucleotide encoding the IL- 12 and the polynucleotide encoding the CTLA-4 binding protein.

[0114] In some embodiments, the polynucleotide encoding IL- 12 is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is the MMLV promoter In some embodiments, the expression cassette further comprises a polyadenylation signal positioned after the polynucleotide encoding IL- 12, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is the US 10- 12 polyA or the US 9- 10 polyA from HSV, such as from HSV-1 or HSV-2. In some embodiments, the US10-12 polyA comprises the nucleotide sequence of a native HSV-1 or HSV-2 US 10- 12 polyA. In some embodiments, the expression cassette further comprises a Kozak sequence positioned between the promoter and the polynucleotide encoding IL- 12.

[0115] In some embodiments, an expression cassette of the disclosure further comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US11 protein; or a polynucleotide encoding a US11 protein and a US 10 protein.

[0116] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding a US 10 protein and/or a polynucleotide encoding a US11 protein. In some embodiments, the polynucleotide encoding the US11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US 11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the polynucleotide encoding the US 10 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US 10 promoter. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hGHpolyA. In some embodiments, the encoded US11 protein is an HSV US11 protein, such as an HSV-1 or HSV-2 US11 protein. In some embodiments, the polynucleotide encoding the US11 protein comprises a native US 11 gene. In some embodiments, the expression cassette comprises, in order, the polynucleotide encoding the US11 protein (e.g., comprising a native US11 gene) and/or the polynucleotide encoding the US 10 protein, the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US11 protein and/or the US 10 protein are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, the US11 protein and/or the US 10 protein. [0117] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding a US11 protein and a US 10 protein. In some embodiments, the polynucleotide encoding the US11 protein and the US 10 protein comprises a nucleic acid sequence encoding the US11 protein, and a nucleic acid sequence encoding the US 10 protein. In some embodiments, at least a portion of the nucleic acid sequence encoding the US11 protein overlaps with at least a portion of the nucleic acid sequence encoding the US 10 protein. In some embodiments, the nucleic acid sequence encoding the US11 protein is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US11 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US 11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the nucleic acid sequence encoding the US 10 protein is operably linked to a promoter. In some embodiments, the promoter is a native US10 promoter from an HSV, such as HSV-1 or HSV-2. In some embodiments, the promoter is embedded within the nucleic acid sequence encoding the US11 protein. In some embodiments, the encoded US11 protein is an HSV US11 protein, such as an HSV-1 or HSV-2 US 11 protein. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the nucleic acid sequence encoding the US 10 protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is a hGHpolyA. In some embodiments, the polynucleotide encoding the US11 protein and the US 10 protein comprises a native US11 gene. In some embodiments, the expression cassette comprises, in order, the polynucleotide encoding the US11 protein and the US 10 protein (e.g., comprising a native US11 gene), the polynucleotide encoding the CTLA-4 binding protein, the polynucleotide that encodes the CD40 agonist, and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US11 and US 10 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US11 and US 10 proteins.

[0118] In some embodiments, an expression cassette of the disclosure further comprises a polynucleotide encoding a US11 protein, wherein the polynucleotide comprises a variant US11 gene. In some embodiments, the variant US11 gene comprises a sequence that is codon optimized for expression of the US11 protein in human cells. In some embodiments, the variant US11 gene is operably linked to a promoter, such as any suitable promoter known in the art or described herein. In some embodiments, the promoter is an endogenous US 12 promoter from an HSV, such as HSV-1 or HSV-2, or a portion thereof. In some embodiments, the endogenous US 12 promoter, or the portion thereof, directs immediate early expression of the US11 protein during viral replication. In some embodiments, the expression cassette further comprises a 5’ untranslated region (UTR) sequence positioned between the promoter and the variant US11 gene.. In some embodiments, the expression cassette further comprises a polynucleotide encoding a US12 protein positioned after the variant US11 gene (e.g., after a stop codon in the variant US11 gene). In some embodiments, the US12 protein is from an HSV, such as HSV-1 or HSV-2. In some embodiments, the polynucleotide encoding the US 12 protein is not operably linked to a promoter. In some embodiments, the encoded US 12 protein is not expressed. In some embodiments, the expression cassette further comprises a spacer sequence and a UTR sequence positioned between the variant US11 gene and the polynucleotide encoding the US 12 protein. In some embodiments, the expression cassette comprises, in order, the variant US11 gene; the polynucleotides encoding the US 10 and/or US11 proteins, or the polynucleotide encoding the US 10 and US11 proteins; the polynucleotide encoding the CTLA-4 binding protein; the polynucleotide that encodes the CD40 agonist; and the polynucleotide encoding the IL- 12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, the IL- 12, and the US 10 and/or US11 proteins are in the same orientation in the expression cassette, and the polynucleotide that encodes the CD40 agonist is in the reverse orientation relative to the polynucleotides encoding the CTLA-4 binding protein, the IL-12, and the US10 and/or US11 proteins.

[0119] In some embodiments, an expression cassette of the disclosure comprises, in order, a promoter (e.g., an HSV US12 promoter) operably linked to the polynucleotide comprising a variant US11 gene; optionally, a 5’ UTR sequence; the polynucleotide comprising the variant US11 gene; a promoter (e.g., a native HSV US11 promoter); the polynucleotide encoding the US11 protein and the US 10 protein; a polyadenylation signal (e.g., a hGHpA poly A) operably linked to the polynucleotide encoding the US11 protein and the US 10 protein; a promoter (e.g., a CMV promoter such as an mCMV promoter) that directs expression of the polynucleotide encoding the CTLA-4 binding protein; optionally, a Kozak sequence for expression of the polynucleotide encoding the CTLA-4 binding protein; the polynucleotide encoding the CTLA-4 binding protein; a polyadenylation signal (e.g., a GAPDH SpA poly A) that is operably linked to the polynucleotide encoding the CTLA-4 binding protein; optionally, an RNA polymerase II pause site (e.g., a C2 pause site); an RNA polymerase II pause site (e.g., an hGT pause site); a polyadenylation signal (e.g., an hBGpA poly A) that is operably linked to the polynucleotide encoding the CD40 agonist; the polynucleotide that encodes the CD40 agonist; optionally, a Kozak sequence for expression of the polynucleotide that encodes the CD40 agonist; a promoter (e.g., an AoHVl promoter) that controls expression of the CD40 agonist; a promoter (e.g., an MMLV promoter) that controls expression of the IL- 12; optionally, a Kozak sequence for expression of the polynucleotide encoding the IL- 12; the polynucleotide encoding the IL- 12; and a polyadenylation signal (e.g., an HSV US 10- 12 poly A) that is operably linked to the polynucleotide encoding the IL- 12.

[0120] In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:201, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:201. In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO:202, or a nucleotide sequence having any of at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence of SEQ ID NO:202. [0121] In some embodiments, an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2. In some embodiments, the cassette is integrated in the US 10- 12 locus of an oncolytic HSV, e.g., an oncolytic HSV-1 or oncolytic HSV-2.

[0122] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) and the polynucleotide encoding the CTLA-4 binding protein, e.g., as described above. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)- (iii).

[0123] In some embodiments, the expression cassette further comprises polynucleotide(s) encoding a US 10 protein and/or a US11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotides encoding the US 10 protein and/or US11 protein. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv). In other embodiments, the expression cassette further comprises a polynucleotide encoding a US 10 protein and a US11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, and (iv) the polynucleotide encoding the US 10 and US11 proteins. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv). In other embodiments, the expression cassette further comprises a polynucleotide encoding a US10 protein and a polynucleotide encoding a US11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, and (v) the polynucleotide encoding the US11 protein. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)- (iii)-(iv)-(v).

[0124] In some embodiments, the expression cassette further comprises a polynucleotide comprising a variant US11 gene, e.g., as described above. In some such embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL-12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide(s) encoding the US 10 protein and/or US11 protein, or the polynucleotide encoding the US 10 and US11 proteins, and (v) the polynucleotide comprising the variant US11 gene. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv)-(v). In other embodiments, the expression cassette comprises a polynucleotide comprising a variant US11 gene, e.g., as described above. In some such embodiments, the expression cassette comprises: (i) the polynucleotide encoding IL- 12, (ii) the polynucleotide encoding the CD40 agonist, (iii) the polynucleotide encoding the CTLA-4 binding protein, (iv) the polynucleotide encoding the US 10 protein, (v) the polynucleotide encoding the US11 protein, (vi) the polynucleotide comprising the variant US11 gene. In some embodiments, the expression cassette is in the orientation relative to the internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of IRS-(i)-(ii)-(iii)-(iv)-(v)-(vi).

Expression cassettes encoding FLT3L and/or TAP inhibitor

[0125] Also provided herein are expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor.

[0126] In some embodiments, an expression cassette of the disclosure comprises a promoter operably linked to the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor. Any suitable promoter may be used in the cassettes of the disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary and non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotine betaherpesvirus 1 (AoHV 1) promoter, the CAG promoter, a CMV hybrid promoter, the EFla promoter, the MMLV 5’ long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and a native HS V promoter sequence.

[0127] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding FLT3L and/or the polynucleotide encoding the TAP inhibitor. Any suitable polyadenylation signal may be used in the cassettes of the disclosure. Exemplary and non-limiting polyadenylation signals (polyA or pA) that may be used include the simian vacuolating virus 40 polyA (SV40pA), the human beta globin polyA (hBGpA), the human growth hormone polyA (hGH polyA), the rabbit beta globin polyA (rBGpA), a bovine growth hormone polyadenylation (BGHpA), a polyA derived from the human GAPDH gene, and a native HSV polyA sequence.

[0128] In some embodiments, the expression cassettes of the disclosure may comprise any suitable promoter and/or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotide encoding FLT3L and/or to the polynucleotide encoding the TAP inhibitor.

[0129] In some embodiments, the TAP inhibitor is derived from herpes virus 1 or herpes virus 2. In some embodiments, the TAP inhibitor is derived from bovine herpes virus 1. In some embodiments, the TAP inhibitor is any of UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5

[0130] In some embodiments, the expression cassette further comprises a polynucleotide encoding a self-cleaving peptide. Any suitable self-cleaving peptide may be used in the cassettes of the disclosure, including, but not limited to, a T2A, P2A, E2A, or F2A peptide. In some embodiments, the encoded self-cleaving peptide is a P2A peptide. In some embodiments, the encoded P2A comprises the amino acid sequence of SEQ ID NO:91In some embodiments, the self-cleaving peptide is positioned between the polynucleotide encoding the FLT3L and the polynucleotide encoding the TAP inhibitor in the expression cassette.

[0131] In some embodiments, the expression cassette comprises a promoter operably linked to the polynucleotide encoding the FLT3L. In some embodiments, the promoter is the hCMV promoter. In some embodiments, the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 107

[0132] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the TAP inhibitor. In some embodiments, the polyadenylation sequence is a BGHpA polyadenylation signal. In some embodiments, the BGHpA polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 102.

[0133] In some embodiments, an expression cassette of the disclosure comprises, in order, a promoter (e.g., an hCMV promoter) operably linked to the polynucleotide encoding the FLT3L; the polynucleotide encoding the FLT3L; the polynucleotide encoding the self-cleaving peptide (e.g., a P2A peptide); the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal (e.g., a BGHpA polyadenylation signal).

[0134] In some embodiments, an expression cassette of the disclosure comprises a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the expression cassette encodes a polypeptide comprising, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the expression cassette further comprises a promoter, e.g., an hCMV promoter, that regulates expression of the polynucleotide encoding the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the expression cassette further comprises a polyadenylation signal, e.g., a BGHpA. In some embodiments, the expression cassette comprises, in order, a promoter, e.g., an hCMV promoter; a polynucleotide encoding, in order, the FLT3L, the self-cleaving peptide (e.g., a P2A peptide), and the TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal, e.g., a BGHpA.

[0135] In some embodiments, an expression cassette of the disclosure comprises the nucleotide sequence of SEQ ID NO: 100, or a nucleotide sequence having any of about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the nucleotide sequence set forth in SEQ ID NO: 100.

[0136] In some embodiments, an expression cassette of the disclosure is integrated into a genome of a virus, such as an oncolytic HSV, e.g., an HSV-1 or HSV-2. In some embodiments, the cassette is integrated into one or two of the native γ34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV-2. In some embodiments, one or two of the native γ34.5 loci of an oncolytic HSV, e.g., an HSV-1 or HSV-2, are rendered inactive by insertion of the expression cassette. In some embodiments, integration of the expression cassette into a γ34.5 locus comprises replacing all or a part of the native γ34.5 locus with the expression cassette. In some embodiments, the TAP inhibitor encoded by the expression cassette is expressed as an immediate-early gene during viral replication.

[0137] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the selfcleaving peptide (such as a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above. In some embodiments, the expression cassette is integrated in (e.g., replaces) the native γ34.5 locus within the long terminal repeat (TRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome. In some embodiments, the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of (i)-(ii)-(iii)-UL.

[0138] In some embodiments, the expression cassette comprises: (i) the polynucleotide encoding the TAP inhibitor (e.g., a UL49.5 protein), (ii) the polynucleotide encoding the selfcleaving peptide (e.g., a P2A peptide), and (iii) the polynucleotide encoding the FLT3L, e.g., as described above. In some embodiments, the expression cassette is integrated in (e.g., replaces) the native γ34.5 locus within the internal long repeat (IRL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome. In some embodiments, the expression cassette is in the orientation relative to the unique long (UL) region of the genome, e.g. an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, of UL-(iii)-(ii)-(i).

Oncolytic viruses, genomes, vectors and cells comprising one or more expression cassettes [0139] Also provided herein is an oncolytic virus (e.g., an oncolytic HSV, such as an oncolytic HSV-1 or oncolytic HSV-2) comprising one or more of the expression cassettes described above (e.g., in Sections FV-A and/or IV-B). In some embodiments, the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section FV-A. In some embodiments, the oncolytic virus comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, the oncolytic virus comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, an oncolytic virus of the disclosure exhibits increased T cell activation relative to an oncolytic virus lacking any one, any two, or all of the polynucleotides encoding the IL- 12 protein, the CD40 agonist, and the CTLA-4 binding protein. T cell activation may be assessed using any suitable method known in the art, such as using an in vitro IL-2 secretion assay. In some embodiments, an oncolytic virus of the disclosure has increased abscopal efficacy relative to an oncolytic virus lacking any one, any two, or any three of the FLT3L, the IL- 12, the CD40 agonist, and the CTLA-4 binding protein. Abscopal efficacy may be assessed using any suitable method known in the art, such as using an in vivo tumor or cancer animal model, herein. In some embodiments, an oncolytic virus of the disclosure is capable of evading an individual’s immune system. In some embodiments, an oncolytic virus of the disclosure reduces or impairs viral antigen loading onto histocompatibility complex (MHC) Class I molecules for display at the cell surface, thereby reducing adaptive immune responses to the virus.

[0140] Also provided herein, is a modified HSV genome (e.g., an HSV-1 or HSV-2 genome) comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B). In some embodiments, the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A. In some embodiments, the modified HSV genome comprises one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B. In some embodiments, the modified HSV genome comprises: (a) one or more expression cassettes comprising a polynucleotide encoding IL- 12, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above in Section IV-A; and (b) one or more expression cassettes comprising a polynucleotide encoding FLT3L and/or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, e.g., as described above in Section IV-B.

[0141] Also provided herein, is a vector comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B). Suitable vectors include, without limitation, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self -replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR.322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector may be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno- associated viruses, HSV viruses, e.g. HSV-1 or HSV-2, retroviruses, and cosmids. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually included, such as ribosome binding sites, translation initiation sites, and stop codons.

[0142] In some embodiments, cells, such as host cells, comprising one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B) are also provided. In some embodiments, the cell is an isolated cell. An isolated cell is a cell that is identified and separated from at least one contaminant cell with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated cell is free of association with all components associated with the production environment. The isolated cell is in a form other than in the form or setting in which it is found in nature. Isolated cells are distinguished from cells existing naturally in tissues, organs, or individuals. JP-OV-2

[0143] JP-OV-2 is a next-generation recombinant HSV Type-l-based oncolytic virus (OV) that has been modified to 1) reduce innate and adaptive antiviral host responses that shut down viral replication to allow greater lysis of target tumor cells and 2) to enhance all stages of the adaptive immune response to the cancer cells through expression of multiple synergistic immune stimulatory payloads.

[0144] In some embodiments, JP-OV-2 comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US 10- 12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHVl promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12.

[0145] In some embodiments, JP-OV-2 comprises a polynucleotide for hFLT3L encoding the amino acid sequence set forth in SEQ ID NO:71.

[0146] In some embodiments, JP-OV-2 comprises a polynucleotide for UL49.5 encoding the amino acid sequence set forth in SEQ ID NO: 82.

[0147] In some embodiments, JP-OV-2 comprises a polynucleotide for IL- 12 encoding the amino acid sequence set forth in SEQ ID NO: 4.

[0148] In some embodiments, JP-OV-2 comprises a polynucleotide for CD40 agonist encoding the amino acid sequence set forth in SEQ ID NO: 25, [0149] In some embodiments, JP-OV-2 comprises a polynucleotide for CTLA-4 binding protein encoding the amino acid sequence set forth in SEQ ID NO: 50.

[0150] In some embodiments, JP-OV-2 comprises a polynucleotide for variant US 11 gene comprising the polynucleotide sequence set forth in SEQ ID NO: 204.

[0151] In some embodiments, JP-OV-2 comprises an additional polynucleotide encoding for US11 encoding the amino acid sequence set forth in SEQ ID NO: 80.

[0152] In some embodiments, JP-OV-2 comprises a polynucleotide for US 10 encoding the amino acid sequence set forth in SEQ ID NOVO.

Methods of making oncolytic viruses

[0153] The oncolytic viruses (such as the oncolytic HSV) described herein may be prepared using any methods known in the art or as described herein. In some embodiments, the oncolytic virus (such as the oncolytic HSV) may be engineered (such as to comprise one or more of the expression cassettes described herein and/or to express one or more of the payload proteins described herein) by modifying a wild-type virus (such as a wild-type HSV-1) genome.

Transgenes and/or expression cassettes, including as otherwise described herein, may be inserted in the native genome or replace native portions of the genome using recombinant cloning techniques well known in the art. Exemplary engineering methods are described herein at Examples 4-7. Engineered oncolytic virus genomes may be propagated in suitable cells and collected from cell media or collected from cell lysates. The virus-containing cell media or viruscontaining cell lysates may then be sterilized, such as by filtration or other suitable means. The virus may be concentrated, such as by ultracentrifugation. Purified virus may be stored by suitable means, including by storage at about -80 °C in DMEM. Titers of virus stocks vary by orders of magnitude, depending upon the viral genotype and the protocol used to prepare and purify them. Purified virus may be titered using assays well known in the art. Viral titer may be expressed in terms of infectious viral units, such as plaque-forming units (pfu). The integrity and sequence of the viral genome may be assessed by techniques well known in the art, including whole-genome sequencing.

METHODS OF TREATING A SOLID TUMOR [0154] Provided herein are methods of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus; and b) administering a therapeutically effective amount of an immunomodulator. In some embodiments, the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, Seneca valley virus, coxsackie virus, reo virus, vesicular stomatitis virus, maraba and rhabdovirus, and parvovirus. In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulator is an immune-stimulating agent. In some embodiments, the immunomodulator is administered systemically. In some embodiments, the oncolytic virus is locally administered directly into the tumor.

[0155] Another aspect provides a method of treating a solid tumor in an individual, comprising: a) administering to the site of the tumor an effective amount of an oncolytic virus; and b) systemically (such as intravenously) administering an effective amount of an immunomodulator. The embodiments described herein as being applicable to local administration of the oncolytic virus are also applicable to the method comprising systemic administration of the oncolytic virus.

[0156] Exemplary viruses that are suitable for use as the oncolytic virus in the present invention include, but are not limited to, herpes simplex virus, for example, JP-OV-2, Talimogene laherparapvec (T-VEC®) and HSV-1716 (SEPREHVIR®); adenovirus, for example, H101 (ONCOCRINE®), CG-TG-102 (Ad5/3-D24-GM-CSF), and CG0070; reo virus, for example, REOLYSIN®; vaccinia virus, for example, JX-594; Seneca valley virus, for example, NTX-010 and SW-001; Newcastle disease virus, for example, NDV-NS1 and GL-ONC1; polio virus, for example, PVS-RIPO; measles virus, for example, MV-NIS; coxsackie virus, for example, CAVATAK™; vesicular stomatitis virus; maraba and rhabdoviruses; parvovirus and mumps virus. In some embodiments, the oncolytic virus is oncolytic herpes simplex virus. In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus is only a part, or parts of the wild type oncolytic virus that can cause infection, inflammation or infection-like effects. In some embodiments, the virus is replication competent. In some embodiments, the virus replicates preferentially in a tumor cell. In some embodiments, the oncolytic virus preferentially replicates in a cancer cell.

[0157] In some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1); and b) administering a therapeutically effective amount of an immunomodulator. In some embodiments, the oncolytic virus is replication competent. In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulator is an immune-stimulating agent. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the immunomodulator is administered intravenously.

[0158] In some embodiments, the oncolytic virus and the immunomodulator discussed above are administered sequentially, i.e., the administration of the oncolytic virus is administered before or after the administration of the immunomodulator. In some embodiments, the oncolytic virus is administered prior to the administration of the immunomodulator. In some embodiments, the oncolytic virus is administered after the administration of the immunomodulator.

[0159] In some embodiments, the oncolytic virus and the immunomodulator are administered simultaneously. In some embodiments, the oncolytic virus and the immunomodulator are administered simultaneously via separate compositions.

[0160] The immunomodulators discussed herein include both immune-stimulating agents and immune checkpoint inhibitors.

[0161] In some embodiments, the immunomodulator is an immune-stimulating agent. In some embodiments, the immune-stimulating agent is a natural or engineered ligand of an immune stimulatory molecule, including, for example, ligands of 0X40 (e.g., OX40L), ligands of 4-1BB (e.g., 4-1BBL, Ultra4- 1BBL), and ligands of CD40 (e.g., CD40L). In some embodiments, the immune-stimulating agent is an antibody selected from the group consisting of anti-OX40 (e.g., MEDI6469, MEDI-0562), anti-GITR (e.g., TRX518, INBRX- 110, NOV- 120301), anti-4-lBB (e.g., BMS-663513, PF-05082566), and anti-CD40 (e.g., CP870,893, BI- 655064, BMS-986090, APX005, APX005M). In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof. [0162] In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an inhibitory immune checkpoint protein. In some embodiments, the immunomodulator is an antibody selected from the group consisting of anti-CTLA-4 (e.g., Ipilimumab, Tremelimumab, KAHR-102, anti- PD-1 (e.g., Cetrelimab, Nivolumab, Pidilizumab, Pembrolizumab, BMS- 936559, Lambrolizumab, MK-3475, AMP-224, AMP-514, STI-Al l 10, TSR-042), and anti-PD- L1 (e.g., KY- 1003 (EP20120194977), MCLA-145, atezohzumab, BMS-936559, durvalumab, MSB0010718C, AUR-012, STT-A1010, PCT/US2001/020964, MPDL3280A, AMP-224, Dapirolizumab pegol (CDP-7657), MEDI-4920). In some embodiments, the antibody is an antagonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof. [0163] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulator is an immune-stimulating agent. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the immunomodulator is administered intravenously. [0164] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the inhibitor of PD-1 is an anti -PD-1 antibody, including, but not limited to, Cetrelimab, Nivolumab, pembrolizumab, pidilizumab, BMS-936559, Lambrolizumab, MK- 3475, AMP- 224, AMP-514, STI-Al l 10, and TSR-042. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-PD- 1 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full-length anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a human, humanized, or chimeric antibody. In some embodiments, the anti-PD-1 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variants or derivatives thereof. In some embodiments, the inhibitor of PD-1 is a natural or engineered ligand of PD-1, such as PD-L1 or PD-L2. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of PD-1/PD-L1 interaction or an inhibitor of PD-1/PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an inhibitor of a PD-1 ligand, such as an inhibitor of PD-L1 (e.g., anti-PD-L1 antibody) or an inhibitor of PD-L2 (e.g., anti-PD-L2 antibody). Any of the inhibitors of interaction between PD- 1 and its ligand may be used in the present invention, see, for example, US20190225689A1, US7709214, US7432059, US7722868, US8217149, US8383796, and US9102725. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224).

[0165] Thus, for example, in some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, for example, Cetrelimab, Nivolumab, Pembrolizumab, or Pidilizumab, or an Fc fusion protein of a PD-1 ligand, for example, AMP-224). In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, for example, Cetrelimab, Nivolumab, Pembrolizumab, or Pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of PD-1/PD-L1 interaction or an inhibitor of PD-1/PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the inhibitor of PD- 1 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered sequentially. In some embodiments, the oncolytic virus is administered prior to (such as immediately prior to) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered simultaneously.

[0166] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, for example, Cetrelimab, Nivolumab, Pembrolizumab, or Pidilizumab, or an Fc fusion protein of a PD-1 ligand, for example, AMP-224). In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, for example, Cetrelimab, Nivolumab, Pembrolizumab, or Pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of PD-1/PD-L1 interaction or an inhibitor of PD-1/PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, the JP-OV-2 is administered directly into the tumor. In some embodiments, the inhibitor of PD-1 is administered systemically. In some embodiments, the JP-OV-2 and the inhibitor of PD-1 are administered sequentially. In some embodiments, the JP-OV-2 is administered prior to (such as immediately prior to) the administration of the inhibitor of PD-1. In some embodiments, the JP-OV-2 is administered after (such as immediately after) the administration of the inhibitor of PD-1. In some embodiments, the JP-OV-2 and the inhibitor of PD-1 are administered simultaneously.

[0167] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. Any of the anti- CTLA-4 antibodies that are known in the art may be used in the present disclosure, including, but not limited to, Ipilimumab, Tremelimumab, and KAHR-102. In some embodiments, the anti- CTLA-4 antibody is YERVOY® (Ipilimumab). In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-CTLA-4 antibody is an antigen- binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full length anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is a human, humanized, or chimeric antibody. In some embodiments, the anti-CTLA-4 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin protein specifically recognizing CTLA-4 (such as an anticalin molecule that specifically binds to CTLA-4). In some embodiments, the inhibitor of CTLA-4 is a natural or engineered ligand of CTLA-4, such as B7.1 or B7.2.

[0168] Thus, for example, in some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, for example Ipilimumab, or an engineered lipocalin protein, for example an anticalin that specifically recognizes CTLA-4. In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, for example Ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin protein, for example an anticalin that specifically recognizes CTLA-4. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the inhibitor of CTLA-4 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the oncolytic virus is administered prior to (such as immediately prior to) the administration of the inhibitor of CTLA- 4. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of CTLA-4. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered simultaneously

[0169] In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, comprising: a) administering a therapeutically effective amount of JP- OV-2; and b) administering a therapeutically effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, for example Ipilimumab, or an engineered lipocalin protein, for example an anticalin that specifically recognizes CTLA-4). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, for example Ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin protein, for example an anticalin that specifically recognizes CTLA-4. In some embodiments, the JP-OV-2 is administered directly into the tumor. In some embodiments, the inhibitor of CTLA-4 is administered intravenously. In some embodiments, the JP-OV-2 and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the JP-OV-2 is administered prior to (such as immediately prior to) the administration of the inhibitor of CTLA-4. In some embodiments, the JP-OV-2 is administered after (such as immediately after) the administration of the inhibitor of CTLA-4. In some embodiments, the JP-OV-2 and the inhibitor of CTLA-4 are administered simultaneously.

[0170] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 ligand (e.g., PD-L1 and/or PD-L2). In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L1 antibody. In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L2 antibody. Exemplary anti-PD-L1 antibodies include, but are not limited to, KY-1003, MCLA-145, RG7446 (also known as atezolizumab), BMS935559 (also known as MDX-1105), MPDL3280A, MEDI4736, Avelumab (also known as MSB0010718C), and SU-A1010. In some embodiments, the anti- PD-L1 or anti- PD-L2 is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-PD-L1 or anti-PD-L2 is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full-length anti-PD-L1 or anti-PD-L2 antibody. In some embodiments, the anti-PD-L1 or anti- PD-L2 antibody is a human, humanized, or chimeric antibody. In some embodiments, the anti- PD-L1 or anti-PD-L2 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variants or derivatives thereof. In some embodiments, the inhibitor of PD-1 ligand is an inhibitor (e.g., peptide, protein or small molecule) of both PD-L1 and PD-L2. Exemplary inhibitors of both PD-L1 and PD-L2 include, but are not limited to, AUR-012, and AMP-224. In some embodiments, the inhibitor of PD-L1 and the inhibitor of PD-L2 can be used interchangeably in any of the methods of treatment described herein.

[0171] In some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an inhibitor of PD-1 ligand (such as an anti- PD-L1 or anti-PD- L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L1 antibody, for example, KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, MEDI4736, Avelumab, or STI- A1010. In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of PD-1 ligand is an inhibitor (e.g., peptide, protein or small molecule) of both PD-L1 and PD-L2, such as AUR-012, and AMP-224. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the inhibitor of PD-1 ligand is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of PD-1 ligand are administered sequentially. In some embodiments, the oncolytic virus is administered prior to (such as immediately prior to) the administration of the inhibitor of PD-1 ligand. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of PD-1 ligand. In some embodiments, the oncolytic virus and the inhibitor of PD-1 ligand are administered simultaneously.

[0172] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an inhibitor of PD- 1 ligand (such as an anti- PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2).

[0173] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an inhibitor of PD-1 ligand (such as an anti- PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L1 antibody, for example, KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, durvalumab, Avelumab, or STLA1010. In some embodiments, the inhibitor of PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of PD-1 ligand is an inhibitor (e.g., peptide, protein or small molecule) of both PD-L1 and PD-L2, such as AUR-012, and AMP-224. In some embodiments, the JP-OV-2 is administered directly into the tumor. In some embodiments, the inhibitor of PD- 1 ligand is administered intravenously. In some embodiments, the JP-OV-2 and the inhibitor of PD-1 ligand are administered sequentially. In some embodiments, the JP-OV-2 is administered prior to (such as immediately prior to) the administration of the inhibitor of PD-1 ligand. In some embodiments, the JP-OV-2 is administered after (such as immediately after) the administration of the inhibitor of PD- 1 ligand. In some embodiments, the JP-OV-2 and the inhibitor of PD-1 ligand are administered simultaneously.

[0174] In some embodiments, the immune-stimulating agent is an activator of CD40. In some embodiments, the activator of CD40 is an agonistic anti-CD40 antibody. Any of the known anti- CD40 antibodies may be used in the present invention, including, but not limited to, CP-870,893, Dacetuzumab (also known as SGN-40), ChiLob 7/4, APX005, and APX005M, BI-655064, and BMS-986090. In some embodiments, the agonistic anti-CD40 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the agonistic anti-CD40 antibody is an antigenbinding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of the full-length anti-CD40 antibody. In some embodiments, the agonistic anti-CD40 antibody is a human, humanized, or chimeric antibody. In some embodiments, the agonistic anti-CD40 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variants or derivatives thereof. In some embodiments, the activator of CD40 is a natural or engineered CD40 ligand, such as CD40L. In some embodiments, the activator of CD40 is an inhibitor of the interaction between CD40 and CD40L. In some embodiments, the activator of CD40 increases the signaling of CD40.

[0175] Thus, for example, in some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an activator of CD40 (such as an agnostic anti- CD40 antibody, for example, CP-870,893, Dacetuzumab, ChiLob 7/4 or APX005M). In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the activator of CD40 is an agnostic anti-CD40 antibody, for example, CP-870,893, Dacetuzumab, ChiLob 7/4 or APX005M. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the activator of CD40 is administered intravenously. In some embodiments, the oncolytic virus and the activator of CD40 are administered sequentially. In some embodiments, the oncolytic virus is administered prior to (such as immediately prior to) the administration of the activator of CD40. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the activator of CD40. In some embodiments, the oncolytic virus and the activator of CD40 are administered simultaneously.

[0176] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an activator of CD40 (such as an agnostic anti-CD40 antibody, for example, CP-870,893, Dacetuzumab, ChiLob 7/4 or APX005M). [0177] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an activator of CD40 (such as an agnostic anti-CD40 antibody, for example, CP-870,893, Dacetuzumab, ChiLob 7/4 or APX005M). In some embodiments, the activator of CD40 is an agnostic anti-CD40 antibody, for example, CP- 870,893, Dacetuzumab, ChiLob 7/4 or APX005M. In some embodiments, the JP-OV-2 is administered directly into the tumor. In some embodiments, the activator of CD40 is administered intravenously. In some embodiments, the JP-OV-2 and the activator of CD40 are administered sequentially. In some embodiments, the JP-OV-2 is administered prior to (such as immediately prior to) the administration of the activator of CD40. In some embodiments, the JP- OV-2 is administered after (such as immediately after) the administration of the activator of CD40. In some embodiments, the JP-OV-2 and the activator of CD40 are administered simultaneously

[0178] In some embodiments, the immune-stimulating agent is an activator of 0X40. In some embodiments, the activator of 0X40 is an agonistic anti-OX40 antibody. Any of the known anti- 0X40 antibodies may be used in the present invention, including, but not limited to, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 and InVivoMAb clone OX-86. In some embodiments, the agonistic anti-OX40 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the agonistic anti-OX40 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigenbinding subsequences of the full-length anti-OX40 antibody. In some embodiments, the agonistic anti-OX40 antibody is a human, humanized, or chimeric antibody. In some embodiments, the agonistic anti-OX40 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variants or derivatives thereof. In some embodiments, the activator of 0X40 is a natural or engineered 0X40 ligand, such as OX40L. In some embodiments, the activator of 0X40 is an inhibitor of the interaction between 0X40 and OX40L. Any of the inhibitors of interaction between 0X40 and OX40L may be used in the present invention, see, for example, U.S Patent No. US8283450, US11867621, US7547438, US7063845, US7537763 and US5801227. In some embodiments, the activator of 0X40 increases the signaling of 0X40.

[0179] Thus, for example, in some embodiments, there is provided a method of treating a solid tumor in an individual (such as a human), comprising: a) administering a therapeutically effective amount of an oncolytic virus (such as oncolytic herpes simplex virus type 1, for example, JP-OV-2); and b) administering a therapeutically effective amount of an activator of 0X40 (such as an agnostic anti- 0X40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), In some embodiments, the oncolytic virus is attenuated (for example through multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in a cancer cell. In some embodiments, the activator of 0X40 is an agnostic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the activator of 0X40 is administered intravenously. In some embodiments, the oncolytic virus and the activator of 0X40 are administered sequentially. In some embodiments, the oncolytic virus is administered prior to (such as immediately prior to) the administration of the activator of 0X40. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the activator of 0X40. In some embodiments, the oncolytic virus and the activator of 0X40 are administered simultaneously.

[0180] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of an JP-OV-2; and b) administering a therapeutically effective amount of an activator of 0X40 (such as an agnostic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86).

[0181] In some embodiments, there is provided a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of JP-OV-2; and b) administering a therapeutically effective amount of an activator of 0X40 (such as an agnostic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86). In some embodiments, the activator of 0X40 is an agnostic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, JP-OV-2 is administered directly into the tumor. In some embodiments, the JP-OV-2 and the activator of 0X40 are administered sequentially. In some embodiments, the JP-OV-2 is administered prior to (such as immediately prior to) the administration of the activator of 0X40. In some embodiments, the JP-OV-2 is administered after (such as immediately after) the administration of the activator of 0X40. In some embodiments, JP-OV-2 and the activator of 0X40 are administered simultaneously.

EXEMPLARY EMBODIMENTS

[0182] The present disclosure may be better understood with reference to the following exemplary embodiments.

[0183] Embodiment 1 is a method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of an oncolytic virus; and b) administering a therapeutically effective amount of an immunomodulator, wherein the oncolytic virus comprises one or more expression cassettes, comprising a polynucleotide encoding a hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding an IL- 12. [0184] Embodiment 2 is the method of embodiment 1, wherein the oncolytic virus preferentially replicates in a cancer cell.

[0185] Embodiment 3 is the method embodiment 1 or embodiment 2, wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, Seneca valley virus, coxsackie virus, reo virus, vesicular stomatitis virus, maraba and rhabdovirus, and parvovirus. [0186] Embodiment 4 is the method of any one of embodiments 1 to 3 wherein the one or more expression cassettes comprises a cassette integrated in one or both of the γ34.5 loci comprising, a polynucleotide encoding hFLT3L protein, and/or a polynucleotide encoding UL49.5.

[0187] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the polynucleotide encoding the hFLT3L encodes the amino acid sequence set forth in SEQ ID NO:71.

[0188] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein the polynucleotide encoding the UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82.

[0189] Embodiment 7 is the method of any one of embodiments 1 to 6, wherein the one or more expression cassettes further comprises a polynucleotide comprising a variant US11 gene.

[0190] Embodiment 8 is the method of any one of embodiments 1 to 7, wherein one of the expression cassettes is inserted at the native US10-US12 locus.

[0191] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the one or more expression cassettes comprises an expression cassette integrated in the US10-12 locus comprising a polynucleotide comprising a variant US11 gene, one or more polynucleotides encoding a native US11 protein, a polynucleotide encoding US 10 protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding IL- 12.

[0192] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the polynucleotide encoding the IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4. [0193] Embodiment 11 is the method of any one of embodiments 1 to 10, wherein the polynucleotide encoding the CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25.

[0194] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the polynucleotide encoding the CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50.

[0195] Embodiment 13 is the method of any one of embodiments 7 to 12, wherein the polynucleotide encoding the variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204. [0196] Embodiment 14 is the method of any one of embodiments 7 to 13, wherein the additional polynucleotide encoding the US11 encodes the amino acid sequence set forth in SEQ ID NO: 80. [0197] Embodiment 15 is the method of any one of embodiments 7 to 14, wherein the polynucleotide encoding the US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90. [0198] Embodiment 16 is the method of any one of embodiments 7 to 15, wherein one or both native γ34.5 genes are inactivated by deletion, substitution, or insertion in the backbone nucleic acid.

[0199] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein a native US 12 gene of the virus is inactivated by deletion, substitution, or insertion in the backbone nucleic acid.

[0200] Embodiment 18 is the method of embodiment 9, wherein the variant US11 gene is operably associated with an immediate-early promoter.

[0201] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein the oncolytic virus further comprises a native late US11 gene.

[0202] Embodiment 20 is the method of any one of embodiments 1 to 19 , wherein the oncolytic virus is an oncolytic herpes simplex type 1 (HSV-1) virus.

[0203] Embodiment 21 is the method of embodiment 20, wherein the oncolytic HSV-1 virus comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US10-12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV 1 promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12, wherein the polynucleotide for hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71, the polynucleotide for UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82, the polynucleotide for IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4 , the polynucleotide for CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25, and the polynucleotide for CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50, the polynucleotide for variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204, the additional polynucleotide encoding for US11 encodes the amino acid sequence set forth in SEQ ID NO: 80, and the polynucleotide for US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90.

[0204] Embodiment 22 is the method of embodiment 20 or embodiment 21, wherein the oncolytic HSV-1 virus is JP-OV-2.

[0205] Embodiment 23 is the method of any one of embodiments 1 to 22, wherein the oncolytic virus is locally administered to the site of the tumor.

[0206] Embodiment 24 is the method of any one of embodiments 1 to 23, wherein the oncolytic virus is administered directly into the tumor.

[0207] Embodiment 26 is the method of any one of embodiments 1 to 25, wherein the immunomodulator is administered systemically.

[0208] Embodiment 27 is the method of any one of embodiments 1 to 26, wherein the immunomodulator is administered intravenously.

[0209] Embodiment 28 is the method of any one of embodiments 1 to 27, wherein the oncolytic virus and the immunomodulator are administered sequentially.

[0210] Embodiment 29 is the method of any one of embodiments 1 to 28, wherein the oncolytic virus and theimmunomodulator are administered simultaneously.

[0211] Embodiment 30 is the method of any one of embodiments 1 to 29, wherein the immunomodulator is a modulator of an immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, PD-L1, PD-L2, and ligands thereof. [0212] Embodiment 31 is the method of any one of embodiments 1 to 30, wherein the immunomodulator is an antibody or an antigen-binding fragment thereof that binds specifically to PD-1.

[0213] Embodiment 32 is the method of embodiment 31, wherein the antibody that specifically binds to PD-1 comprises a VH comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 500, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 501, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 502; and/or a VL comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 503, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 504, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 505.

[0214] Embodiment 33 is the method of embodiment 34 or embodiment 35, wherein the antibody or an antigen-binding that binds specifically to PD-1 is cetrelimab.

[0215] Embodiment 34 is the method of any one of embodiments 1 to 29, wherein the immunomodulator is an immune- stimulating agent selected from the group consisting of 4- IBB, 0X40, and CD40.

[0216] Embodiment 35 is the method of any one of embodiments 1 to 34, wherein the solid tumor is lung cancer or other cancer susceptible to oncolytic virus intratumoral injection.

[0217] Embodiment 36 is the method of any one of embodiments 1 to 35, wherein the treatment triggers an abscopal effect.

[0218] Embodiment 37 is the method of any one of embodiments 1 to 36, wherein the treatment triggers immunological memory of the solid tumor.

EXAMPLES

[0219] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.

Example 1 : Oncolytic HSV-1 virus in combination with an anti-PDl antibody in an MC-38 5 AG Tumor Model.

[0220] This example describes an oncolytic HSV-1 virus in combination with an anti-PD-1 antibody. The genome architecture of the mouse surrogate HSV-1 virus, mJP-OV-2, and the human HSV-1 virus, JP-OV-2, are provided in FIGs. 1 A-1B. Briefly, the mJP-OV-2 virus was engineered to express several immunomodulatory payloads including an anti-CTLA-4 antagonist (maCTLA-4), a CD40 agonist (mCD40ag), IL- 12 (mscIL-12), and hFLT3L. The virus was also engineered to express and UL49.5, codon-optimized US11 (hCoUSl 1) using the US12 immediate early promoter, and endogenous US11 using late US11 promoter. The mouse surrogate HSV-1 virus mJP-OV-2 was engineered with 4 immunomodulatory payloads: hFLT3L, mCD40 ag, αmCTLA4, and mscIL12.8 known to activate all stages of adaptive immunity. hFLT3L was retained in mJP-OV-2, as this pay load is cross-reactive between mouse and human.

[0221] FIG. 2A depicts a diagram of the experimental workflow to assess the treatment with an oncolytic HSV-1 virus in combination with immune checkpoint therapies. The general experimental design was to implant tumor cells bilaterally, followed by intratumoral treatment of established tumors on the right flank. Treated and untreated tumors were monitored to assess if the treatment inhibited growth of the primary tumor as well as an abscopal effect on growth of the secondary untreated tumor, which would indicate that the mice had developed an adaptive immune response to tumor antigens.

[0222] Female C57BL/6 mice were obtained from Charles River Laboratories and were enrolled when they were approximately 8 weeks of age with an average body weight of 18 to 20 g. The mouse syngeneic cancer cell line, MC-38 5 AG, was obtained from Janssen R&D, Spring House, PA, and was grown in complete culture medium. MC-38 5 AG cells were harvested during exponential growth on Day 0, using TrypLE. Cells were washed twice in cold DPBS and resuspended in cold DPBS at a concentration of 5x106 cells/mL. Mice were implanted bilaterally in each flank by SC injection with 0.1 mL of the cell suspension (ie, 5xl0⁵ cells in each flank). In all studies, Day 0 was the day of tumor cell implantation and study initiation. Cells were implanted SC in both the left and right flanks, just below the ribcage. Mice were randomized by the Multi Task method in Studylog software (Studylog Systems, Version 4.3) according to tumor volume (TV), using the right side as the primary tumor, followed by the left side as the secondary tumor, such that the p value was as close to 1 as possible and the percent difference and standard deviations were similar among the groups within each cohort. All intratumoral injections were in 0.05 mL and were performed on the right tumor. The vehicle control group was dosed with formulation buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 130 mM NaCl, 10% sucrose, pH 7.4) in each study.

[0223] Mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP-OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti-PDl antibody RMP1 14 according to the study design.

[0224] At study end, as determined by twice the median survival day of the vehicle control group, naive mice (n=10) were challenged, and any cured mice with CRs were rechallenged with 5× 10⁵ MC-38 5 AG cells implanted in the left flank. All mice were monitored until a median survival day was assessed from the naive group, at which point a study end date was determined for the rechallenge. A summary of the study design described above is provided in Table 1. [0225] Significant antitumor effect was observed for the tumors treated with the oncolytic HSV- 1 virus with or without anti-PDl antibody RMP1 14, or with anti-PDl alone, as compared to vehicle control mice over time to Day 29 post tumor implantation (p≤0.001; FIG. 2B). In addition, a significant abscopal effect on the untreated tumors was observed for mice treated with the oncolytic HSV-1 virus alone or in combination with anti-PDl (p≤0.001). Survival analysis of these groups demonstrated that the oncolytic HSV-1 virus, with or without anti-PDl, significantly enhanced overall survival (p≤0.001; FIG. 2C). On Day 61, complete responses (CRs) were observed for 5 of 10 mice treated with the oncolytic HSV-1 virus and 10 of 10 mice treated with the oncolytic HSV-1 virus + anti-PDl. Anti-PDl also significantly increased survival (p≤0.001), but no CRs were observed in this group on Day 61. These results indicate that the combination of the oncolytic HSV-1 virus + anti-PDl was more effective than the single agents to inhibit tumor growth and prolong survival.

[0226] These results suggest that activity of the anti-PDl therapy to block the interaction of PD1 and programmed death-ligand 1 (PDL1), enhances the antitumor activity of the oncolytic HSV-1 virus by abolishing the inhibition of CD8+ T cells. Further, the combination of the oncolytic HSV-1 virus and PD1 antibody was effective for treating solid tumors and that the two agents act synergistically to provide a stronger anti-tumor effect than either agent individually. Further, the tumor reduction observed on the untreated tumor demonstrate that treatment with the oncolytic HSV-1 virus and a PD1 antibody resulted in a significant abscopal effect.

Example 2: Oncolytic HSV-1 virus in combination with an anti-CTLA4 antibody in an MC-38 5AG Tumor Model.

[0227] This example describes the effect of treatment with an oncolytic HSV-1 virus in combination with an anti-CTLA4 antibody. This example follows the same experimental design described in Example 1 and depicted in FIG. 2A.

[0228] Briefly, mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP-OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti- CTLA4 antibody 9D9 according to the study design.

[0229] At study end, as determined by twice the median survival day of the vehicle control group, naive mice (n=10) were challenged, and any cured mice with CRs were rechallenged with 5× 10⁵ MC-38 5 AG cells implanted in the left flank. All mice were monitored until a median survival day was assessed from the naive group, at which point a study end date was determined for the rechallenge. A summary of the study design described above is provided in Table 1.

[0230] Significant antitumor activity was observed for the tumors treated with the oncolytic HSV-1 virus alone or in combination with anti-CTLA4 antibody 9D9, or with anti-CTLA4 alone, as compared to vehicle control mice on Day 29 post tumor implantation (p≤0.001; FIG. 2D). In addition, each treatment induced a significant abscopal effect on the untreated tumors (p≤0.001). Survival analysis of this study demonstrated that the oncolytic HSV-1 virus with and without anti-CTLA4, or anti-CTLA4 alone, significantly enhanced overall survival (p≤0.001; FIG. 2E). On Day 61, CRs were observed for 5 of 10 mice treated with the oncolytic HSV-1 virus, 10 of 10 mice treated with mJP-OV-2 + anti-CTLA4, and 3 of 10 mice treated with anti- CTLA4. These results indicate that the combination of the oncolytic HSV-1 virus + anti-CTLA4 was more effective than the single agents to inhibit tumor growth and prolong survival.

[0231] These results suggest that the combination of the oncolytic HSV-1 virus and a CTLA4 antibody is effective for treating solid tumors and that the two agents act synergistically to provide a stronger anti-tumor effect than either agent individually. Further, the tumor reduction observed on the untreated tumor demonstrate that treatment with the oncolytic HSV-1 virus and a CTLA4 antibody resulted in a significant abscopal effect.

Example 3: Oncolytic HSV-1 virus in combination with an anti-OX40 antibody in an MC-38 5AG Tumor Model.

[0232] This example describes treatment with an oncolytic HSV-1 virus in combination with an anti-OX40 antibody. This example follows the same experimental design described in Example 1 and depicted in FIG. 2A.

[0233] Briefly, mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP-OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti- 0X40 antibody OX-86 according to the study design.

[0234] At study end, as determined by twice the median survival day of the vehicle control group, survival analysis was assessed. A summary of the study design described above is provided in Table 2.

MC-38

Table 2: Study design to test effect of an oncolytic HSV-1 virus and anti-OX40, anti-4- IBB, and anti-GITR antibody therapy in MC-38 5 AG Tumor Model.

[0235] Significant antitumor activity was observed for the tumors treated with the oncolytic HSV-1 virus alone or in combination with anti-OX40 antibody OX-86, as compared to vehicle control mice on Day 29 post tumor implantation (p≤0.001; FIG. 2F). In addition, each treatment induced a significant abscopal effect on the untreated tumors (p≤0.05 and p≤0.001, respectively). Survival analysis of this study demonstrated that the oncolytic HSV-1 virus with and without anti-OX40, significantly enhanced overall survival (p≤0.001; FIG. 2G). On Day 64, CRs were observed for 2 of 10 mice treated with the oncolytic HSV-1 virus and 5 of 10 mice treated with mJP-OV-2 + anti-OX40. These results indicate that the combination of the oncolytic HSV-1 virus + anti-OX40 was more effective than the single agents to inhibit tumor growth and prolong survival.

[0236] These results demonstrate that the combination of the oncolytic HSV-1 virus and an 0X40 antibody is effective for treating solid tumors and that the two agents act synergistically to provide a stronger anti-tumor effect than either agent individually. Further, the tumor reduction observed on the untreated tumor demonstrate that treatment with the oncolytic HSV-1 virus and an 0X40 antibody resulted in a significant abscopal effect.

Example 4: Oncolytic HSV-1 virus in combination with an anti-4-lBB antibody in an MC-38 5AG Tumor Model.

[0237] This example describes treatment with an oncolytic HSV-1 virus in combination with an anti-4-lBB antibody. This example follows the same experimental design described in Example 1 and depicted in FIG. 2A. [0238] Briefly, mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP-OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti-4- 1BB antibody LOB 12.3 according to the study design.

[0239] At study end, as determined by twice the median survival day of the vehicle control group, survival analysis was assessed. A summary of the study design described above is provided in Table 2. MC-38

[0240] Significant antitumor activity was observed for the tumors treated with the oncolytic HSV-1 virus alone or in combination with anti-4- IBB antibody LOB 12.3, or with anti-4- IBB alone, as compared to vehicle control mice on Day 29 post tumor implantation (p≤0.001; FIG. 2H). In addition, each treatment induced a significant abscopal effect on the untreated tumors (p≤0.05, p≤0.001 and p≤0.05, respectively). Survival analysis of this study demonstrated that the oncolytic HSV-1 virus with and without anti-4-lBB, significantly enhanced overall survival (p≤0.001; FIG. 21). On Day 64, CRs were observed for 2 of 10 mice treated with the oncolytic HSV-1 virus, 7 of 10 mice treated with mJP-OV-2 + anti-4-lBB, and 2 of 10 mice treated with anti-4-lBB. These results indicate that the combination of the oncolytic HSV-1 virus + anti-4- 1BB was more effective than the single agents to inhibit tumor growth and prolong survival.

Example 5: Oncolytic HSV-1 virus in combination with an anti-GITR antibody in an MC-38 5AG Tumor Model.

[0241] This example describes treatment with an oncolytic HSV-1 virus in combination with an anti-GITR antibody. This example follows the same experimental design described in Example 1 and depicted in FIG. 2A.

[0242] Briefly, mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to tumor volume (TV), with mean right and left TV of 26 mm3 and 24 mm3, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or mJP-OV-2. On Days 8, 12, 15, and 19, mice received intraperitoneal (IP) injections of anti-GITR antibody DTA-1 according to the study design. [0243] At study end, as determined by twice the median survival day of the vehicle control group, survival analysis was assessed. A summary of the study design described above is provided in Table 2.

[0244] Significant antitumor activity was observed for the tumors treated with the oncolytic HSV-1 virus alone or in combination with anti-GITR antibody DTA-1, or with anti-GITR alone, as compared to vehicle control mice on Day 29 post tumor implantation (p≤0.001, p≤0.001 and p≤0.01; FIG. 2J). In addition, each treatment induced a significant abscopal effect on the untreated tumors (p≤0.05, p≤0.001 and p≤0.05, respectively, respectively). Survival analysis of this study demonstrated that the oncolytic HSV-1 virus with and without anti- GITR, significantly enhanced overall survival (p<0.001; FIG. 2K). On Day 64, CRs were observed for 2 of 10 mice treated with the oncolytic HSV-1 virus and 6 of 10 mice treated with mJP-OV-2 + anti- GITR. These results indicate that the combination of the oncolytic HSV-1 virus + anti- GITR was more effective than the single agents to inhibit tumor growth and prolong survival.

Example 6: Rechallenge of Cured Mice After Treatment with mJP-OV-2, Alone and Combined with Immunomodulatory agents

[0245] Several mice exhibited CRs after treatment with the oncolytic HSV-1 virus + anti-PDl antibody, or with the oncolytic HSV-1 virus and/or anti-CTLA4 antibody (FIGs. 2B-2E). To determine if the mice developed a systemic antitumor memory immune response, a rechallenge study was conducted in which the cured mice were injected in the left flank with the same number of MC-38 5 AG tumor cells as originally injected (FIG. 3 A). As a control, naive mice were challenged with single tumors of MC-38 5 AG (n=10), and most mice in this group grew tumors that reached the tumor burden criteria for sacrifice. Cured mice from the previously treated groups were completely protected from challenge with MC-38 5 AG cells up to Day 52 post rechallenge, or overall Day 113 of the study. This result demonstrated that treatment with the oncolytic HSV-1 virus, alone or in combination with immunomodulatory agents, produced a durable, specific, and protective antitumor response.

Example 7: Effect of Treatment with Different Dose Levels of mJP-OV-2 + Anti-PDl Antibody on MC-38 5 AG Tumor Growth and Mouse Survival. [0246] This example tests the effect of treatment with different dose levels of the oncolytic HSV-1 virus in combination with anti-PDl antibody treatment. This example uses a similar experimental design as described in FIG. 2A. Briefly, female C57BL/6 mice were randomized into groups of 10 animals each, 7 days post MC-38 5 AG cell implantation according to TV, with mean right and left TV of 32 mm³ and 31 mm³, respectively. On Days 8, 11, and 14, mice received intratumoral injections of vehicle or the oncolytic HSV-1 virus at different doses in 0.05 mL, in the right tumor. On Days 8, 12, 15, and 19, mice were treated IP with the anti-PDl antibody RMP1 14 at a fixed dose of 200 pg. A summary of the study design is shown below (Table 3).

[0247] For the mice treated with HSV-1 virus with anti-PDl antibody, significant TGI was observed upon treatment at all dose levels tested (p<0.001; FIG. 4C). A significant abscopal effect was demonstrated for the untreated tumors in all dose levels of oncolytic HSV-1 virus groups + anti PD1 (either p<0.001 or p<0.01), while anti-PDl alone did not induce significant abscopal effects. Survival analysis of these groups demonstrated significantly increased survival over the control group, and the extent of survival correlated with the dose of virus (FIG. 4D). In addition, the group treated with the high dose of oncolytic HSV-1 virus + anti-PDl exhibited 6 CRs, 2 PRs, and a median lifespan not reached on Day 64, providing further evidence of a prolonged abscopal effect. A dose-response was also observed in the medium- and low-dose combination groups, with 4 CRs / 1 PR, and 1 CR / 1 PR, respectively, and prolonged lifespan (Table 4). An abscopal effect was observed at all doses tested in combination with anti-PDl, with a maximal effect at the highest dose tested of 5x106 pfu (1 x108 pfu/mL). [0248] For the mice treated with the oncolytic HSV-1 virus alone, significant TGI was observed upon treatment at all dose levels tested (FIG. 4A). A significant abscopal effect was demonstrated for the untreated tumors in the high-dose oncolytic HSV-1 virus group, but not for the groups treated with the oncolytic HSV-1 virus alone at the lower doses. Survival analysis of these groups demonstrated a dose-response for mice treated with the oncolytic HSV-1 virus alone, with the high-dose group exhibiting the greatest enhancement of survival compared with the control group, and 1 PR on Day 64 (FIG. 4B, Table 4). The groups treated with lower doses of the oncolytic HSV-1 virus exhibited less survival improvement and no CRs or PRs by Day 64. The minimum efficacious dose for a significant abscopal effect for the oncolytic HSV-1 virus alone was determined to the high dose of 5×10⁶ pfu (1 ×10⁸ pfu/mL).

Conclusions

[0249] An oncolytic HSV-1 virus, administered with systemic immune checkpoint therapies comprising anti-PDl, anti-CTLA4, anti- 0X40, anti-4-lBB, or anti-GITR antibodies, led to improved activity of injected and abscopal tumors compared with the oncolytic HSV-1 virus alone. Cured mice remained protected upon rechallenge. Taken together, these results demonstrated that treatment with the oncolytic HSV-1 virus caused direct and abscopal TGI that resulted in prolonged survival, and sustained immune protection upon tumor rechallenge. SEQUENCES

Table 6: CD40 Agonist Payload Amino Acid Sequences.

Table 7: CTLA-4 Antagonist Pay load Amino Acid Sequences.

Table 8: Human FLT3L Payload Amino Acid Sequences.

Table 9: Immune Stealth Protein Amino Acid Sequences.

Table 12: US10-12 Locus Cassete Sequences.

Table 13: Additional Nucleotide Sequences.

Claims

What is claimed is:

1. A method of treating a solid tumor in an individual, comprising: a) administering a therapeutically effective amount of an oncolytic virus; and b) administering a therapeutically effective amount of an immunomodulator, wherein the oncolytic virus comprises one or more expression cassettes, comprising a polynucleotide encoding a hFLT3L protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding an IL- 12.

2. The method of claim 1, wherein the oncolytic virus preferentially replicates in a cancer cell.

3. The method claim 1 or claim 2, wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, Seneca valley virus, coxsackie virus, reo virus, vesicular stomatitis virus, maraba and rhabdovirus, and parvovirus.

4. The method of any one of claims 1 to 3 wherein the one or more expression cassettes comprises a cassette integrated in one or both of the γ34.5 loci comprising, a polynucleotide encoding hFLT3L protein, and/or a polynucleotide encoding UL49.5.

5. The method of any one of claims 1 to 4, wherein the polynucleotide encoding the hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71.

6. The method of any one of claims 1 to 5, wherein the polynucleotide encoding the UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82.

7. The method of any one of claims 1 to 6, wherein the one or more expression cassettes further comprises a polynucleotide comprising a variant US11 gene.

8. The method of any one of claims 1 to 7, wherein one of the expression cassettes is inserted at the native US10-US12 locus.

9. The method of any one of claims 1 to 8, wherein the one or more expression cassettes comprises an expression cassette integrated in the US 10- 12 locus comprising a polynucleotide comprising a variant US11 gene, one or more polynucleotides encoding a native US11 protein, a polynucleotide encoding US 10 protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and/or a polynucleotide encoding IL- 12.

10. The method of any one of claims 1 to 9, wherein the polynucleotide encoding the IL- 12 encodes the amino acid sequence set forth in SEQ ID NO: 4.

11. The method of any one of claims 1 to 10, wherein the polynucleotide encoding the CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25.

12. The method of any one of claims 1 to 11, wherein the polynucleotide encoding the CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50.

13. The method of any one of claims 7 to 12, wherein the polynucleotide encoding the variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204.

14. The method of any one of claims 7 to 13, wherein the additional polynucleotide encoding the US11 encodes the amino acid sequence set forth in SEQ ID NO: 80.

15. The method of any one of claims 7 to 14, wherein the polynucleotide encoding the US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90.

16. The method of any one of claims 7 to 15, wherein one or both native γ34.5 genes are inactivated by deletion, substitution, or insertion in the backbone nucleic acid.

17. The method of any one of claims 1 to 16, wherein a native US 12 gene of the virus is inactivated by deletion, substitution, or insertion in the backbone nucleic acid.

18. The method of claim 9, wherein the variant US11 gene is operably associated with an immediate-early promoter.

19. The method of any one of claims 1 to 18, wherein the oncolytic virus further comprises a native late US 11 gene.

20. The method of any one of claims 1 to 19 , wherein the oncolytic virus is an oncolytic herpes simplex type 1 (HSV-1) virus.

21. The method of claim 20, wherein the oncolytic HSV-1 virus comprises: a. a cassette integrated in one or both of the γ34.5 loci comprising in order, from upstream to downstream, a CMV promoter, a polynucleotide encoding hFLT3L, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. another cassette integrated in the US10-12 locus comprising in order, from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding native US11 protein, an additional polynucleotide encoding native US11 protein, a polynucleotide encoding US 10 protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the US 10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal that is operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal that is operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV 1 promoter that controls expression of the CD40 agonist, an MMLV promoter that controls expression of an IL- 12, a polynucleotide encoding an IL- 12, and a polyadenylation signal that is operably linked to the polynucleotide encoding the IL- 12, wherein the polynucleotide for hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71, the polynucleotide for UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82, the polynucleotide for IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4 , the polynucleotide for CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25, and the polynucleotide for CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50, the polynucleotide for variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204, the additional polynucleotide encoding for US11 encodes the amino acid sequence set forth in SEQ ID NO: 80, and the polynucleotide for US 10 encodes the amino acid sequence set forth in SEQ ID NO: 90.

22. The method of claim 20 or claim 21, wherein the oncolytic HSV-1 virus is JP-OV-2.

23. The method of any one of claims 1 to 22, wherein the oncolytic virus is locally administered to the site of the tumor.

24. The method of any one of claims 1 to 23, wherein the oncolytic virus is administered directly into the tumor.

25. The method of any one of claims 1 to 24, wherein the immunomodulator is administered systemically.

26. The method of any one of claims 1 to 25, wherein the immunomodulator is administered intravenously.

27. The method of any one of claims 1 to, wherein the oncolytic virus and the immunomodulator are administered sequentially.

28. The method of any one of claims 1 to 27, wherein the oncolytic virus and the immunomodulator are administered simultaneously.

29. The method of any one of claims 1 to 28, wherein the immunomodulator is a modulator of an immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, PD-L1, PD-L2, and ligands thereof.

30. The method of any one of claims 1 to 29, wherein the immunomodulator is an antibody or an antigen-binding fragment thereof that binds specifically to PD-1.

31. The method of claim 30, wherein the antibody that specifically binds to PD-1 comprises a VH comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 500, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 501, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 502; and/or a VL comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 503, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 504, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 505.

32. The method of claim 30 or claim 31, wherein the antibody or an antigen-binding that binds specifically to PD-1 is cetrelimab.

33. The method of any one of claims 1 to 28, wherein the immunomodulator is an immune- stimulating agent selected from the group consisting of 4- IBB, 0X40, and CD40.

34. The method of any one of claims 1 to 33, wherein the solid tumor is lung cancer or other cancer susceptible to oncolytic virus intratumoral injection.

35. The method of any one of claims 1 to 34, wherein the treatment triggers an abscopal effect.

36. The method of any one of claims 1 to 35, wherein the treatment triggers immunological memory of the solid tumor.