Oncolytic viruses and uses thereof

Abstract

Provided herein are oncolytic viruses containing payload genes, including genes encoding IL-12, FLT3L, CD40 agonists, and / or CTLA-4 antibodies. Expression cassettes, pharmaceutical compositions, and methods of treatment using these viruses are also provided. Additionally, expression cassettes and CD40 agonist molecules are also provided in the present disclosure.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Patent Application No. 63 / 433,781, filed December 20, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing, which has been submitted electronically via EFS-Web as an XML format Sequence Listing having a filename "JBI6709WOPCT1Seqlist.xml" with a creation date of December 18, 2023, and a size of 209 KB. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present invention relates to oncolytic viruses, including those derived from herpes simplex virus 1 (HSV-1), expression cassettes, and proteins expressed from the oncolytic viruses and / or expression cassettes. [Background technology]

[0004] Oncolytic virotherapy is a form of immunotherapy that utilizes the cytotoxicity and / or vector capabilities of viruses to selectively target and destroy tumor cells. Oncolytic virotherapy can also stimulate immune responses against the target tumor or tumors distal to the target tumor. Safety concerns have limited the use of live, infectious viruses in cancer patients, but the development of robust genetic engineering has matured this field by developing improved viruses. Kelly and Russell, "History of oncolytic viruses: genesis to genetic engineering," Mol. Ther. 2007 Apr;15(4):651-9. Oncolytic viruses can be selectively enhanced against tumor cells (e.g., by enhanced cytotoxicity in cancer cells and / or reduced cytotoxicity in normal cells) and engineered to express therapeutic payloads such as immunostimulatory proteins.

[0005] Herpes simplex virus (HSV) is a candidate for the emergence of additional oncolytic viruses. HSV has a broad host cell range in humans, a short replication cycle, a large genome suitable for multiple payload genes, and effective antiviral options for controlling infection. Sanchala et al., "Oncolytic Herpes Simplex Viral Therapy: A Strike toward Selective Targeting of Cancer Cells," Front. Pharmacol. 2017;8:270.

[0006] There is a need in the art for next generation oncolytic viruses, including those derived from HSV, that exhibit improved safety and improved efficacy.

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] Provided herein are oncolytic viruses comprising 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, and / or a polynucleotide encoding an FLT3 ligand. Also provided herein are expression cassettes comprising such polynucleotides. In some embodiments, provided herein are methods of treating cancer in an individual comprising administering to the individual an oncolytic virus provided herein. In some embodiments, the oncolytic viruses provided herein induce an abscopal response against distant tumors and / or induce immunological memory of the tumor. [Brief explanation of the drawings]

[0009] The present application can be understood by reference to the following description taken in conjunction with the accompanying drawings. [Figure 1A] The left panel shows the design of a viral genome mimicking a previously reported genetically engineered oncolytic herpesvirus with only a single copy of IE-US11, and the right panel shows the design of an engineered natural stealth virus with expression of both endogenous L-US11 and IE-US11. IRL, internal repeat long; IRS, internal repeat short; TRL, terminal repeat long; TRS, terminal repeat short; UL, unique long; US, unique short; β-Gluc, β-glucuronidase. The hexagonal S represents a stop codon. Co-US11 represents US11 codon-optimized as an IE gene. The star represents the promoter or transcription start site. The arrowhead originating from the star represents the RNA transcript. [Figure 1B]Figure 1 shows the results of Western blot experiments measuring US11 and phosphorylated (p-)eIF2α levels in A549 human lung cancer cells (MOI = 5) infected with wild-type (WT) HSV-1, Δγ34.5 HSV-1, a virus mimicking a previously reported genetically engineered oncolytic herpesvirus, and an engineered congenital stealth virus expressing both immediate-early (IE) and late (L) US11 (see Figure 1A and Example 1 herein). ICP27 viral protein and actin were used as virus infection and loading controls, respectively. MOI, multiplicity of infection. 6h = 6 hours post-infection; 12h = 12 hours post-infection; 18h = 18 hours post-infection. [Figure 2A]

[0023] Figure 1 shows the design of a viral genome expressing the model antigens SIINFEKL (SEQ ID NO: 319) (SIINFEKL (SEQ ID NO: 319)) and UL49.5 (left panel: "41T_2B"), as well as a viral genome that is genetically identical to 41T_2B except for the presence of a stop codon inserted within UL49.5 to prevent its expression (right panel: "41T_4B"). iRFP, near-infrared fluorescent protein. The figure discloses "SIINFEKL" as SEQ ID NO: 319. [Figure 2B] 1 provides the results of two independent flow cytometry experiments (Run 1 and Run 2, indicated by circles and triangles, respectively) using MB49 cells infected with the indicated viruses (MOI=10) and stained for H2Kb (MHC) and SIINFEKL-H2Kb (MHC-SIINFEKL ("SIINFEKL" disclosed as SEQ ID NO: 319)). The top panel shows the percentage of live cells expressing MHC, 41T_2B, or 41T_4B infected (iRFP+) cells. The bottom panel shows the percentage of live 41T_2B or 41T_4B infected cells presenting the SIINFEKL (SEQ ID NO: 319) antigen in the context of MHC. [Figure 3]FIG. 1 shows the rationale for the selection of four immunomodulatory payloads for HSV-1 OV development as described in Example 2 herein. APC, antigen-presenting cell; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; CD, cluster of differentiation; DC, dendritic cell; FLT3L, fms-like tyrosine kinase 3 ligand; IL, interleukin; Th, T helper; TME, tumor microenvironment. [Figure 4] This shows that bivalency of anti-CTLA-4 molecules is required to block CTLA-4 interaction with CD80:CD86. The indicated anti-CTLA-4 molecules (i.e., anti-CTLA-4 mAb, Fab'2, Fab, scFv, scFv-G1m1, and scFv-G1m (17)) were tested for their ability to block CTLA-4 interaction with CD80:CD86 using a reporter assay. Reporter luminescence (RLU) indicates the ability to block CTLA-4 interaction with CD80:CD86. Fab, fragment antigen binding; mAb, monoclonal antibody; RLU, relative light units; scFv, single-chain variable fragment; anti-CTLA-4 mAb is an anti-CTLA-4 monoclonal antibody. [Figure 5A]This indicates that anti-CTLA-4 payloads for use with HSV-1 OVs should have an active Fc region (capable of binding to Fc-γ receptors) for in vivo activity. Figure 5A shows the effect of the indicated anti-CTLA-4 molecules on tumor growth at the indicated days after tumor implantation (assessed as mean tumor volume, mm + standard error of the mean (SEM)). Anti-CTLA-4 molecules were administered intratumorally (IT) every other week for a total of four treatments (biw × 4). Figure 5B shows the effect of the indicated anti-CTLA-4 molecules on Treg depletion (assessed by the percentage of CD4+ T cells expressing FoxP3 and CD25 using flow cytometry) or T cell activation (assessed by the percentage of CD8+ T cells expressing IFNγ and TNFα using flow cytometry after restimulation with tumor antigen peptides) in tumors from mice treated similarly to the experiment shown in Figure 5A. The anti-CTLA-4 molecules shown in Figures 5A-5B are a positive (+) control anti-CTLA4 mAb, an scFv containing the variable region of the anti-CTLA4 mAb fused to IgG1-Fc, and an Fc-less construct (i.e., Ipi-Fab tandem-scFv) composed of two scFvs containing the variable regions of the anti-CTLA mAb linked by a linker. [Figure 5B]This indicates that anti-CTLA-4 payloads for use with HSV-1 OVs should have an active Fc region (capable of binding to Fc-γ receptors) for in vivo activity. Figure 5A shows the effect of the indicated anti-CTLA-4 molecules on tumor growth at the indicated days after tumor implantation (assessed as mean tumor volume, mm + standard error of the mean (SEM)). Anti-CTLA-4 molecules were administered intratumorally (IT) every other week for a total of four treatments (biw × 4). Figure 5B shows the effect of the indicated anti-CTLA-4 molecules on Treg depletion (assessed by the percentage of CD4+ T cells expressing FoxP3 and CD25 using flow cytometry) or T cell activation (assessed by the percentage of CD8+ T cells expressing IFNγ and TNFα using flow cytometry after restimulation with tumor antigen peptides) in tumors from mice treated similarly to the experiment shown in Figure 5A. The anti-CTLA-4 molecules shown in Figures 5A-5B are a positive (+) control anti-CTLA4 mAb, an scFv containing the variable region of the anti-CTLA4 mAb fused to IgG1-Fc, and an Fc-less construct (i.e., Ipi-Fab tandem-scFv) composed of two scFvs containing the variable regions of the anti-CTLA mAb linked by a linker. [Figure 6A]Diagrams of the anti-CTLA-4 and CD40 agonist payloads selected for inclusion in HSV-1 OVs are provided. Figure 6A shows the design of the selected anti-CTLA-4 antagonist payload, designated hαCTLA-4. The anti-CTLA-4 antagonist is an anti-CTLA-4 single-chain variable fragment (scFv) fused to the N-terminus of the heavy chain of human IgG1_G1m (17). Figure 6B shows the design of the selected CD40 agonist payload, designated hCD40ag. The CD40 agonist is a trimerization C-terminal fusion protein containing a trimeric bundle of human CD40L ectodomain, which binds to the CD40 receptor protein. hCD40ag contains a 27-amino acid trimerization motif derived from the fibritin protein of T4 phage fused to a glycine / serine linker and the N-terminal CD40L ectodomain trimeric bundle. The CD40L ectodomain trimer bundle consists of individual CD40L peptides fused together by a glycine / serine linker from the C-terminus of one peptide to the N-terminus of the subsequent peptide to form a single polypeptide trimer bundle. Figures 6A and 6B disclose SEQ ID NOS: 23, 22, and 320, respectively, in order of appearance. [Figure 6B]Diagrams of the anti-CTLA-4 and CD40 agonist payloads selected for inclusion in HSV-1 OVs are provided. Figure 6A shows the design of the selected anti-CTLA-4 antagonist payload, designated hαCTLA-4. The anti-CTLA-4 antagonist is an anti-CTLA-4 single-chain variable fragment (scFv) fused to the N-terminus of the heavy chain of human IgG1_G1m (17). Figure 6B shows the design of the selected CD40 agonist payload, designated hCD40ag. The CD40 agonist is a trimerization C-terminal fusion protein containing a trimeric bundle of human CD40L ectodomain, which binds to the CD40 receptor protein. hCD40ag contains a 27-amino acid trimerization motif derived from the fibritin protein of T4 phage fused to a glycine / serine linker and the N-terminal CD40L ectodomain trimeric bundle. The CD40L ectodomain trimer bundle consists of individual CD40L peptides fused together by a glycine / serine linker from the C-terminus of one peptide to the N-terminus of the subsequent peptide to form a single polypeptide trimer bundle. Figures 6A and 6B disclose SEQ ID NOS: 23, 22, and 320, respectively, in order of appearance. [Figure 6C]Figure 6A provides a diagram of the FLT3L and IL-12t payloads selected for inclusion in HSV-1 OVs and shows the crystal structure of a selected FLT3L payload, designated hFLT3L, a full-length human FLT3L molecule. This molecule features a native signal peptide, allowing efficient secretion to the plasma membrane, where it is anchored by a transmembrane helix. FLT3L forms functional dimers and can be processed into a soluble version. Figure 6D shows the crystal structure of a selected IL-12 payload, designated human single-chain IL-12 (hscIL-12). The hscIL-12 payload is a human IL-12 fusion protein containing the human p40 subunit, also known as IL-12 subunit β (Uniprot: P29460), at the N-terminus of human p35, also known as IL-12 subunit α (Uniprot: P29459), from IL-12 fused with a glycine / serine linker. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) was added to the C-terminus of the p35 subunit to aid in purification of the recombinant protein by Ni-NTA resin. Figures 6C and 6C disclose SEQ ID NOs: 3 and 320, respectively, in order of appearance. [Figure 6D]Figure 6A provides a diagram of the FLT3L and IL-12t payloads selected for inclusion in HSV-1 OVs and shows the crystal structure of a selected FLT3L payload, designated hFLT3L, a full-length human FLT3L molecule. This molecule features a native signal peptide, allowing efficient secretion to the plasma membrane, where it is anchored by a transmembrane helix. FLT3L forms functional dimers and can be processed into a soluble version. Figure 6D shows the crystal structure of a selected IL-12 payload, designated human single-chain IL-12 (hscIL-12). The hscIL-12 payload is a human IL-12 fusion protein containing the human p40 subunit, also known as IL-12 subunit β (Uniprot: P29460), at the N-terminus of human p35, also known as IL-12 subunit α (Uniprot: P29459), from IL-12 fused with a glycine / serine linker. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) was added to the C-terminus of the p35 subunit to aid in purification of the recombinant protein by Ni-NTA resin. Figures 6C and 6C disclose SEQ ID NOs: 3 and 320, respectively, in order of appearance. [Figure 7A]We demonstrate that an Fc-less CD40 agonist, designated hCD40ag, is bioactive in vitro. Figure 7A shows the effect of CD40 agonists on CD40 pathway activity using a reporter cell line that emits a signal after CD40 pathway activation. The CD40 agonists tested were hCD40ag (an Fc-less trimer of CD40L trimeric bundles) and hCD40ag2 (containing bivalent CD40L trimeric bundles linked to Fc). Two positive controls (positive controls 1 and 2) and a negative control of medium alone were tested. CD40 pathway activation is expressed as optical density (OD) at 650 nm (y-axis) using the concentrations of CD40 agonists indicated on the x-axis (ng / mL). Figure 7B shows the effect of the indicated CD40 agonists on dendritic cell (DC) activation, assessed by CD86 expression using flow cytometry. CD40 agonists tested included hCD40ag and several controls: positive controls 1 and 2, wild-type CD40 agonist (soluble monomer), and an IgG1 isotype control antibody. Each CD40 agonist or control was used at the concentration, gMFI, or geometric mean fluorescence intensity indicated on the x-axis. [Figure 7B]We demonstrate that an Fc-less CD40 agonist, designated hCD40ag, is bioactive in vitro. Figure 7A shows the effect of CD40 agonists on CD40 pathway activity using a reporter cell line that emits a signal after CD40 pathway activation. The CD40 agonists tested were hCD40ag (an Fc-less trimer of CD40L trimeric bundles) and hCD40ag2 (containing bivalent CD40L trimeric bundles linked to Fc). Two positive controls (positive controls 1 and 2) and a negative control of medium alone were tested. CD40 pathway activation is expressed as optical density (OD) at 650 nm (y-axis) using the concentrations of CD40 agonists indicated on the x-axis (ng / mL). Figure 7B shows the effect of the indicated CD40 agonists on dendritic cell (DC) activation, assessed by CD86 expression using flow cytometry. CD40 agonists tested included hCD40ag and several controls: positive controls 1 and 2, wild-type CD40 agonist (soluble monomer), and an IgG1 isotype control antibody. Each CD40 agonist or control was used at the concentration, gMFI, or geometric mean fluorescence intensity indicated on the x-axis. [Figure 8A]Figure 8 shows the in vivo antitumor effects of several CD40 agonists using MC38-5AG tumors in human CD40 knock-in mice. The CD40 agonists shown in Figure 8A were injected IT on days 1, 4, and 7 to mimic expression from OVs, and tumor volumes (mm3) were measured on the indicated days after treatment. The agonists tested were hCD40ag (i.e., Fc-less trimers of CD40L trimeric bundles), hCD40ag2 (i.e., bivalent CD40L trimeric bundles linked to Fc), and WT CD40 agonist (CD40L) monomer. An isotype control antibody (IgG1 mAb) was also tested. Confidence interval (CI) values ​​are the lower and upper limits of the 95% CI for tumor growth inhibition (TGI) values, and p values ​​compared to the isotype control were calculated for each construct tested using InVivoLDA version 4.8. Figure 8B provides a comparison of the mean tumor volumes measured in mice treated with hCD40ag antibody or isotype control antibody (IgG1 mAb). The arrows in Figure 8B indicate the days on which mice were treated with hCD40ag or isotype control antibody. [Figure 8B] Figure 8 shows the in vivo antitumor effects of several CD40 agonists using MC38-5AG tumors in human CD40 knock-in mice. The CD40 agonists shown in Figure 8A were injected IT on days 1, 4, and 7 to mimic expression from OVs, and tumor volumes (mm3) were measured on the indicated days after treatment. The agonists tested were hCD40ag (i.e., Fc-less trimers of CD40L trimeric bundles), hCD40ag2 (i.e., bivalent CD40L trimeric bundles linked to Fc), and WT CD40 agonist (CD40L) monomer. An isotype control antibody (IgG1 mAb) was also tested. Confidence interval (CI) values ​​are the lower and upper limits of the 95% CI for tumor growth inhibition (TGI) values, and p values ​​compared to the isotype control were calculated for each construct tested using InVivoLDA version 4.8. Figure 8B provides a comparison of the mean tumor volumes measured in mice treated with hCD40ag antibody or isotype control antibody (IgG1 mAb). The arrows in Figure 8B indicate the days on which mice were treated with hCD40ag or isotype control antibody. [Figure 9A] The functionality and biological activity of a selected FLT3L payload, designated hFLT3L, are shown. Figure 9A shows the levels of soluble FLT3L in the supernatants of the indicated human tumor cell lines (A375, A549, H1299, HT29, and 22Rv1) infected with three viruses expressing hFLT3L. Each bar within a group of three bars for each tumor cell line on the x-axis corresponds to a different HSV-1 virus expressing hFLT3L. Figure 9B shows the effect of human WT FLT3L protein on DC differentiation. Differentiation of DCs (MHCII+CD11c+) from mouse bone marrow was assessed by flow cytometry upon treatment with supernatants from Vero cells infected (MOI = 1) with recombinant human FLT3L (rhFLT3L) or either an HSV-1 virus expressing hFLT3L or a negative control virus with a similar architecture but not expressing hFLT3L. The amount of hFLT3L in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The activity of rhFLT3L was assessed alone or spiked into supernatant from cells infected with a negative control virus. [Figure 9B] The functionality and biological activity of a selected FLT3L payload, designated hFLT3L, are shown. Figure 9A shows the levels of soluble FLT3L in the supernatants of the indicated human tumor cell lines (A375, A549, H1299, HT29, and 22Rv1) infected with three viruses expressing hFLT3L. Each bar within a group of three bars for each tumor cell line on the x-axis corresponds to a different HSV-1 virus expressing hFLT3L. Figure 9B shows the effect of human WT FLT3L protein on DC differentiation. Differentiation of DCs (MHCII+CD11c+) from mouse bone marrow was assessed by flow cytometry upon treatment with supernatants from Vero cells infected (MOI = 1) with recombinant human FLT3L (rhFLT3L) or either an HSV-1 virus expressing hFLT3L or a negative control virus with a similar architecture but not expressing hFLT3L. The amount of hFLT3L in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The activity of rhFLT3L was assessed alone or spiked into supernatant from cells infected with a negative control virus. [Figure 10A] Figure 1 shows the results of an enzyme-linked immunosorbent assay (ELISA) used to measure IFNγ (pg / mL) produced by human peripheral blood mononuclear cells (PBMCs) treated with recombinant hIL-12 (2 subunits) or selected hscIL-12 payloads at the concentrations indicated on the x-axis. Bars corresponding to selected hscIL-12 payloads are marked with an arrow; bars corresponding to recombinant hIL-12 (commercially available rhIL-12) are unmarked. [Figure 10B] This figure shows the results of an ELISA used to measure IFNγ (pg / mL) produced by human PBMCs treated with suboptimal PHA / PMA stimulation for T cell activation followed by supernatants from Vero cells infected (MOI = 1) with recombinant hIL-12 (rhIL-12), a virus expressing an hscIL-12 payload, or a negative control virus not expressing an hscIL-12 payload. The amount of hIL-12 in the supernatant was determined by ELISA, as indicated on the x-axis. The activity of rhIL-12 was assessed either alone or spiked into supernatants from cells infected with the negative control virus. Bars corresponding to rhIL-12 are marked with an "R," bars corresponding to viruses expressing an hscIL-12 payload are indicated by an arrow, bars corresponding to rhIL-12 spiked into supernatants from cells infected with the negative control virus are marked with an "R / -," and bars corresponding to the negative control virus are marked with a "-." [Figure 11] We demonstrate that anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL-12 (hscIL-12) payloads have a synergistic effect on T cell activation. Human PBMCs were incubated with a fixed concentration of T cell-activating superantigen in the presence of increasing concentrations of hαCTLA-4 alone or the dual combination of hαCTLA-4 and hscIL-12 (Figure 11, left). PBMCs were then exposed to a fixed concentration of hCD40ag to activate APCs, which were then combined with increasing concentrations of hαCTLA-4 or the combination of hαCTLA-4 and hscIL-12 (Figure 11, right). T cell activation was measured by IL2 secretion into supernatants collected on day 5. [Figure 12A] Figure 1 shows the architecture of a cassette with eight different promoter combinations and orientations, each of which contains a promoter selected from mCMV, MMLV, AoHV1, and Pbidir3 (described in WO 2016 / 166088). [Figure 12B] Ten sets of polyadenylation signal pairs (pA1 and pA2) are shown, resulting in 80 reporter cassettes. Using the reporter system, 80 designed cassettes were screened. The payload was replaced with two fluorescent proteins (i.e., DsRed and TagBFP2) and a plasma membrane marker (i.e., mThy1.1) that is easily detectable by flow cytometry. The positions of the reporter genes (i.e., mThy1.1, DsRed, and TagBFP2) were fixed, as was the 3' most common polyadenylation signal (US9-10pA). [Figure 13A] Schematic diagram of payload expression cassettes 17E, 37E and 75E. [Figure 13B] The expression levels of the anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL-12 (hscIL-12) payloads for cassettes 17E, 37E, and 75E, respectively, in the supernatants of two different cell lines (HEK293T and H1299) collected after 24 hours (H1299) and 48 hours (HEK293T) of transient transfection are shown (mean ± SD, n = 2 technical replicates). Payload expression levels were assessed by blocking assays, secreted alkaline phosphatase assays, and ELISA assays. The pUC57 plasmid vector backbone (containing no expression cassette) was used as a negative control. [Figure 14]Figure 1 shows the expression levels of hFLT3L, anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL-12 (hscIL-12) payloads from the indicated OVs upon infection of five different tumor cell lines. The OVs tested were JP-OV-1 (containing the 17E cassette), JP-OV-2 (containing the 37E cassette), and the parental OV expressing hFLT3L. Payload expression was quantified by ELISA (mean ± SD, n = 3 technical replicates) from the culture medium of five cancer cell lines infected at an MOI of 1 24 h postinfection. The parental virus (expressing hFLT3L) was used as a positive control for hFLT3L and as a negative control for the three other payloads. To account for differences in total cell number at the same culture cell density, the concentrations of payloads were expressed per 10 cells (at the time of infection). * indicates a concentration below the detection level of the assay. The bar corresponding to the parent OV is marked with a "P", the bar corresponding to JP-OV-1 is marked with a "1", and the bar corresponding to JP-OV-2 is marked with a "2". [Figure 15A] Figure 15A shows the multi-step construction of the oncolytic virus JP-OV-2. Figure 15A shows the genomic structure of the ΔXN1 parent virus used to generate JP-OV-2. β-Gluc represents the replacement of the neurovirulence gene γ34.5 with β-glucuronidase at the γ34.5 locus. US12, along with its intron sequence (shown as a latch), was deleted from the US10-12 locus and is shown in light gray. This virus expresses US11 as an immediate-early gene using the US12 promoter. Figure 15B shows the genomic structure of the ΔXN1-GFP virus. The virus expresses eGFPS from the γ34.5 locus using the CMV promoter and US11 from the US10-12 locus using the US12 promoter. [Figure 15B]Figure 15A shows the multi-step construction of the oncolytic virus JP-OV-2. Figure 15A shows the genomic structure of the ΔXN1 parent virus used to generate JP-OV-2. β-Gluc represents the replacement of the neurovirulence gene γ34.5 with β-glucuronidase at the γ34.5 locus. US12, along with its intron sequence (shown as a latch), was deleted from the US10-12 locus and is shown in light gray. This virus expresses US11 as an immediate-early gene using the US12 promoter. Figure 15B shows the genomic structure of the ΔXN1-GFP virus. The virus expresses eGFPS from the γ34.5 locus using the CMV promoter and US11 from the US10-12 locus using the US12 promoter. [Figure 15C] Additional figures depicting the multi-step construction of oncolytic virus JP-OV-2. Figure 15C shows the genomic structure of the step 1 virus. The virus was engineered to express hFLT3L and UL49.5 transgenes using the CMV promoter from the γ34.5 locus. US11 is expressed using the immediate-early US12 promoter. Figure 15D shows the genomic structure of the step 2 virus. The US10-12 locus was engineered to express eGFP. The US12 intron (shown as a dark latch) was added back into the US10-12 locus. US11 is expressed using the US12 promoter. [Figure 15D] Additional figures depicting the multi-step construction of oncolytic virus JP-OV-2. Figure 15C shows the genomic structure of the step 1 virus. The virus was engineered to express hFLT3L and UL49.5 transgenes using the CMV promoter from the γ34.5 locus. US11 is expressed using the immediate-early US12 promoter. Figure 15D shows the genomic structure of the step 2 virus. The US10-12 locus was engineered to express eGFP. The US12 intron (shown as a dark latch) was added back into the US10-12 locus. US11 is expressed using the US12 promoter. [Figure 15E]The genomic structure of the step 3 virus, the final viral construct of JP-OV-2, is shown. The virus was engineered to express anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL-12 (hscIL-12) payloads from the US10-12 locus, as well as hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon-optimized US11 (hCoUS11) using the US12 immediate-early promoter and endogenous US11 using the late US11 promoter. The hexagonal "S" represents a stop codon between hCoUS11 and US12, preventing US12 expression. The gray US12 indicates that the gene is not expressed. IRL, internal repeat long; IRS, internal repeat short; TRL, terminal repeat long; TRS, terminal repeat short; UL, unique long; US, unique short. [Figure 16A] Immunoblots for ICP27, US11, UL49.5, and actin expression from Vero cell lysates infected with the indicated viruses are shown. ICP27 and actin were used as controls. [Figure 16B] Quantification of the immunoblot shown in Figure 16A is provided. US11 and UL49.5 protein levels were quantified and normalized to ICP27 and actin. The relative fold expression of UL49.5 and US11 from step 3 clones 1-4 was compared to either Δγ34.5 or step 2 virus. [Figure 17]Figure 1 shows the expression of hFLT3L, anti-CTLA-4 antagonist (hαCTLA-4), CD40 agonist (hCD40ag), and IL-12 (hscIL-12) payload proteins from Vero cells infected with the indicated viruses at an MOI of 1. Cell culture supernatants were analyzed for payload protein expression by ELISA. Δγ34.5 virus served as a negative control for hFLT3L and hscIL-12 expression, and step 2 virus served as a parental control virus that does not encode the hαCTLA-4, hCD40ag, and hscIL-12 payloads (i.e., within the 37E cassette). PY23_40A_P2_1_1 served as a positive control for hscIL-12 and hαCTLA-4 expression. PY16_26C_1_1, an OV HSV-1 virus, served as a positive control for hCD40ag expression. [Figure 18] Cytotoxicity of step 3 virus clones 1-4, as assessed by cell viability of virus-infected cells, is shown. HT29 cells (top panel), which are stringent for virus replication, or Vero cells (bottom panel), which are highly permissive and used to ensure similar amounts of each virus were used in the experiments, were infected with the indicated viruses (MOI = 0.01). Virus-induced cytopathic effects were monitored using the xCelligence system at 6-hour intervals for 120 hours post-infection. Cell cytotoxicity and percent viability in the presence of virus were calculated based on the cell index of mock-infected cells. Results shown represent the average of six replicates. [Figure 19] Genetic stability of JP-OV-2 over multiple cell culture replication cycles, as assessed by key protein expression levels, is shown. Vero cells were infected (MOI = 5) with JP-OV-2 collected after replication cycles 6, 7, and 8. Expression of ICP27, US11, UL49.5, and hCD40ag payloads was detected by Western blot in whole cell extracts and culture supernatants (hCD40ag only) 6 hours postinfection. Δγ34.5 virus was used as a negative control, and early-passage JP-OV-2 was also used as a control (denoted as "JP-OV-2"). [Figure 20] Genetic stability of JP-OV-2 over multiple cell culture replication cycles, as assessed by immunomodulatory payload protein expression levels, was shown. Expression of hFLT3L, CD40 agonist (hCD40ag), anti-human CTLA-4 antagonist (hαCTLA-4), and single-chain human IL-12 (hscIL-12) payload proteins was determined by ELISA (mean ± SD; * indicates concentrations below the detection level of the assay) in culture supernatants of Vero cells infected with JP-OV-2 (MOI = 1, 24 h postinfection) collected after replication cycles 6, 7, and 8. Δγ34.5 virus was used as a negative control, and early-passage JP-OV-2 was also used as a control (denoted as "JP-OV-2"). [Figure 21] Figure 1 shows the functional stability of JP-OV-2 over multiple passages (cycles) on Vero cells as determined by virus-induced cancer cell killing. HT29 cells, which are stringent for OV replication (top two panels), or Vero cells, which are highly permissive for OV replication and were used to ensure similar amounts of each virus were used in the experiments (bottom two panels), were infected for multiple replication cycles (cycles 6, 7, and 8; MOI = 0.01) and compared to early-passage JP-OV-2 (denoted as "JP-OV-2") for virus-induced cell death as monitored by xCelligence (% viability, y-axis). [Figure 22A] Figure 22A shows an acyclovir dose-response experiment using wild-type HSV-1 (WT-HSV), a previously reported genetically engineered oncolytic herpesvirus mimic, and JP-OV-2 virus. Viral replication in Vero cells was assessed based on virus-induced cell death as measured by xCelligence. Figure 22B provides a comparison of viral replication in the absence of acyclovir in Vero cells, as assessed by xCelligence, to confirm that similar amounts of each virus were used in the experiment shown in Figure 22A. [Figure 22B]Figure 22A shows an acyclovir dose-response experiment using wild-type HSV-1 (WT-HSV), a previously reported genetically engineered oncolytic herpesvirus mimic, and JP-OV-2 virus. Viral replication in Vero cells was assessed based on virus-induced cell death as measured by xCelligence. Figure 22B provides a comparison of viral replication in the absence of acyclovir in Vero cells, as assessed by xCelligence, to confirm that similar amounts of each virus were used in the experiment shown in Figure 22A. [Figure 23A] Figure 23A shows a dose-response experiment of IFNβ-mediated inhibition of cell death induced by wild-type HSV-1 (WT-HSV), a virus deleted only for γ34.5 (Δγ34.5), a previously reported genetically engineered oncolytic herpesvirus mimic, and JP-OV-2 (MOI = 0.01) in normal human fibroblasts (HFFs). Virus-induced cell death was measured by xCelligence. Figure 23B provides a comparison of virus replication in Vero cells in the absence of IFNβ, as measured by xCelligence, to confirm that similar amounts of each virus were used in the experiment shown in Figure 23A. [Figure 23B] Figure 23A shows a dose-response experiment of IFNβ-mediated inhibition of cell death induced by wild-type HSV-1 (WT-HSV), a virus deleted only for γ34.5 (Δγ34.5), a previously reported genetically engineered oncolytic herpesvirus mimic, and JP-OV-2 (MOI = 0.01) in normal human fibroblasts (HFFs). Virus-induced cell death was measured by xCelligence. Figure 23B provides a comparison of virus replication in Vero cells in the absence of IFNβ, as measured by xCelligence, to confirm that similar amounts of each virus were used in the experiment shown in Figure 23A. [Figure 24] These results demonstrate that JP-OV-2 has tumor growth inhibitory (TGI) activity in vivo. Mice bearing H1299 human xenograft tumors were treated intratumorally (IT) with vehicle control or JP-OV-2 at 5 x 10 or 5 x 10 PFU / injection, once every three days (q3dx3) for three doses. Tumor volume measurements were obtained twice weekly throughout the study period. TGI was calculated on day 45 (D45), when 70% of animals remained in all groups. Treatment is indicated by black bars on the graphs in the upper panel (below the x-axis). P values ​​(lower panel) were calculated by linear mixed-effects (LME) analysis. [Figure 25] This shows that hFLT3L expressed from JP-OV-2 has biological activity as assessed using a DC differentiation assay. Differentiation of DCs (MHCII+CD11c+) from mouse bone marrow was assessed by flow cytometry upon treatment with the supernatant of Vero cells infected (MOI = 1) with recombinant hFLT3L (rhFLT3L), JP-OV-2, or a negative control virus with a similar architecture but lacking hFLT3L. The amount of hFLT3L in the supernatant was determined by ELISA. The activity of rhFLT3L was assessed alone or spiked into the supernatant from cells infected with the negative control virus. [Figure 26] This shows that the hscIL-12 payload expressed from JP-OV-2 has biological activity as assessed by IFNγ production by human PBMCs. IFNγ produced by PBMCs was assessed by ELISA upon suboptimal PHA / PMA stimulation for T cell activation followed by treatment with recombinant hIL-12 (rhIL-12) or supernatants (MOI=1) of Vero cells infected with JP-OV-2 or negative control step 2 virus (lacking hIL-12). The amount of hIL-12 in the supernatants was determined by ELISA. The activity of rhIL-12 was assessed alone or spiked into supernatants from cells infected with the negative control virus (rhIL-12 + step 2 virus). [Figure 27A]Figure 27A provides a diagram of three payloads selected for inclusion in mJP-OV-2, the murine surrogate HSV-1 OV described in Example 6 herein. Figure 27A is a diagram of the murine surrogate CD40 agonist payload (referred to as "mCD40ag"). mCD40ag is a trimerization C-terminal fusion protein consisting of a trimeric bundle of murine CD40L ectodomain. This molecule features a 27-amino acid trimerization motif derived from the fibritin protein of T4 phage fused to a glycine / serine linker and an N-terminal CD40L ectodomain bundle. The CD40L ectodomain trimeric bundle consists of individual CD40L protomers fused to each other by glycine / serine linkers from the C-terminus of one protomer to the N-terminus of the subsequent protomer to form a single polypeptide trimeric bundle. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) was added to the C-terminus of the final CD40L protomer to aid in purification of the recombinant protein on Ni-NTA resin, but this feature is not present in the final viral construct. The figures disclose SEQ ID NOs: 27, 22, and 320, respectively, in order of appearance. Figure 27B is the crystal structure of the murine surrogate IL-12 payload (mscIL-12). The murine surrogate payload is a murine IL-12 fusion protein containing the murine p40 subunit from IL-12 fused to the N-terminus of murine p35 with a glycine / serine linker. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) at the C-terminus of the p35 subunit was added to aid in purification of the recombinant protein on Ni-NTA resin. However, this purification tag is not present in the final viral construct. Figures 27A and 27B disclose SEQ ID NOs: 3 and 320, respectively, in order of appearance. [Figure 27B]Figure 27A provides a diagram of three payloads selected for inclusion in mJP-OV-2, the murine surrogate HSV-1 OV described in Example 6 herein. Figure 27A is a diagram of the murine surrogate CD40 agonist payload (referred to as "mCD40ag"). mCD40ag is a trimerization C-terminal fusion protein consisting of a trimeric bundle of murine CD40L ectodomain. This molecule features a 27-amino acid trimerization motif derived from the fibritin protein of T4 phage fused to a glycine / serine linker and an N-terminal CD40L ectodomain bundle. The CD40L ectodomain trimeric bundle consists of individual CD40L protomers fused to each other by glycine / serine linkers from the C-terminus of one protomer to the N-terminus of the subsequent protomer to form a single polypeptide trimeric bundle. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) was added to the C-terminus of the final CD40L protomer to aid in purification of the recombinant protein on Ni-NTA resin, but this feature is not present in the final viral construct. The figures disclose SEQ ID NOs: 27, 22, and 320, respectively, in order of appearance. Figure 27B is the crystal structure of the murine surrogate IL-12 payload (mscIL-12). The murine surrogate payload is a murine IL-12 fusion protein containing the murine p40 subunit from IL-12 fused to the N-terminus of murine p35 with a glycine / serine linker. A hexameric polyhistidine sequence (6x-His tag (SEQ ID NO: 320)) at the C-terminus of the p35 subunit was added to aid in purification of the recombinant protein on Ni-NTA resin. However, this purification tag is not present in the final viral construct. Figures 27A and 27B disclose SEQ ID NOs: 3 and 320, respectively, in order of appearance. [Figure 27C] FIG. 1 is a diagram of the design of a murine surrogate anti-CTLA-4 antagonist payload called mαCTLA-4, an antibody that binds to murine CTLA-4 and comprises an anti-CTLA-4 VHH fused to the heavy chain of murine IgG2a Fc. [Figure 28]These results demonstrate that the murine surrogate CD40 agonist payload (mCD40ag) had comparable biological activity to the human CD40 agonist payload (hCD40ag). The biological activity of purified hCD40ag and mCD40ag was assessed using the HEK-Blue reporter assay, as described in Example 6 herein. CD40 agonist activity results in the production of a colorimetric readout assessed by optical density at 655 nm (OD655). Recombinant WT mouse CD40L (rmCD40L) and recombinant WT human CD40L (rhCD40L) were included as controls. [Figure 29] This shows that human IL-12 payload (hscIL-12) and murine surrogate IL-12 payload (mscIL-12) had comparable biological activity. The biological activity of purified hscIL-12 and mscIL-12 payloads was assessed using the hIL-12 receptor HEK-Blue reporter assay, as described in Example 6 herein. IL-12 activity results in the production of a colorimetric readout assessed by optical density at 655 nm (OD). Recombinant WT murine IL-12 (rmIL-12) and recombinant WT human IL-12 (rhIL-12) were included as controls. [Figure 30] This figure shows that mαCTLA-4, a murine surrogate of the human anti-CTLA-4 antagonist payload, is effective in vivo, as evaluated in the MC38-5AG syngeneic tumor model. MC38-5AG is an MC38 line containing neoantigenic mutations and confirmed as described in Yadav et al., Nature 515:572-76 (2014). WT mice were implanted with a single MC38-5AG tumor and treated IT once every three days for four doses (q3dx4) with purified mαCTLA-4 payload. Tumor volume (mm3; mean ± SEM) was monitored at the days after tumor implantation indicated on the x-axis. ***p<0.001. [Figure 31]Diagram of the genomic structure and transgene cassette of the murine surrogate virus mJP-OV-2. The virus was engineered to express anti-CTLA-4 antagonist (mαCTLA-4), CD40 agonist (mCD40ag), and IL-12 (mscIL-12) payloads from the US10-12 locus, as well as hFLT3L and UL49.5 from the γ34.5 locus. The virus expresses codon-optimized US11 (hCoUS11) using the US12 immediate-early promoter and endogenous US11 using the late US11 promoter. The "S" indicated by a hexagon represents a stop codon between hCoUS11 and US12, preventing US12 expression. The gray US12 indicates that the gene is not expressed. IRL, internal repeat long; IRS, internal repeat short; TRL, terminal repeat long; TRS, terminal repeat short; UL, unique long; US, unique short. [Figure 32A] Immunoblots showing ICP27, US11, UL49.5, and β-actin levels in whole cell extracts from Vero cells infected with the indicated viruses (MOI = 5, 6 hours post-infection) are provided. [Figure 32B] Immunoblots showing mCD40ag payload and actin levels in whole-cell extracts (upper panels) and culture supernatants (lower panels, mCD40ag only) from Vero cells infected with the indicated viruses (MOI = 5 or 6 h postinfection). In Figures 32A-32B, Δγ34.5 was used as a negative control for immediate-early expression of US11, ΔXN1 virus was used as a negative control for UL49.5 expression, step 2 virus was the parental virus and was used as a negative control for mCD40ag payload expression, and a virus containing a cassette expressing mCD40L was used as a positive control for mCD40ag payload. For whole-cell extracts, the viral protein ICP27 was used as an infection control, and β-actin was used as a protein loading control. [Figure 33]Expression levels of the hFLT3L, mscIL-12, mαCTLA-4, and mCD40ag murine surrogate payload proteins in cells infected with the indicated viruses are shown. Vero cells were infected with control Δγ34.5, precursor step 2 virus, and murine step 3 clones 1–4 for 24 h (MOI = 1). Supernatants from infected cells were analyzed by ELISA. Δγ34.5 virus was used as a negative control for hFLT3L expression (upper left panel), and step 2 virus was used as a positive control for hFLT3L and a negative control for the other payloads. Expression levels of the four payload proteins are presented as the mean ± standard deviation (SD). [Figure 34] Cytotoxicity of the murine surrogate virus mJP-OV-2 was assessed based on cell viability of virus-infected cells. HT29 cells (top panel), which are stringent for virus replication, or Vero cells (bottom panel), which are highly permissive and used to ensure similar amounts of each virus were used in the experiments, were infected with the indicated viruses (MOI = 0.01). Virus-induced cell death was monitored by xCelligence. The unarmed virus ΔXN1 and the attenuated virus Δγ34.5 were used as positive and negative controls, respectively. [Figure 35] These results demonstrate that the mCD40ag murine surrogate payload protein is active when expressed from the murine surrogate virus mJP-OV-2. The bioactivity of mCD40ag in supernatant medium from Vero cells infected with mJP-OV-2 (MOI = 1) was compared to the bioactivity of recombinant WT murine CD40L (rmCD40L) or purified mCD40ag protein diluted in either medium or supernatant from cells infected with the negative control step 2 virus. CD40 agonist activity was assessed using a hCD40 receptor HEK-Blue reporter assay. CD40 agonist activity results in the production of a colorimetric readout assessed by optical density at 655 nm (OD655). [Figure 36]These results demonstrate that the mscIL-12 murine surrogate payload protein is active when expressed from the murine surrogate virus mJP-OV-2. The bioactivity of mscIL-12 in supernatant medium from Vero cells infected with the murine surrogate virus mJP-OV-2 (MOI = 1) was compared to the bioactivity of purified mscIL-12 protein diluted in either medium or supernatant from cells infected with recombinant wild-type IL-12 (rmIL-12) or a negative control step 2 virus. Bioactivity was assessed using the hIL-12 receptor HEK-Blue reporter assay. IL-12 activity results in the production of a colorimetric readout assessed by optical density at 655 nm (OD655). [Figure 37A] Efficacy of murine payload proteins against in vivo treated tumors is shown. One of two tumors in the MC38-5AG bilateral syngeneic mouse tumor model was treated intratumorally (IT) with 5x106 PFU / step 2 virus injection (expressing the hFLT3L payload) alone or in combination with a commercially available anti-mouse CD40 agonist antibody (FGK4.5, 5µg / injection). Tumors were treated once every three days (q3dx3) at three doses. Treated or untreated (contralateral) tumor volumes were monitored over time (mm3; shown as mean±SEM). Differences in tumor growth inhibition (ΔTGI) compared to vehicle controls are shown for treated tumors 31 days after tumor implantation. *p<0.05, ***p<0.001. Arrows indicate the day of administration. [Figure 37B] Efficacy of murine payload proteins against in vivo treated tumors is shown. One of two tumors in the MC38-5AG bilateral syngeneic mouse tumor model was treated intratumorally (IT) with 5x106 PFU / step 2 virus injection (expressing the hFLT3L payload) alone or in combination with purified murine surrogate mscIL-12 protein (0.5ng / injection). Tumors were treated once every three days (q3dx3) at three doses. Treated or untreated (contralateral) tumor volumes were monitored over time (mm3; shown as mean ± SEM). Differences in tumor growth inhibition (ΔTGI) compared to vehicle controls are shown for treated tumors 27 days after tumor implantation. *p<0.05, ***p<0.001. Arrows indicate the day of administration. [Figure 37C] Figure 1 shows the efficacy of murine payload proteins against in vivo treated tumors. One of two tumors in the MC38-5AG bilateral syngeneic mouse tumor model was treated intratumorally (IT) with 5x106 PFU / injection of Step 2 virus (expressing the hFLT3L payload), alone or in combination with a commercially available anti-murine CTLA-4 antibody (9H10, 20µg / injection). F. Tumors were treated at three doses, once every three days (q3dx3). Treated or untreated (contralateral) tumor volumes were monitored over time (mm3; shown as mean ± SEM). Differences in tumor growth inhibition (ΔTGI) compared to vehicle controls are shown for treated tumors 27 days after tumor implantation. *p<0.05, ***p<0.001. Arrows indicate the day of administration. [Figure 38A] Figure 38A shows the synergistic effect of murine immunomodulatory payload proteins on treated and abscopal tumors in vivo. One of two tumors in the MC38-5AG bilateral syngeneic tumor model mouse was treated with 5 x 10 PFU / injection of step 2 virus (expressing the hFLT3L payload) alone or in combination with purified mscIL-12 (50 ng / injection), mCD40ag (13 μg / injection), and mαCTLA-4 (10.8 μg / injection) murine surrogate payloads. Tumors were treated once every other day for six doses (q2dx6, indicated by the arrows in Figure 38A). Figure 38A shows tumor volume (mm; mean ± SEM; ***p<0.001) at the indicated times after tumor implantation for the treated tumor and the contralateral tumor. [Figure 38B] Shown is a survival analysis of the mice used in the experiment shown in Figure 38A. Mice were followed for twice the median survival time of vehicle controls. [Figure 39A]The synergistic effect of murine immunomodulatory payload proteins on in vivo treated tumors and / or abscopal tumors using multiple doses of payload protein is shown. WT mice were implanted bilaterally into each flank and treated IT every other day with six doses (q2dx6; indicated by arrows) of step 2 virus (expressing hFLT3L) alone or in combination with the remaining murine surrogate payloads (i.e., mαCTLA-4, mCD40ag, and mscIL-12 payloads). The amounts of virus and mscIL-12 were kept constant (5 x 10 PFU and 50 ng, respectively). A dose response was performed with the remaining two payloads administered at high doses (13 μg mCD40ag and 10.8 μg mαCTLA-4). Tumor volumes (mm3) of treated and untreated (contralateral) tumors were monitored over time and are shown as mean ± SEM. ***p<0.001. [Figure 39B] This figure shows the synergistic effect of murine immunomodulatory payload proteins on in vivo treated tumors and / or abscopal tumors using multiple doses of payload protein. WT mice were implanted bilaterally into each flank and treated IT every other day with six doses (q2dx6; indicated by arrows) of step 2 virus (expressing hFLT3L) alone or in combination with the remaining murine surrogate payloads (i.e., mαCTLA-4, mCD40ag, and mscIL-12 payloads). The amounts of virus and mscIL-12 were kept constant (5 x 10 PFU, 50 ng, respectively). A dose response was performed for the remaining two payloads, which were administered at intermediate doses (1.3 μg mCD40ag and 1.1 μg mαCTLA-4). Tumor volumes (mm3) of treated and untreated (contralateral) tumors were monitored over time and are shown as mean ± SEM. ***p<0.001. [Figure 39C]The synergistic effects of murine immunomodulatory payload proteins on in vivo treated tumors and / or abscopal tumors using multiple doses of payload protein are shown. MC38-5AG tumor cells were implanted bilaterally into each flank of wild-type mice and treated IT every other day with six doses (q2dx6; indicated by arrows) of step 2 virus (expressing hFLT3L) alone or in combination with the remaining murine surrogate payloads (i.e., mαCTLA-4, mCD40ag, and mscIL-12 payloads). The amounts of virus and mscIL-12 were kept constant (5 x 10 PFU, 50 ng, respectively). A dose response was performed with the remaining two payloads administered at lower doses (0.13 μg mCD40ag and 0.11 μg mαCTLA-4). Tumor volumes (mm3) of treated and untreated (contralateral) tumors were monitored over time and are shown as mean ± SEM. ***p<0.001. [Figure 40A] Survival of mice administered the high payload dose (i.e., 13 μg mCD40ag and 10.8 μg mαCTLA-4) is shown. *p<0.05, ***p<0.001. [Figure 40B] Survival of mice receiving the medium payload dose (1.3 μg mCD40ag and 1.1 μg mαCTLA-4) is shown. *p<0.05, ***p<0.001. [Figure 40C] Survival of mice receiving low payload doses (0.13 μg mCD40ag and 0.11 μg mαCTLA-4) is shown. *p<0.05, ***p<0.001. [Figure 41]This shows that all mouse surrogate payload combinations resulted in durable and specific antitumor responses in mice that protected against rechallenge with the same tumor type. Naive mice, or mice previously cured of bilateral MC38-5AG tumors by treatment with step 2 virus in combination with all remaining mouse surrogate payloads (i.e., mαCTLA-4, mCD40ag, and mscIL-12 payloads), were challenged or rechallenged with either MC38-5AG or AE17 single tumors. Tumor volume was monitored over time, and mice were sacrificed when they reached endpoint tumor burden. Survival over time is shown on the y-axis with days after tumor implantation shown on the x-axis. [Figure 42A] Figure 1 shows the antitumor effect of the murine surrogate virus mJP-OV-2 on both in vivo treated and abscopal tumors. Mice bearing bilateral syngeneic MC38-5AG tumors were treated with mJP-OV-2 using a dosing regimen of once every three days for three doses (q3dx3). The efficacy of mJP-OV-2 was compared to that of a similar virus that does not contain an immune payload, termed a non-armed backbone virus. The vehicle group was treated q2dx6. Tumor volume (mm3) of treated and untreated (contralateral) tumors was monitored over time and is shown as the mean ± SEM with days after tumor implantation indicated on the x-axis. Arrows indicate administration of the corresponding virus or vehicle control. *p<0.05, ***p<0.001. [Figure 42B]Figure 1 shows the antitumor effect of the murine surrogate virus mJP-OV-2 on both in vivo treated and abscopal tumors. Mice bearing bilateral syngeneic MC38-5AG tumors were treated with mJP-OV-2 using a dosing regimen: once every other day (q2dx6) for six doses. The efficacy of mJP-OV-2 was compared with that of a similar virus lacking an immune payload, termed a non-armed backbone virus, and a previously reported genetically engineered oncolytic herpesvirus mimic. The vehicle group was treated q2dx6. Tumor volumes (mm3) of treated and untreated (contralateral) tumors were monitored over time and are shown as the mean ± SEM with days post-tumor implantation indicated on the x-axis. Arrows indicate administration of the corresponding virus or vehicle control. *p<0.05, ***p<0.001. [Figure 43A] Figures 42A-42B show the survival of mice used in the experiment shown. Figure 43A shows the survival of mice treated with the indicated viruses once every three days for three doses (q3dx3), and Figure 43B shows the survival of mice treated with the indicated viruses once every other day for six doses (q2dx6). In Figures 43A-43B, the vehicle group was treated q2dx6. Survival was monitored over time. **p<0.01, ***p<0.001. [Figure 43B] Figures 42A-42B show the survival of mice used in the experiment shown. Figure 43A shows the survival of mice treated with the indicated viruses once every three days for three doses (q3dx3), and Figure 43B shows the survival of mice treated with the indicated viruses once every other day for six doses (q2dx6). In Figures 43A-43B, the vehicle group was treated q2dx6. Survival was monitored over time. **p<0.01, ***p<0.001. [Figure 44A]Figure 1 shows the expression levels of hFLT3L, mαCTLA-4, mCD40ag, and mscIL-12 payloads in bilaterally implanted MC38-5AG mouse tumors and IT-treated with mJP-OV-2 (5x106 PFU / injection in one tumor). Four treated tumors were harvested at the indicated times post-injection (x-axis), homogenized, and payload expression was quantified by ELISA. Expression levels are shown as mean ± SEM. Dotted lines were added to visualize payload expression levels over time. [Figure 44B] Figure 1 shows expression levels of hFLT3L, hαCTLA-4, hCD40ag, and hscIL-12 payloads in H1299 human tumors implanted as single tumors in nude mice and treated IT with JP-OV-2 (5x106 PFU / injection). Five treated tumors were harvested at the indicated times post-injection (x-axis), homogenized, and payload expression quantified by ELISA. Expression levels are shown as mean ± SEM. Dotted lines have been added to visualize payload expression levels over time. Detailed Description of the Invention

[0010] The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0011] To assist the reader of this application, the description is separated into various paragraphs or sections. These separations should not be considered to separate the material in one paragraph or section from the material in another paragraph or section. On the contrary, this specification encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated.

[0012] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined below. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0013] Although certain features of the disclosure are described for clarity in the context of separate embodiments, it is understood that they may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that 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 relating to specific method steps, reagents, or conditions are specifically embraced by the disclosure and are disclosed herein just as if each and every combination were individually and expressly disclosed.

[0014] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a basis for prior limitation over the use of exclusive terminology such as "only," "only," and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0015] Reference herein to "about" a value or parameter refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) aspects related to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

[0016] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include 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).

[0017] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0018] As used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0019] Unless otherwise defined, 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 pertains. 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 those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0020] Units, prefixes, and symbols are shown in their Systeme International d'Unites (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of this disclosure which may 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 as a whole.

[0021] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures (immunoglobulin molecules, fragments of immunoglobulin molecules, or derivatives of any of these that have the ability to specifically bind to an antigen under typical physiological conditions), including, but not limited to, monoclonal antibodies, four-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 "four-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 a "heavy-chain-only antibody" or "HCAb," refers to a functional antibody that contains two heavy chains but lacks the two light chains typically found in four-chain antibodies. Camelids (camels, llamas, and alpacas) are known to produce HCAbs.

[0022] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens, such as homologous antigens from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0023] An "antibody fragment" comprises a portion of an antibody, preferably the antigen-binding or variable region of the antibody. Examples of antibody fragments include VHH, single domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. 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 a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of an immunoglobulin, the variable domain, that contains the antigen-binding site. The constant domain is the C of the heavy chain. H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or CL ) domain.

[0024] As used herein, the terms "binding," "binding," or "specifically binding" in reference to the binding of an antibody to a predetermined antigen typically refer to a binding activity of about 10, as determined by, for example, biolayer interferometry (BLI) techniques on an Octet HTX instrument using the antibody as the ligand and the antigen as the analyte. 6 M or less, e.g. 10 7 M or less, for example, about 10 8 M or less, for example, about 10 9 M or less, about 10 10 M or less, or about 10 11 K below M D and antibodies bind with an affinity corresponding to a K for binding to nonspecific antigens other than the designated antigen or closely related antigens (e.g., BSA, casein). D at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, D binds to a given antigen with an affinity corresponding to the K D The lower the amount of antibody D Since it depends on the antibody's K D If the K of binding to the antigen is very low, D is the K for nonspecific antigen binding D The amount lower can be at least 10,000-fold (ie, the antibody is highly specific).

[0025] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. As used herein, affinity and K D are inversely related, i.e., higher affinity corresponds to lower K D A lower affinity is intended to refer to a higher K D is intended to refer to.

[0026] "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most variable regions within 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 have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and can therefore adopt various conformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Both terms are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within canonical antibody variable regions have been determined by comparison of multiple 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 hypervariable regions varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c, etc. next to the residue number in the standard variable region numbering scheme. Al-Lazikani et al., supra (1997). Such nomenclature is similarly well known to those skilled in the art.

[0027] As used herein, the term "Fc region" is used to define the 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 can vary, the Fc region of a human IgG heavy chain is often defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering a nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0028] 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 an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (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 to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen and then screened against 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).

[0029] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally distinct loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts"). Generally, four-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).

[0030] Numerous HVR delineations are in use and are encompassed herein. Exemplary HVRs of the four-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) the 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) the CDRs present 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) antigenic 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).

[0031] Single domain antibodies (such as VHHs) are derived from VHH 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 by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23;240(1-2):185-195). H The domains may be numbered according to the conventional numbering system. According to this numbering system, FR1 of VHH comprises amino acid residues at positions 1 to 30, CDR1 of VHH comprises amino acid residues at positions 31 to 35, FR2 of VHH comprises amino acid residues at positions 36 to 49, CDR2 of VHH comprises amino acid residues at positions 50 to 65, FR3 of VHH comprises amino acid residues at positions 66 to 94, CDR3 of VHH comprises amino acid residues at positions 95 to 102, and FR4 of VHH comprises amino acid residues at positions 103 to 113. In this regard, V H It should be noted that, as is well known in the art for VHH domains and 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 Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering).

[0032] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, framework, "FR" residues) are numbered herein according to Kabat et al.

[0033] The terms "cassette," "expression cassette," or "gene cassette" refer to a sequence of DNA capable of carrying and directing the expression of one or more genes of interest between a set of one or more restriction sites. This can be transferred from one DNA sequence (usually a vector) to another by "cutting" the fragment using restriction enzymes and "pasting" it into a new context (such as a viral genome). Typically, the DNA fragment (nucleic acid sequence) is operably associated with expression control sequence elements that provide for proper transcription and translation of the target nucleic acid sequence (gene). Such sequence elements may include a promoter and polyadenylation signal.

[0034] A sequence that "encodes" an expression product such as a polypeptide is the minimal nucleotide sequence that, when expressed, results in the production of that polypeptide.

[0035] The term "exogenous" refers to a combination of elements that do not occur in nature. For example, an "exogenous gene" refers to a gene introduced into the genome of a virus that is not normally found in the genome of the virus or is a homolog of a gene expressed in a virus from a different species (e.g., the bovine herpesvirus UL49.5 gene, which encodes a TAP inhibitor, is exogenous when inserted into a viral genome that does not naturally encode UL49.5).

[0036] As used herein, the term "herpes simplex virus" or "HSV" refers to a member of the Herpesviridae family. Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), also known by the taxonomic names human alphaherpesvirus I and human alphaherpesvirus 2, are two members of the Human Herpesviridae family, a set of viruses that cause viral infections in the majority of humans.

[0037] "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 to the amino acid residues in the compared polypeptide, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, 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 necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, and 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.

[0038] A coding sequence is "under the control of" or "operably 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.

[0039] As used herein, the terms "specific binding" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or a particular polypeptide target means, for example, at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 K against M or more targets D In some embodiments, the term "specific binding" refers to binding by a molecule to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. D 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 the binding of a molecule compared to the binding of a control molecule, which is a molecule of similar structure that generally does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, for example, excess unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0040] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as rhesus and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is human.

[0041] "Cancer" refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" can include tumors. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to "originate" from the pre-metastatic tumor. As used herein, "cancer" refers to solid tumors, including sarcomas, carcinomas, and lymphomas.

[0042] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the intent to cure, reverse, alleviate, ameliorate, inhibit, slow, or prevent the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. In some embodiments, the disease is cancer.

[0043] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any quantity of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of disability or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0044] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal being treated therewith.

[0045] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as 1 / 10 and hundredths of integers), unless otherwise specified. The recitation of endpoints includes ranges between all disclosed endpoints. For example, recitation of 1, 2, or 3 includes the ranges 1 to 2, 2 to 3, and 1 to 3.

[0046] II. Oncolytic Viruses Oncolytic virus cancer immunotherapy is an emerging and mature treatment modality that uses replication-competent viruses that selectively infect and damage tumor cells and, preferably, induce immunological responses that can control both the target tumor and distant tumors. Each type of oncolytic virus has a different cellular tropism, which helps determine which tissues are preferentially infected. Viral engineering can expand, restrict, or regulate this host range. Various species of viruses are being investigated for use in oncolytic therapy, including those derived from HSV, vaccinia, and reovirus.

[0047] Thus, the present application provides oncolytic viruses effective for treating cancer. Non-limiting examples of oncolytic viruses include those derived from herpes simplex virus, vaccinia virus, adenovirus, reovirus, or vesicular stomatitis virus. Preferentially, oncolytic viruses (such as oncolytic HSV) preferentially induce an immune response that leads to the killing of tumor cells. As used herein, a virus "preferentially kills" tumor cells if a particular infectious dose of the virus is more likely to kill tumor cells than adjacent healthy cells (e.g., if a given dose is at least two-fold more likely to kill tumor cells than adjacent healthy cells). Preferentially, the oncolytic virus expresses one or more payload proteins described below. Preferentially, the oncolytic virus induces an immune response against tumors and, in some embodiments, kills tumor cells at sites distal to the site of infection. Preferably, the oncolytic virus is capable of evading an individual's immune system after administration to the individual. As used herein, evading an individual's immune system means that the oncolytic virus can replicate preferentially in tumor cells. In some embodiments, the oncolytic viruses provided herein are more susceptible to innate antiviral responses than wild-type viruses, allowing them to replicate preferentially in tumor cells. In some embodiments, the oncolytic viruses provided herein have moderate resistance to interferon.

[0048] II-A. Oncolytic Herpes Simplex Virus Herpes simplex virus (HSV) replicates in a variety of cell types, including epithelial cells and fibroblasts. Two members of the human herpesvirus family are HSV-1 and HSV-2. Natural HSV establishes a lifelong latent infection in neuronal cell bodies within the sensory ganglia of infected individuals. During the productive phase, HSV genes are classified into three broad classes based on the temporal order of their expression: immediate-early (IE), early (E), and late (L). Late genes can be further divided into two subclasses: leaky late genes, which are expressed at low levels early after infection and upregulated after infection, and true late genes, which are expressed exclusively after and dependent on DNA replication.

[0049] Wild-type mature HSV contains a linear, double-stranded DNA genome of approximately 152 kb encoding at least 74 genes enclosed in an icosahedral capsid composed of 162 capsomers derived from six different viral proteins, 20–23 different viral target proteins, and an envelope containing different glycoproteins. During adsorption to host cells, the glycoproteins interact with surface receptors (and each other) to promote fusion of the viral envelope with the host membrane, allowing the virus to enter the cell.

[0050] In one aspect, the present disclosure relates to oncolytic herpes simplex viruses (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 US12. 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 native HSV proteins in a different temporal order, such as expressing immediate-early US11. The oncolytic HSV can be a component of a pharmaceutical composition described herein. Oncolytic HSV or pharmaceutical compositions comprising oncolytic HSV can be administered to an individual according to the methods described herein (e.g., the treatment methods described herein). In some embodiments, the oncolytic HSV preferentially elicits an immune response that leads to tumor cell killing compared to the wild-type HSV from which it is derived. In some embodiments, the oncolytic HSV can elicit an immune response that triggers tumor cell killing at one or more sites distal to the target site.

[0051] III. Viral Payload In some embodiments, an oncolytic virus (such as an oncolytic HSV) or a gene cassette described elsewhere herein comprises one or more genes encoding one or more payload molecules. The payload molecules are generally intended to enhance the therapeutic efficacy of the oncolytic virus (such as an oncolytic HSV). For example, the payload molecules may enhance an immune response (e.g., against a tumor target) or enhance the cytotoxicity of the oncolytic virus.

[0052] III-A.IL-12 In some embodiments, the oncolytic virus (such as oncolytic HSV) or expression cassette described elsewhere herein comprises a polynucleotide encoding interleukin-12 (IL-12).

[0053] IL-12 is a heterodimeric protein containing two subunits: p35 and p40. The native p35 subunit is linked to the p40 subunit by a disulfide bond. 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, JJ, 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 (containing both subunits in a heterodimer) functions to differentiate naive T cells into Th1 cells, promote the cytotoxic activity of NK cells and T cells, and block angiogenesis.

[0054] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere 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 the p40 subunit of IL-12 are human. In some embodiments, the p35 subunit and / or the p40 subunit of IL-12 are murine. In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere 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 the p35 subunit of IL-12 and the p40 subunit of IL-12 linked by a peptide linker.

[0055] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as otherwise described herein, comprises a polynucleotide encoding the human p35 subunit of IL-12 and / or a polynucleotide encoding the 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% identity 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% identity 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 NO:9 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% identity to the amino acid sequence set forth in SEQ ID NO:9.In some embodiments, an oncolytic virus (such as an oncolytic HSV) or an expression cassette described elsewhere 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 the human p35 subunit of IL-12 and the human p40 subunit of IL-12 linked by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising a glycine residue and a serine residue. 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:7 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:7.In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO:4 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% identity to the amino acid sequence set forth in SEQ ID NO:4. In 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% identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0056] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as otherwise described herein, comprises a polynucleotide encoding the murine p35 subunit of IL-12 and / or a polynucleotide encoding the 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% identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the mouse 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% identity to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the mouse 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% identity to the amino acid sequence set forth in SEQ ID NO:11.In some embodiments, an oncolytic virus (such as an oncolytic HSV) or an expression cassette described elsewhere 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 the murine p35 subunit of IL-12 and the murine p40 subunit of IL-12 linked by a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence comprising a glycine residue and a serine residue. 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:7 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:7.In some embodiments, the IL-12 heterodimer comprises the amino acid sequence of SEQ ID NO:8 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% identity to the amino acid sequence set forth in SEQ ID NO:8. In 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% identity to the amino acid sequence set forth in SEQ ID NO: 12.

[0057] III-B. CD40 agonists Cluster of differentiation 40 (CD40) is a costimulatory polypeptide expressed on numerous cell types, from antigen-presenting cells (APCs) to epithelial cells. It is also present on various cancer cells. CD40 agonist, also known as cluster of differentiation 154 (CD154), is a type II membrane glycopeptide containing 261 amino acids and expressed on the surface of activated T cells. Natural CD40 agonist promotes B cell maturation. It is also essential for immunoglobulin class switching, as its absence is associated with hyper-IgM syndrome. CD40 agonist exists as a membrane-bound form in which the extracellular domain forms a homotrimer, and as a proteolytically cleaved soluble form, which has been shown to be biologically active.

[0058] 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 described elsewhere herein comprises a polynucleotide encoding a 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 trimer formation of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is fused to an Fc region, e.g., to direct trimer formation of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the Fc region is an IgG Fc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions, insertions, or deletions that abrogate binding of the Fc region to another Fc region, e.g., an IgG Fc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region comprises a substitution of an IgG interacting domain with an IgA interacting domain. In some embodiments, each of the three single-chain trimeric CD40 ligand ectodomains is bivalent. In some embodiments, the CD40 agonist is an agonistic antibody.

[0059] 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% identity 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 ectodomain comprises a polypeptide comprising three human CD40 ligand ectodomains linked by a peptide linker. In some embodiments, the single-chain trimeric human CD40 ligand ectodomain polypeptide comprises a first human CD40 ligand ectodomain connected to a second human CD40 ligand ectodomain, which is connected to a third human CD40 ligand ectodomain by a peptide linker. In some embodiments, the peptide linker comprises a glycine residue and a serine residue. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:22 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: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 or an amino acid sequence having 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% identity to 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 including a glycine and / or serine residue. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:23.In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having 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% identity to the amino acid sequence set forth in 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: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% identity 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% identity to the amino acid sequence set forth in SEQ ID NO:30.In some embodiments, the CD40 agonist forms 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% identity to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the CD40 agonist forms 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% identity to the amino acid sequence set forth in SEQ ID NO:30.

[0060] 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% identity 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 mouse CD40 ligand ectodomain comprises a polypeptide comprising three mouse CD40 ligand ectodomains linked by a peptide linker. In some embodiments, the single-chain trimeric mouse CD40 ligand ectodomain polypeptide comprises a first mouse CD40 ligand ectodomain connected to a second mouse CD40 ligand ectodomain, which is connected to a third mouse CD40 ligand ectodomain by a peptide linker. In some embodiments, the peptide linker comprises a glycine residue and a serine residue. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:22 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: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 or an amino acid sequence having 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% identity to 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 including leucine, glycine, and / or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:23.In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having 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% identity to the amino acid sequence set forth in 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 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% identity to the amino acid sequence set forth in 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% identity 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 an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the CD40 agonist forms a trimer comprising three polypeptides 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% identity to the amino acid sequence set forth in SEQ ID NO:29.

[0061] III-C. CTLA-4 binding protein Cytotoxic T lymphocyte-associated protein 4 (CTLA-4 or CTLA-4), also known as cluster of differentiation 152 (CD152), 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. Different isoforms have been characterized. CTLA-4 is constitutively expressed in regulatory T cells but is only upregulated in conventional T cells after activation, contributing 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 to induce its inhibitory function on T cells.

[0062] In some embodiments, the oncolytic virus (such as oncolytic HSV) or expression cassette described elsewhere 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, mouse CTLA-4, or both human CTLA-4 and mouse CTLA-4.

[0063] 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 IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or murine IgG1, IgG2, IgG3, or IgG4 constant domain. In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a single-chain variable fragment (scFv). In some embodiments, the anti-CTLA-4 scFv is fused to the N-terminus of an IgG1, IgG2, IgG3, or IgG4 constant domain, e.g., a human or murine IgG1, 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 IgG1, IgG2, IgG3, or IgG4 Fc, e.g., a human or murine IgG1, IgG2, IgG3, or IgG4 Fc.

[0064] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof, e.g., 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 an IgG1 constant domain, e.g., a human IgG1 constant domain. In some embodiments, the human IgG1 is a variant human IgG1 containing a C220S substitution, where residue numbering is according to EU numbering. In some embodiments, the human IgG1 is G1m(17)IgG1. In some embodiments, the anti-CTLA-4 antibody causes depletion of regulatory T (Treg) cells.

[0065] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an 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) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 40; (b) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 41; (c) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and / or the VL comprises one or more of: (a) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 43; (b) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 44; and (c) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises a VH and a VL, wherein the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0066] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., 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% identity 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% identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the variable heavy chain and variable light chain are linked via a linker sequence. In some embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0067] In some embodiments, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an anti-CTLA-4 scFv) comprises a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VH amino acid sequence of SEQ ID NO: 46, wherein the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises a VL comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VL amino acid sequence of SEQ ID NO: 47, and the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an anti-CTLA-4 scFv) comprises a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VH amino acid sequence of SEQ ID NO: 46, and contains substitutions (e.g., conservative substitutions, insertions, or deletions relative to the reference sequence), but wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the VH amino acid sequence of SEQ ID NO: 46. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in the VH amino acid sequence of SEQ ID NO: 46.In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FR regions). In some embodiments, the substitutions, insertions, or deletions occur in the FR regions. Optionally, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises the VH sequence of SEQ ID NO: 46, including post-translational modifications of the sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 40, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an anti-CTLA-4 scFv) comprises a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VL amino acid sequence of SEQ ID NO: 47, and contains substitutions (e.g., conservative substitutions, insertions, or deletions relative to the reference sequence), but wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the VL amino acid sequence of SEQ ID NO: 47. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in the VL amino acid sequence of SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., the FR regions). In some embodiments, the substitutions, insertions, or deletions occur in the FR region. Optionally, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises the VL sequence of SEQ ID NO: 47, including post-translational modifications of the sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 43, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 45.In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:46 and a VL comprising the amino acid sequence set forth in SEQ ID NO:47.

[0068] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 scFv) comprises an IgG1 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% identity 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 NO: 48, with or without the C-terminal lysine.

[0069] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., 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% identity to the amino acid sequence set forth in SEQ ID NO:60.

[0070] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., 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., 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% identity to the amino acid sequence set forth in SEQ ID NO: 50.

[0071] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof, e.g., anti-CTLA-4 VHH, specifically binds to mouse CTLA-4. In some embodiments, the anti-CTLA-4 VHH is fused to the heavy chain of mouse IgG2a Fc.

[0072] In some embodiments, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an 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, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an 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., an 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% identity to the amino acid sequence set forth in SEQ ID NO:54. In some embodiments, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an anti-CTLA-4 VHH) comprises a VH comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VH amino acid sequence of SEQ ID NO: 54, 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, an anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., an anti-CTLA-4 VHH) comprises a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to the VH amino acid sequence of SEQ ID NO: 54, and contains substitutions (e.g., conservative substitutions, insertions, or deletions relative to the reference sequence), but wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the VH amino acid sequence of SEQ ID NO: 54. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in the VH amino acid sequence of SEQ ID NO: 54. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., the FR regions). In some embodiments, the substitutions, insertions, or deletions occur in the FR region. Optionally, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., anti-CTLA-4 VHH) comprises the VH sequence of SEQ ID NO: 54, including post-translational modifications of the sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 51, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and 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., anti-CTLA-4 VHH) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 54.

[0073] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., 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% identity 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., 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% identity to the amino acid sequence set forth in SEQ ID NO:59.

[0074] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof (e.g., 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., anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO: 56, 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% identity to 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., anti-CTLA-4 VHH) comprises the amino acid sequence set forth in SEQ ID NO:57, 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% identity to the amino acid sequence set forth in SEQ ID NO:57.

[0075] III-D. FLT 3 Ligand In some embodiments, an oncolytic virus (such as an oncolytic HSV) or an expression cassette described elsewhere herein comprises a polynucleotide encoding fms-like tyrosine kinase 3 (FLT3) ligand (FLT3L), a growth and differentiation factor that enhances and expands dendritic cells (DCs) and recruits DCs to the tumor microenvironment. Intratumoral DCs (IT DCs) have been identified as important mediators of antitumor T cell responses by processing tumor antigens and restimulating effector T cells in tertiary lymphoid structures or priming naive T cells in draining lymph nodes (Broz et al., Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell. 2014;26(5):638-652. doi:10.1016 / j.ccell.2014.09.007; and Cueto and Sancho, The Flt3L / Flt3 axis in dendritic cell biology and cancer immunotherapy. Cancers (Basel). 2021;13(7):1525. Published 2021 Mar 26. doi:10.3390 / cancers13071525). Multiple clinical trials are investigating the use of systemic FLT3L to boost antitumor T cell responses.

[0076] FLT3L naturally functions as a cytokine and growth factor. It binds to FLT3 (CD135). The human FLT3L polynucleotide encodes a 235-amino acid type I transmembrane protein. Human FLT3L contains an N-terminal 26-residue signal peptide, a 156-residue extracellular domain, a 23-residue transmembrane domain, and a 30-residue cytoplasmic domain. FLT3L can be released from the cell membrane by proteolytic cleavage. Soluble FLT3L naturally forms a noncovalent dimer through the interaction of six cysteine ​​residues.

[0077] In some embodiments, an oncolytic virus (such as an oncolytic HSV) or an expression cassette described elsewhere herein comprises a polynucleotide encoding 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% identity to the amino acid sequence set forth in SEQ ID NO: 72.

[0078] In some embodiments, human FLT3L comprises a signal peptide that directs secretion to the plasma membrane. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, 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% identity to the amino acid sequence set forth in SEQ ID NO: 71.

[0079] In some embodiments, FLT3L, for example, human FLT3L, is a homodimer. In some embodiments, human FLT3L is proteolytically processed into soluble FLT3L. In some embodiments, soluble FLT3L forms a homodimer.

[0080] III-E. Other payload molecules In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere herein, comprises one or more polynucleotides encoding a US11 protein, such as a US11 protein from an HSV, e.g., HSV-1 or HSV-2.

[0081] The protein kinase R (PKR) pathway is a component of the host cell's innate antiviral response. PKR is activated in response to binding of double-stranded RNA (dsRNA), a by-product of viral replication, leading to the phosphorylation and inactivation of the translation initiation factor eukaryotic translation initiation factor 2 subunit 1 (eIF2α). Phosphorylated eIF2α prevents translation initiation, a cellular defense mechanism aimed at blocking viral protein production. The US11 protein is thought to bind to and capture dsRNA, preventing activation of the PKR pathway in host cells and allowing enhanced viral replication.

[0082] 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% identity to the amino acid sequence set forth in SEQ ID NO:80.

[0083] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere herein, comprises a polynucleotide encoding a US11 protein, wherein the polynucleotide comprises a native US11 gene nucleotide sequence from an HSV, e.g., HSV-1 or HSV-2. In some embodiments, the native US11 gene is a native US11 late gene, and the US11 protein is expressed in late stages of viral replication. In some embodiments, the native US11 late gene is under the control of an endogenous US11 promoter from an HSV, e.g., HSV-1 or HSV-2.

[0084] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere 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 from an HSV, e.g., HSV-1 or 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, hi some embodiments, the promoter is an endogenous US12 promoter from HSV, such as HSV-1 or HSV-2, or a portion thereof.

[0085] In some embodiments, an oncolytic virus (such as an oncolytic HSV), or an expression cassette as described elsewhere herein, comprises both a polynucleotide encoding a US11 protein and a polynucleotide 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.

[0086] In some embodiments, an oncolytic virus (e.g., oncolytic HSV) or an expression cassette described elsewhere herein comprises a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor, such as a viral TAP inhibitor. Generally, 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 presentation on 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):e1004743). While TAP inhibition disrupts the transport of newly expressed MHC molecules to the cell surface, it does not block pre-existing antigen display. Thus, TAP inhibition by a TAP inhibitor can prevent the display of viral antigens on the cell surface, preventing premature elimination of infected cells and allowing viral persistence through multiple rounds of viral replication.

[0087] In some embodiments, the TAP inhibitor is derived from herpesvirus 1 or herpesvirus 2. In some embodiments, the TAP inhibitor is derived from bovine herpesvirus 1. In some embodiments, the TAP inhibitor is either 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% identity 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 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% identity to the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the TAP inhibitor is expressed during the immediate early stage of viral replication, i.e., 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.

[0088] IV. Expression Cassettes 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 a FLT3 ligand (FLT3L), or any combination thereof.

[0089] IV-A. Expression cassettes encoding IL-12, CD40 agonists, and / or CTLA-4 binding proteins 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.

[0090] In some embodiments, the expression cassette of the present disclosure comprises a promoter operably linked to each of the polynucleotides encoding IL-12, the CD40 agonist, and / or the CTLA-4 binding protein. Any suitable promoter may be used in the cassette of the present disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary, non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotin beta herpesvirus 1 (AoHV 1) promoter, the CAG promoter, the CMV hybrid promoter, the EF1a promoter, the MMLV 5' long terminal repeat (LTR) derived from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and native HSV promoter sequences, such as the HSV-1 or HSV-2 US12 promoter, or the HSV-1 or HSV-2 US11 promoter.

[0091] In some embodiments, the expression cassette of the present disclosure comprises a polyadenylation signal operably linked to each of the polynucleotides encoding IL-12, CD40 agonists, and / or CTLA-4 binding proteins. Any suitable polyadenylation signal can be used in the cassette of the present disclosure. Exemplary, non-limiting polyadenylation signals (polyA or pA) that can be used include simian vacuolating virus 40 polyA (SV40pA), human β-globin polyA (hBGpA), human growth hormone polyA (hGH polyA), rabbit β-globin polyA (rBGpA), bovine growth hormone polyadenylation (BGHpA), polyA from the human GAPDH gene, and native HSV polyA sequences such as US10-12 polyA or US9-10 polyA from HSV-1 or HSV-2.

[0092] In some embodiments, the expression cassettes of the present disclosure may comprise any suitable promoter and / or polyadenylation signal known in the art or described herein operably linked to either a polynucleotide encoding IL-12, a polynucleotide encoding a CD40 agonist, and / or a polynucleotide encoding a CTLA-4 binding protein.

[0093] In some embodiments, the expression cassettes of the present disclosure comprise an RNA polymerase II transcription pausing signal located after each of the polynucleotides encoding IL-12, CD40 agonists, and / or CTLA-4 binding proteins. Any suitable RNA polymerase II transcription pausing signal may be used in the cassettes of the present disclosure. Exemplary, non-limiting RNA polymerase II transcription pausing signals include the human complement C2 protein terminator (C2) and the human gastrin terminator (hGT).

[0094] In some embodiments, an expression cassette of the present disclosure comprises, in order, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL-12. In some embodiments, the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding IL-12 are in the same orientation in the expression cassette, and the polynucleotide encoding the CD40 agonist is in the opposite orientation relative to the polynucleotide encoding the CTLA-4 binding protein and the polynucleotide encoding IL-12.

[0095] 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 mCMV promoter comprises the nucleotide sequence of SEQ ID NO:210 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:210. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, e.g., any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is polyA derived from the human GAPDH gene.In some embodiments, the polyadenylation signal is a GAPDH_SPA polyadenylation signal comprising the nucleotide sequence of SEQ ID NO:213 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:213. In some embodiments, the expression cassette further comprises a Kozak sequence located between the promoter and the polynucleotide encoding the CTLA-4 binding protein. In some embodiments, the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:211, or a nucleotide sequence having any of 5, 4, 3, 2, or 1 base substitutions relative to the nucleotide sequence of SEQ ID NO:211. In some embodiments, the expression cassette further comprises an RNA polymerase II transcription pausing signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcription pausing signal known in the art or described herein. In some embodiments, the RNA polymerase II transcription pausing signal is a C2 RNA polymerase II transcription pausing signal.In some embodiments, the C2 RNA polymerase II transcription pausing signal comprises the nucleotide sequence of SEQ ID NO:214 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:214. 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. In some specific embodiments, the encoded CTLA-4 binding protein 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% identity to the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the CTLA-4 binding protein comprises the amino acid sequence set forth in SEQ ID NO: 50, with or without the C-terminal lysine.In some specific embodiments, the encoded CTLA-4 binding protein 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% identity to the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the encoded CTLA-4 binding protein comprises the amino acid sequence set forth in SEQ ID NO: 60 and does not include the C-terminal lysine. In other embodiments, the encoded CTLA-4 binding protein comprises the amino acid sequence set forth in SEQ ID NO:56, 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% identity to the amino acid sequence set forth in SEQ ID NO:56. In other embodiments, the encoded CTLA-4 binding protein comprises the amino acid sequence set forth in SEQ ID NO:57, 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% identity to the amino acid sequence set forth in SEQ ID NO:57.In other embodiments, the encoded CTLA-4 binding protein 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% identity to the amino acid sequence set forth in SEQ ID NO:58. In other embodiments, the encoded CTLA-4 binding protein 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% identity to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the polynucleotide encoding the CTLA-4 binding protein comprises the nucleotide sequence of SEQ ID NO:212 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:212.In some embodiments, the polynucleotide encoding the CTLA-4 binding protein comprises the nucleotide sequence of SEQ ID NO:303 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:303.

[0096] 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 AOHV1 promoter comprises the nucleotide sequence of SEQ ID NO:219 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:219. In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the CD40 agonist, e.g., any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is hBGpA.In some embodiments, hBGpA comprises the nucleotide sequence of SEQ ID NO:216 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:216. In some embodiments, the expression cassette further comprises a Kozak sequence located between the promoter and the polynucleotide encoding the CD40 agonist. In some embodiments, the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:218, or a nucleotide sequence having any of 5, 4, 3, 2, or 1 base substitutions relative to the nucleotide sequence of SEQ ID NO:218. In some embodiments, the expression cassette further comprises an RNA polymerase II transcription pausing signal positioned after the polyadenylation signal, such as any suitable RNA polymerase II transcription pausing signal known in the art or described herein. In some embodiments, the RNA polymerase II transcription pausing signal is an hGT RNA polymerase II transcription pausing signal.In some embodiments, the hGT RNA polymerase II transcription pausing signal comprises the nucleotide sequence of SEQ ID NO:215 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:215. In some embodiments, the encoded CD40 agonist is any of the CD40 agonists described herein, e.g., in Section III-B or V herein. In some specific embodiments, the encoded CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having about 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% identity to the amino acid sequence set forth in SEQ ID NO:25.In some specific embodiments, the encoded 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% identity to the amino acid sequence set forth in SEQ ID NO:30. In other embodiments, the encoded CD40 agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:28, 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% identity to the amino acid sequence set forth in SEQ ID NO:28. In other embodiments, the encoded 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% identity to the amino acid sequence set forth in SEQ ID NO:29.In some embodiments, the polynucleotide encoding the CD40 agonist comprises the nucleotide sequence of SEQ ID NO:217 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:217. In some embodiments, the polynucleotide encoding the CD40 agonist comprises the nucleotide sequence of SEQ ID NO:308 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:308. In some embodiments, the polynucleotide encoding the CD40 agonist is in a reverse orientation within the expression cassette compared to the polynucleotide encoding IL-12 and the polynucleotide encoding the CTLA-4 binding protein.

[0097] 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 an MMLV promoter. In some embodiments, the MMLV promoter comprises the nucleotide sequence of SEQ ID NO:220 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:220. In some embodiments, the expression cassette further comprises a polyadenylation signal positioned after the polynucleotide encoding IL-12, e.g., any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is US10-12 polyA or US9-10 polyA from HSV, such as HSV-1 or HSV-2.In some embodiments, the US9-10 polyA comprises the nucleotide sequence of SEQ ID NO:314 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:314. In some embodiments, the US10-12 polyA has the nucleotide sequence of a native HSV-1 or HSV-2 US10-12 polyA, or a native HSV-1 or HSV-2 The expression cassette comprises 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 US10-12 poly A. In some embodiments, the expression cassette further comprises a Kozak sequence located between the promoter and the polynucleotide encoding IL-12. In some embodiments, the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:221 or a nucleotide sequence having any of 5, 4, 3, 2, or 1 base substitutions relative to the nucleotide sequence of SEQ ID NO:221.In some embodiments, the encoded IL-12 is any of the IL-12 proteins described herein, e.g., in Section III-A, above. In some specific embodiments, the encoded IL-12 comprises the amino acid sequence of SEQ ID NO:4, 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% identity to the amino acid sequence set forth in SEQ ID NO:4. In some specific embodiments, the encoded IL-12 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% identity to the amino acid sequence set forth in SEQ ID NO:10. In other embodiments, the encoded IL-12 comprises the amino acid sequence of SEQ ID NO:8 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% identity to the amino acid sequence set forth in SEQ ID NO:8.In other embodiments, the encoded IL-12 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% identity to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the polynucleotide encoding IL-12 comprises the nucleotide sequence of SEQ ID NO:222 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:222.In some embodiments, the polynucleotide encoding IL-12 comprises the nucleotide sequence of SEQ ID NO:313 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:313.

[0098] In some embodiments, the expression cassette of the present disclosure further comprises a polynucleotide encoding a US10 protein and / or a polynucleotide encoding a US11 protein; or a polynucleotide encoding a US11 protein and a US10 protein.

[0099] In some embodiments, an expression cassette of the present disclosure comprises a polynucleotide encoding a US10 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 HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the US11 promoter comprises the nucleotide sequence of SEQ ID NO:207 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:207. In some embodiments, the polynucleotide encoding the US10 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 the endogenous US10 promoter. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the polynucleotide encoding the US10 protein, such as any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is hGH polyA.In some embodiments, the hGH polyA comprises the nucleotide sequence of SEQ ID NO:209 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:209. 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 encoded US11 protein comprises the amino acid sequence set forth in 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% identity to the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the encoded US10 protein is an HSV US10 protein, such as an HSV-1 or HSV-2 US10 protein.In some embodiments, the encoded US10 protein comprises the amino acid sequence set forth in SEQ ID NO: 90, 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% identity to the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the polynucleotide encoding the US11 protein comprises a native US11 gene. In some embodiments, the expression cassette comprises, in order, a polynucleotide encoding a US11 protein (e.g., including a naturally occurring US11 gene) and / or a polynucleotide encoding a US10 protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL-12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, IL-12, US11 protein, and / or US10 protein are in the same orientation within the expression cassette, and the polynucleotide encoding the CD40 agonist is in the opposite orientation to the polynucleotides encoding the CTLA-4 binding protein, IL-12, US11 protein, and / or US10 protein.

[0100] In some embodiments, the expression cassette of the present disclosure comprises a polynucleotide encoding a US11 protein and a US10 protein. In some embodiments, the polynucleotide encoding the US11 protein and the US10 protein comprises a nucleic acid sequence encoding a US11 protein and a nucleic acid sequence encoding a US10 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 US10 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 HSV, such as HSV-1 or HSV-2. In some embodiments, the endogenous US11 promoter directs late expression of the US11 protein during viral replication. In some embodiments, the US11 promoter comprises the nucleotide sequence of SEQ ID NO: 207 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: 207. In some embodiments, a nucleic acid sequence encoding a US10 protein is operably linked to a promoter. In some embodiments, the promoter is the native US10 promoter from HSV, such as HSV-1 or HSV-2. In some embodiments, the promoter is integrated 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 US11 protein. In some embodiments, the encoded US11 protein comprises the amino acid sequence set forth in 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% identity to the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the encoded US10 protein is an HSV US10 protein, such as an HSV-1 or HSV-2 US10 protein. In some embodiments, the encoded US10 protein comprises the amino acid sequence set forth in SEQ ID NO:90, 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% identity to the amino acid sequence set forth in SEQ ID NO:90.In some embodiments, the polynucleotides encoding the US10 and US11 proteins comprise the nucleotide sequence of SEQ ID NO:208 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:208. In some embodiments, the expression cassette comprises a polyadenylation signal operably linked to the nucleic acid sequence encoding the US10 protein, e.g., any suitable polyadenylation signal known in the art or described herein. In some embodiments, the polyadenylation signal is hGH polyA. In some embodiments, the hGH polyA comprises the nucleotide sequence of SEQ ID NO:209 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:209.In some embodiments, the polynucleotides encoding the US11 and US10 proteins comprise a native US11 gene. In some embodiments, the expression cassette comprises, in order, polynucleotides encoding the US11 and US10 proteins (e.g., comprising a native US11 gene), a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL-12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, IL-12, and the US11 and US10 proteins are in the same orientation in the expression cassette, and the polynucleotide encoding the CD40 agonist is in the opposite orientation to the polynucleotides encoding the CTLA-4 binding protein, IL-12, and the US11 and US10 proteins.

[0101] In some embodiments, the expression cassette of the present 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 US12 promoter from HSV, such as HSV-1 or HSV-2, or a portion thereof. In some embodiments, the endogenous US12 promoter or a portion thereof directs immediate-early expression of the US11 protein during viral replication. In some embodiments, the endogenous US12 promoter comprises the nucleotide sequence of SEQ ID NO:203 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:203.In some embodiments, the encoded US11 protein comprises the amino acid sequence set forth in 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% identity to the amino acid sequence set forth in SEQ ID NO:80. 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 expression cassette further comprises a 5' untranslated region (UTR) sequence located between the promoter and the variant US11 gene.In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO:223 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:223. In some embodiments, the expression cassette further comprises a polynucleotide encoding a US12 protein located after the variant US11 gene (e.g., after the stop codon in the variant US11 gene). In some embodiments, the US12 protein is derived from HSV, such as HSV-1 or HSV-2. In some embodiments, the polynucleotide encoding the US12 protein is not operably linked to a promoter. In some embodiments, the encoded US12 protein is not expressed.In some embodiments, the polynucleotide encoding the US12 protein comprises the nucleotide sequence of SEQ ID NO:206 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:206. In some embodiments, the expression cassette further comprises a spacer sequence and a UTR sequence located between the variant US11 gene and the polynucleotide encoding the US12 protein. In some embodiments, the spacer sequence and the UTR sequence comprise the nucleotide sequence of SEQ ID NO:205 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:205.In some embodiments, the expression cassette comprises, in order, a variant US11 gene, a polynucleotide encoding a US10 and / or US11 protein, or a polynucleotide encoding a US10 and US11 protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL-12. In some embodiments, the polynucleotides encoding the CTLA-4 binding protein, IL-12, and the US10 and / or US11 protein are in the same orientation within the expression cassette, and the polynucleotide encoding the CD40 agonist is in the opposite orientation to the polynucleotides encoding the CTLA-4 binding protein, IL-12, and the US10 and / or US11 protein.

[0102] In some embodiments, an expression cassette of the disclosure comprises, in order, a promoter (e.g., an HSV US12 promoter) operably linked to a polynucleotide comprising a variant US11 gene; optionally, a 5'UTR sequence; a polynucleotide comprising a variant US11 gene; a promoter (e.g., a native HSV US11 promoter); a polynucleotide encoding a US11 protein and a US10 protein; a polyadenylation signal (e.g., an hGHpA polyA) operably linked to the polynucleotide encoding the US11 protein and the US10 protein; a promoter (e.g., a CMV promoter, e.g., an mCMV promoter) directing expression of a polynucleotide encoding a CTLA-4 binding protein; optionally, a Kozak sequence for expression of a polynucleotide encoding a CTLA-4 binding protein; a polynucleotide encoding a CTLA-4 binding protein; a polyadenylation signal (e.g., GAPDH_SpA polyA) operably linked to the polynucleotide encoding the CTLA-4 binding protein; and optionally, an RNA polymerase II pause site ( For example, a C2 pause site; an RNA polymerase II pause site (e.g., an hGT pause site); a polyadenylation signal (e.g., hBGpA polyA) operably linked to a polynucleotide encoding a CD40 agonist; a polynucleotide encoding a CD40 agonist; optionally, a Kozak sequence for expression of a polynucleotide encoding a CD40 agonist; a promoter (e.g., an AoHV1 promoter) that controls expression of a CD40 agonist; a promoter (e.g., an MMLV promoter) that controls expression of IL-12; optionally, a Kozak sequence for expression of a polynucleotide encoding IL-12; a polynucleotide encoding IL-12; and a polyadenylation signal (e.g., HSV US10-12 polyA) operably linked to a polynucleotide encoding IL-12.

[0103] In some embodiments, an expression cassette of the present 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 present 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.

[0104] In some embodiments, the expression cassette of the present disclosure is integrated into the genome of a virus, such as an oncolytic HSV, e.g., oncolytic HSV-1 or oncolytic HSV-2. In some embodiments, the cassette is integrated into the US10-12 locus of an oncolytic HSV, e.g., oncolytic HSV-1 or oncolytic HSV-2.

[0105] In some embodiments, the expression cassette comprises (i) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, and (iii) a polynucleotide encoding a CTLA-4 binding protein, e.g., as described above. In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, at IRS-(i)-(ii)-(iii).

[0106] In some embodiments, the expression cassette further comprises a polynucleotide encoding a US10 protein and / or a US11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, (iii) a polynucleotide encoding a CTLA-4 binding protein, and (iv) a polynucleotide encoding a US10 protein and / or a US11 protein. In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, in IRS-(i)-(ii)-(iii)-(iv). In other embodiments, the expression cassette further comprises polynucleotides encoding a US10 protein and a US11 protein, e.g., as described above. In some such embodiments, the expression cassette comprises (i) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, (iii) a polynucleotide encoding a CTLA-4 binding protein, and (iv) a polynucleotide encoding US10 and US11 proteins. In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., an oncolytic HSV genome, such as an HSV-1 or HSV-2 genome, in 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) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, (iii) a polynucleotide encoding a CTLA-4 binding protein, (iv) a polynucleotide encoding a US10 protein, and (v) a polynucleotide encoding a US11 protein.In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., IRS-(i)-(ii)-(iii)-(iv)-(v) of an oncolytic HSV genome (such as an HSV-1 or HSV-2 genome).

[0107] 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) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, (iii) a polynucleotide encoding a CTLA-4 binding protein, (iv) a polynucleotide encoding a US10 protein and / or a US11 protein, or a polynucleotide encoding a US10 protein and a US11 protein, and (v) a polynucleotide comprising a variant US11 gene. In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., IRS-(i)-(ii)-(iii)-(iv)-(v) of an oncolytic HSV genome (such as an HSV-1 or HSV-2 genome). 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) a polynucleotide encoding IL-12, (ii) a polynucleotide encoding a CD40 agonist, (iii) a polynucleotide encoding a CTLA-4 binding protein, (iv) a polynucleotide encoding a US10 protein, (v) a polynucleotide encoding a US11 protein, or (vi) a polynucleotide comprising a variant US11 gene. In some embodiments, the expression cassette is oriented relative to an internal short repeat (IRS) region of the genome, e.g., IRS-(i)-(ii)-(iii)-(iv)-(v)-(vi) of an oncolytic HSV genome (such as an HSV-1 or HSV-2 genome).

[0108] IV-B. Expression cassettes encoding FLT3L and / or TAP inhibitors Also provided herein are expression cassettes comprising a polynucleotide encoding FLT3L and / or a polynucleotide encoding a transporter associated with antigen processing (TAP) inhibitor.

[0109] In some embodiments, the expression cassette of the present disclosure comprises a promoter operably linked to a polynucleotide encoding FLT3L and / or a polynucleotide encoding a TAP inhibitor. Any suitable promoter may be used in the cassette of the present disclosure, so long as the promoter drives expression of the associated polynucleotide. Exemplary, non-limiting promoters that may be used include the human cytomegalovirus (hCMV) promoter, the murine cytomegalovirus (mCMV) promoter, the Aotin beta herpesvirus 1 (AoHV 1) promoter, the CAG promoter, the CMV hybrid promoter, the EF1a promoter, the MMLV 5' long terminal repeat (LTR) from the Moloney murine leukemia virus promoter (i.e., the MMLV promoter), the Pbidir3 promoter, and the native HSV promoter sequence.

[0110] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding FLT3L and / or the polynucleotide encoding a TAP inhibitor. Any suitable polyadenylation signal can be used in the cassettes of the present disclosure. Exemplary, non-limiting polyadenylation signals (polyA or pA) that can be used include simian vacuolating virus 40 polyA (SV40pA), human beta globin polyA (hBGpA), human growth hormone polyA (hGH polyA), rabbit beta globin polyA (rBGpA), bovine growth hormone polyadenylation (BGHpA), polyA from the human GAPDH gene, and the native HSV polyA sequence.

[0111] In some embodiments, the expression cassettes of the present disclosure may comprise any suitable promoter and / or polyadenylation signal known in the art or described herein operably linked to any of the polynucleotides encoding FLT3L and / or a polynucleotide encoding a TAP inhibitor.

[0112] In some embodiments, the encoded FLT3L is any of the FLT3L proteins described herein, e.g., in Section III-D, above. In one particular embodiment, the encoded 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% identity to the amino acid sequence set forth in SEQ ID NO:71. In one specific embodiment, the encoded 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% identity to the amino acid sequence set forth in SEQ ID NO:72. In some embodiments, the polynucleotide encoding FLT3L comprises the nucleotide sequence of SEQ ID NO: 105, or a nucleotide 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 nucleotide sequence set forth in SEQ ID NO: 105.

[0113] In some embodiments, the TAP inhibitor is derived from herpesvirus 1 or herpesvirus 2. In some embodiments, the TAP inhibitor is derived from bovine herpesvirus 1. In some embodiments, the TAP inhibitor is either UL49.5, US6, or ICP47. In some embodiments, the TAP inhibitor is UL49.5. In some embodiments, the encoded 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% identity 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 encoded 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% identity to the amino acid sequence set forth in SEQ ID NO: 82.In some embodiments, the polynucleotide encoding the TAP inhibitor comprises the nucleotide sequence of SEQ ID NO:103, or a nucleotide 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% identity to the nucleotide sequence set forth in SEQ ID NO:103.

[0114] In some embodiments, the expression cassette further comprises a polynucleotide encoding a self-cleaving peptide. Any suitable self-cleaving peptide can be used in the cassettes of the present disclosure, including, but not limited to, T2A, P2A, E2A, or F2A peptides. 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:91 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:91. In some embodiments, the polynucleotide encoding the self-cleaving peptide comprises the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having about 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 nucleotide sequence set forth in SEQ ID NO: 104. In some embodiments, the self-cleaving peptide is located in the expression cassette between the polynucleotide encoding FLT3L and the polynucleotide encoding the TAP inhibitor.

[0115] In some embodiments, the expression cassette comprises a promoter operably linked to the polynucleotide encoding FLT3L, hi some embodiments, the promoter is an hCMV promoter. In some embodiments, the hCMV promoter comprises the nucleotide sequence of SEQ ID NO:107 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:107.

[0116] In some embodiments, the expression cassette further comprises a polyadenylation signal operably linked to the polynucleotide encoding the TAP inhibitor, hi 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 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:102.

[0117] In some embodiments, the expression cassette of the present disclosure comprises, in order, a promoter (e.g., an hCMV promoter) operably linked to a polynucleotide encoding FLT3L; a polynucleotide encoding FLT3L; a polynucleotide encoding a self-cleaving peptide (e.g., a P2A peptide); a polynucleotide encoding a TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal (e.g., a BGHpA polyadenylation signal).

[0118] In some embodiments, an expression cassette of the present disclosure comprises a polynucleotide that sequentially encodes FLT3L, a self-cleaving peptide (e.g., a P2A peptide), and a TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence having 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% identity to the nucleotide sequence set forth in SEQ ID NO: 106. In some embodiments, the expression cassette encodes a polypeptide comprising, in order, FLT3L, a self-cleaving peptide (e.g., a P2A peptide), and a TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 92.In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 93, 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% identity to the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the expression cassette further comprises a promoter, e.g., an hCMV promoter, that regulates expression of a polynucleotide encoding FLT3L, a self-cleaving peptide (e.g., a P2A peptide), and a TAP inhibitor (e.g., a UL49.5 protein). In some embodiments, the expression cassette further comprises a polyadenylation signal, e.g., BGHpA. In some embodiments, the expression cassette includes, in order, a promoter, e.g., an hCMV promoter; polynucleotides encoding, in order, FLT3L, a self-cleaving peptide (e.g., a P2A peptide), and a TAP inhibitor (e.g., a UL49.5 protein); and a polyadenylation signal, e.g., BGHpA.

[0119] In some embodiments, an expression cassette of the present disclosure comprises the nucleotide sequence of SEQ ID NO:100, or a nucleotide sequence having about 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 nucleotide sequence set forth in SEQ ID NO:100.

[0120] In some embodiments, the expression cassette of the present disclosure is integrated into the genome of a virus, such as an oncolytic HSV, e.g., HSV-1 or HSV-2. In some embodiments, the cassette is integrated into one or two of the native γ34.5 loci of the oncolytic HSV, e.g., HSV-1 or HSV-2. In some embodiments, the cassette is integrated into both of the native γ34.5 loci of the oncolytic HSV, e.g., HSV-1 or HSV-2. In some embodiments, one or two of the native γ34.5 loci of the oncolytic HSV, e.g., HSV-1 or HSV-2, are inactivated by insertion of the expression cassette. In some embodiments, both of the native γ34.5 loci of the oncolytic HSV, e.g., HSV-1 or HSV-2, are inactivated by insertion of the expression cassette. In some embodiments, integration of the expression cassette into the γ34.5 locus comprises replacing all, substantially all, or a portion 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.

[0121] In some embodiments, the expression cassette comprises (i) a polynucleotide encoding a TAP inhibitor (e.g., a UL49.5 protein), (ii) a polynucleotide encoding a self-cleaving peptide (such as a P2A peptide), and (iii) a polynucleotide encoding FLT3L, e.g., as described above. In some embodiments, the expression cassette comprises a polynucleotide encoding a genomic long terminal repeat (TR) L In some embodiments, the expression cassette is oriented relative to the oncolytic HSV genome, such as the HSV-1 or HSV-2 genome, in the unique long (UL) region of the genome, e.g., (i)-(ii)-(iii)-UL, of the native γ34.5 locus.

[0122] In some embodiments, the expression cassette comprises (i) a polynucleotide encoding a TAP inhibitor (e.g., a UL49.5 protein), (ii) a polynucleotide encoding a self-cleaving peptide (e.g., a P2A peptide), and (iii) a polynucleotide encoding FLT3L, e.g., as described above. In some embodiments, the expression cassette comprises a polynucleotide encoding a genomic internal long repeat (IR) L In some embodiments, the expression cassette is oriented relative to the unique long (UL) region of the genome, e.g., the oncolytic HSV genome, such as the HSV-1 or HSV-2 genome, e.g., UL-(iii)-(ii)-(i) of the native γ34.5 locus within the UL-(iii)-(ii)-(i) region.

[0123] IV-C. Oncolytic Viruses, Genomes, Vectors, and Cells Containing One or More Expression Cassettes Also provided herein (e.g., Sections IV-A and / or IV-B) are oncolytic viruses (e.g., oncolytic HSV, e.g., oncolytic HSV-1 or oncolytic HSV-2) comprising one or more of the above-described expression cassettes. 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 IV-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, the oncolytic viruses of the present disclosure exhibit increased T cell activation compared to oncolytic viruses 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 can be assessed using any suitable method known in the art, such as, for example, using an in vitro IL-2 secretion assay, as described in Example 2 herein. In some embodiments, the oncolytic viruses of the present disclosure have increased abscopal efficacy compared to oncolytic viruses lacking any one, any two, or any three of FLT3L, IL-12, CD40 agonist, and CTLA-4 binding protein.Abscopal efficacy can be assessed using any suitable method known in the art, such as using an in vivo tumor or cancer animal model, as described in Example 7 herein. In some embodiments, the oncolytic viruses of the present disclosure can evade an individual's immune system. In some embodiments, the oncolytic viruses of the present disclosure reduce or impair viral antigen loading onto histocompatibility complex (MHC) class I molecules for presentation on the cell surface, thereby reducing the adaptive immune response to the virus.

[0124] Also provided herein are modified HSV genomes (e.g., HSV-1 or HSV-2 genomes) comprising one or more of the above-described expression cassettes (e.g., those described 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 in Section IV-A above. 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 in Section IV-B above. 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 in Section IV-A above; 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 in Section IV-B above.

[0125] Also provided herein are vectors containing one or more of the expression cassettes described above (e.g., in Sections IV-A and IV-B). Suitable vectors include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or can be selected from the numerous cloning vectors available in the art. While the cloning vector selected can vary depending on the host cell intended for use, useful cloning vectors generally possess the ability to autonomously replicate, may have a single target for a particular restriction endonuclease, and / or may have a gene 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 DNA, 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. An expression vector is generally a replicable polynucleotide construct containing a nucleic acid of the present disclosure. Expression vectors can be replicable in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, HSV viruses, such as HSV-1 or HSV-2, retroviruses, and cosmids. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and appropriate transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also usually included.

[0126] In some embodiments, cells, e.g., host cells, comprising one or more of the above expression cassettes (e.g., 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 contaminating cell with which it is normally associated in the environment in which it is produced. In some embodiments, an isolated cell is free from association with all components associated with the production environment. An isolated cell is in a form other than the form or setting in which it is found in nature. An isolated cell is distinguished from cells naturally occurring in a tissue, organ, or individual. In some embodiments, the host cell is a eukaryotic organism, e.g., a Chinese hamster ovary (CHO) cell, a human cell such as a HELA cell, HEK293 cell, or a lymphoid cell (e.g., a YO, NSO, or Sp20 cell). Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells. In some embodiments, the cell is a mammalian cell.

[0127] V. CD40 agonist CD40 agonist proteins are further provided herein. In some embodiments, the CD40 agonist proteins of the present disclosure are agonists of the Cluster of Differentiation 40 (CD40) protein. 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 the formation of a trimer of the three single-chain trimeric CD40 ligand ectodomains. In some embodiments, the CD40 agonist is a trimer, i.e., a trimer comprising three single-chain trimeric CD40 ligand ectodomains.

[0128] 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% identity 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 ectodomain comprises a polypeptide comprising three human CD40 ligand ectodomains, e.g., linked by a peptide linker. In some embodiments, the single-chain trimeric human CD40 ligand ectodomain polypeptide comprises a first human CD40 ligand ectodomain connected to a second human CD40 ligand ectodomain, which is connected to a third human CD40 ligand ectodomain by a peptide linker. In some embodiments, the peptide linker comprises a glycine residue and a serine residue. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:22 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:22.In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to 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 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence including leucine, glycine, and / or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:23.In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to 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: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% identity 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% identity 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.

[0129] 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% identity 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 mouse CD40 ligand ectodomain comprises a polypeptide comprising three mouse CD40 ligand ectodomains, e.g., linked by a peptide linker. In some embodiments, the single-chain trimeric mouse CD40 ligand ectodomain polypeptide comprises a first mouse CD40 ligand ectodomain connected to a second mouse CD40 ligand ectodomain, which is connected to a third mouse CD40 ligand ectodomain by a peptide linker. In some embodiments, the peptide linker comprises a glycine residue and a serine residue. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:22 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:22.In some embodiments, the CD40 agonist comprises a trimerization motif operably linked to 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 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the trimerization motif is linked to the three single-chain trimeric CD40 ligand ectodomains by a peptide linker. In some embodiments, the peptide linker connecting the trimerization motif to the three single-chain trimeric CD40 ligand ectodomains comprises an amino acid sequence including leucine, glycine, and / or serine residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:23.In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the CD40 agonist further comprises a signal peptide sequence operably linked to 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 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% identity to the amino acid sequence set forth in 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% identity to the amino acid sequence set forth in SEQ ID NO:29.In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides comprising the amino acid sequence of SEQ ID NO:28 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:28. In some embodiments, the CD40 agonist forms or is a trimer comprising three polypeptides 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.

[0130] In some embodiments, the CD40 agonists provided herein are any of the CD40 agonists described in Section III-B above.

[0131] In some embodiments, CD40 agonists of the present disclosure induce or enhance CD40 signaling, activate dendritic cells, and / or inhibit tumor growth. In some embodiments, CD40 signaling can be assessed in vitro using any suitable method, such as using CD40 reporter cells, e.g., reporter cells that emit a detectable signal upon activation of the CD40 signaling pathway. In some embodiments, CD40 signaling can be assessed using a HEK-Blue reporter assay (see, e.g., www.invivogen.com / hek-blue-cd40l). In an exemplary HEK-Blue reporter assay, HEK293 reporter cells stably transfected with a CD40 gene, e.g., a human CD40 gene, and an NFκB-inducible secreted alkaline phosphatase (SEAP) construct are contacted with a CD40 agonist. The reporter cells respond to CD40 agonist binding by producing a colorimetric readout. In some embodiments, dendritic cell activation can be assessed using any suitable method, such as by assessing the level of CD86 activation marker expressed on one or more cells in vitro using, for example, flow cytometry. In some embodiments, tumor growth inhibition can be assessed using any suitable method, such as using an in vivo mouse tumor model, for example, as described in Example 2 herein.

[0132] Also provided herein are nucleic acids, e.g., isolated nucleic acids, having a nucleotide sequence encoding any of the CD40 agonists of the present disclosure. Such nucleic acids can encode the amino acid sequence of a CD40 ligand ectodomain, e.g., the human or mouse CD40 ligand ectodomain described above. In some embodiments, the nucleic acids of the present disclosure encode an amino acid sequence comprising three single-chain trimeric human CD40 ligand ectodomains, e.g., as described above. In other embodiments, the nucleic acids of the present disclosure encode an amino acid sequence comprising three single-chain trimeric human or mouse CD40 ligand ectodomains, wherein the amino acid sequences of the three single-chain trimeric human CD40 ligand ectodomains are operably linked (e.g., via a linker) to the amino acid sequence of a trimerization motif, such as the T4 fibritin trimerization motif, e.g., as described above. In some embodiments, the nucleic acids of the present disclosure further encode a signal peptide sequence fused to the amino acid sequence of the three single-chain trimeric human CD40 ligand ectodomains and the trimerization motif, e.g., as described above.

[0133] Also provided herein are one or more vectors (e.g., cloning vectors or expression vectors) containing any of the nucleic acids of the present disclosure, e.g., encoding any of the CD40 agonists of the present disclosure. Suitable vectors containing a nucleic acid sequence encoding any of the CD40 agonists of the present disclosure or fragments thereof include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or can be selected from the numerous cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended for use; however, useful cloning vectors generally have the ability to autonomously replicate, may have a single target for a particular restriction endonuclease, and / or may have a gene 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 DNA, 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 are generally replicable polynucleotide constructs containing a nucleic acid of the present disclosure. Expression vectors may be replicable in a host cell either as an episome 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 such as HSV-1 or HSV-2, retroviruses, and cosmids. Vector components may generally include one or more of the following, but are not limited to: a signal sequence; an origin of replication; one or more marker genes; and appropriate transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also usually included.

[0134] In some embodiments, host cells containing any of the nucleic acids or vectors of the present disclosure are also provided. In some embodiments, the host cell is an isolated host cell. An isolated cell is a cell that has been identified and separated from at least one contaminating cell with which it is normally associated in the environment in which it is produced. In some embodiments, an isolated cell is free from association with all components associated with the production environment. An isolated cell is in a form other than the form or setting in which it is found in nature. An isolated cell is distinguished from cells naturally occurring in a tissue, organ, or individual. In some embodiments, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell, a human cell such as a HELA cell, HEK293 cell, or a lymphoid cell (e.g., a YO, NSO, or Sp20 cell). Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0135] The CD40 agonists of the present disclosure can be produced using recombinant methods and compositions. In some embodiments, methods of making the CD40 agonists of the present disclosure are provided. In some embodiments, the methods include culturing a host cell of the present disclosure containing a nucleic acid encoding a CD40 agonist of the present disclosure under conditions suitable for expression of the CD40 agonist. In some embodiments, the CD40 agonist is then recovered from the host cell (or host cell culture medium). For recombinant production of a CD40 agonist of the present disclosure, the nucleic acid encoding the CD40 agonist is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to a sequence encoding the CD40 agonist). The vectors or nucleic acids of the present disclosure can be introduced into host cells by any of several suitable means, including transfection using electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other agents; microprojectile bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introducing the vector or nucleic acid often depends on characteristics of the host cell.

[0136] The CD40 agonists of the present disclosure can be incorporated into various formulations for therapeutic administration by combining the CD40 agonist with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into solid, semi-solid, liquid, or gaseous forms. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Depending on the desired formulation, the pharmaceutical composition may contain a pharmaceutically acceptable, non-toxic carrier for the diluent, which is a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include, but are not limited to, distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical compositions or formulations of the present disclosure may further contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The compositions may also contain additional substances to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, and detergents. Pharmaceutical compositions may also contain any of a variety of stabilizers, such as antioxidants. When a pharmaceutical composition contains a polypeptide, such as a CD40 agonist of the present disclosure, the polypeptide may be complexed with a variety of well-known compounds that enhance the polypeptide's in vivo stability or otherwise enhance its pharmacological properties (e.g., increasing the polypeptide's half-life, reducing its toxicity, enhancing solubility or uptake). Examples of such modifying or complexing agents include, but are not limited to, sulfate, gluconate, citrate, and phosphate. Polypeptides of the compositions may also be complexed with molecules that enhance their in vivo attributes. Such molecules include, but are not limited to, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.Further examples of formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0137] VI. Methods for producing oncolytic viruses The oncolytic viruses described herein (such as oncolytic HSV) can be prepared using any method known in the art or described herein. In some embodiments, oncolytic viruses (such as oncolytic HSV) can be engineered by modifying a wild-type viral (such as wild-type HSV-1) genome (to contain one or more of the expression cassettes described herein and / or to express one or more of the payload proteins described herein). Transgenes and / or expression cassettes, including those otherwise described herein, can be inserted into the native genome or replace native portions of the genome using recombinational cloning techniques well known in the art. Exemplary engineering methods are described in Examples 4-7 herein. Engineered oncolytic viral genomes can be propagated in appropriate cells and harvested from cell culture media or from cell lysates. Purified viruses can be titered using assays well known in the art. Viral titers can be expressed in terms of infectious viral units, such as plaque-forming units (pfu). The integrity and sequence of the viral genome can be assessed by techniques well known in the art, including whole genome sequencing.

[0138] VII. Pharmaceutical Compositions Further provided herein are pharmaceutical compositions comprising any of the oncolytic viruses described herein (such as any of the oncolytic HSVs) and, optionally, a pharmaceutically acceptable excipient, carrier, and / or stabilizer. The pharmaceutical composition can be prepared by mixing an oncolytic virus described herein (such as any of the oncolytic HSVs) having a desired purity with a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

[0139] The pharmaceutical composition may be administered by any suitable route, including intratumoral or intravesical.

[0140] IX.How to use Further provided are methods of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an oncolytic virus described herein or a pharmaceutical composition comprising an oncolytic virus described herein. Further provided are methods of killing tumor cells in an individual, comprising administering a therapeutically effective amount of an oncolytic virus described herein or a pharmaceutical composition comprising an oncolytic virus described herein. In some embodiments, the oncolytic virus comprises one or more of the expression cassettes described herein. In some embodiments, the oncolytic virus expresses one or more of the payload proteins described herein. In some embodiments, the oncolytic virus is an oncolytic HSV described herein. In some embodiments, the cancer being treated is a solid tumor. In some embodiments, the cancer being treated is a metastatic cancer. In some embodiments, the cancer being treated is a recurrent cancer. In some embodiments, the cancer is partially progressed.

[0141] In some embodiments, provided herein are methods for expressing genes in vivo, e.g., in a tumor microenvironment. In some embodiments, the method includes delivering a viral vector containing the genes. In some embodiments, the one or more genes elicit an immunological response. In some embodiments, expression of one or more of the genes results in the recruitment of dendritic cells to the tumor microenvironment. In some embodiments, one or more of the genes triggers the maturation of dendritic cells into licensed antigen-presenting cells (APCs). In some embodiments, one or more of the genes enhance T cell effector cytokine production. In some embodiments, one or more of the genes promote a CD4+ T helper (Th)1 response that sustains cytotoxic CD8+ T cells. In some embodiments, one or more of the genes drive T cell activation. In some embodiments, one or more of the genes promotes the depletion of regulatory T cells (Tregs). In some embodiments, the genes are selected from the group consisting of FLT3 ligand, CD40 agonist, IL-1, and CTLA-4 antagonist.

[0142] In some embodiments, administration of an oncolytic virus to an individual results in one or more therapeutic effects in the subject. In one embodiment, the one or more therapeutic effects is a reduction in the size of a tumor derived from cancer. In one embodiment, the one or more therapeutic effects is lysis of tumor cells in an individual. In some embodiments, the oncolytic virus preferentially lyses tumor cells. As used herein, selectively lysing tumor cells means that the oncolytic virus preferentially lyses tumor cells compared to adjacent non-tumor cells. It is understood that the oncolytic virus may still lyse adjacent healthy cells, but may lyse them less efficiently compared to the target tumor cells. In one embodiment, the one or more therapeutic effects is a reduction in tumor size.

[0143] Administration of an oncolytic virus described herein or a pharmaceutical composition comprising an oncolytic virus described herein can result in an abscopal response in a subject. In an abscopal response, administration of an oncolytic virus or pharmaceutical composition to a first site (such as a primary tumor) can kill tumor cells at both the first site (primary effect) and one or more additional sites, such as a secondary site (such as a distant tumor site or a metastatic tumor site; abscopal effect). Such an abscopal effect can be mediated by an immune response to the oncolytic virus at the first site (e.g., the site to which the virus is initially delivered). Thus, in some embodiments, an oncolytic virus described herein or a pharmaceutical composition comprising an oncolytic virus described herein is administered to a first site, and the oncolytic virus elicits an immune response that results in the killing of tumor cells at a second site, such as a distant tumor or metastasis. In some embodiments, administration of an oncolytic virus or a pharmaceutical composition comprising an oncolytic virus results in an immune response in a subject. In some embodiments, the immune response results in tumor growth inhibition at either or both the first site (where the virus is delivered) and the second site. In some embodiments, the immune response is enhanced by expression of one or more payload proteins described herein. Expression of an immunomodulatory payload protein following oncolytic virus treatment can result in dendritic cell infiltration into tumors, dendritic cells, monocytes, and / or macrophages, T cell infiltration, T cell activation, T cell expansion, regulatory cell depletion, NK cell infiltration, interferon-γ production, and a reduction in suppressive immune populations such as myeloid-derived suppressor cells and tumor-associated macrophages. In some embodiments, the oncolytic virus induces a sustained anti-tumor immune response. In some embodiments, the immune system develops memory of tumor antigens and is able to recognize and eliminate tumor cells days, weeks, months, or longer after administration.

[0144] In some embodiments, the oncolytic virus enhances T cell function. In some embodiments, the oncolytic virus depletes regulatory T cells (Tregs) in the tumor microenvironment. In some embodiments, the oncolytic virus recruits dendritic cells to the tumor microenvironment. In some embodiments, the oncolytic virus matures dendritic cells.

[0145] In the context of oncolytic HSV, in some embodiments, the oncolytic HSV expresses immediate-early US11. In some embodiments, the oncolytic HSV expresses both immediate-early US11 and native late US11. In some embodiments, the oncolytic HSV expresses UL49.5, a TAP inhibitor from bovine herpesvirus 1. In some embodiments, expression of UL49.5 inhibits TAP from transporting peptides into the lumen of the endoplasmic reticulum, thus impairing loading onto major histocompatibility complex (MHC) class I molecules for presentation on the cell surface and reducing the adaptive immune response to the virus. In some embodiments, this impairment of MHC class I loading is specific to the target cell; adjacent uninfected cells are not impaired for MHC class I loading.

[0146] Exemplary Embodiments The present disclosure may be better understood with reference to the following exemplary embodiments.

[0147] Embodiment 1. An oncolytic herpes simplex virus type 1 (HSV-1), comprising: a. A cassette integrated into one or both of the γ34.5 loci, the cassette 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 into the US10-12 locus, the another cassette comprising, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding a native US11 protein, an additional polynucleotide encoding a native US11 protein, a polynucleotide encoding a US10 protein, a polyadenylation signal operably linked to the polynucleotide encoding the US10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV1 promoter controlling expression of the CD40 agonist, an MMLV promoter controlling expression of IL-12, a polynucleotide encoding IL-12, and a polyadenylation signal operably linked to the polynucleotide encoding IL-12. Including, the polynucleotide of hFLT3L encodes the amino acid sequence set forth in SEQ ID NO:71, the polynucleotide of UL49.5 encodes the amino acid sequence set forth in SEQ ID NO:82, the polynucleotide of IL-12 encodes the amino acid sequence set forth in SEQ ID NO:4, the polynucleotide of the CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO:25, the polynucleotide of the CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO:50, the polynucleotide of the variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO:204, a further polynucleotide of US11 encodes the amino acid sequence set forth in SEQ ID NO:80, and the polynucleotide of US10 encodes the amino acid sequence set forth in SEQ ID NO:90; Oncolytic herpes simplex virus type 1 (HSV-1). 2. The oncolytic HSV-1 of embodiment 1, wherein the bGH polyadenylation signal is SEQ ID NO: 102, the hGH polyadenylation signal is SEQ ID NO: 209, the GAPDH synthetic polyadenylation signal is SEQ ID NO: 213, the C2 transcriptional pause site is SEQ ID NO: 214, the hGT transcriptional pause site is SEQ ID NO: 215, and the hBG polyadenylation signal is SEQ ID NO: 216. 3. The oncolytic HSV-1 of claim 1, wherein expression of a variant US11 gene encoding the native US11 protein is under the control of the native US12 immediate-early promoter, expression of an additional polynucleotide encoding the native US11 protein is under the control of its native promoter, and expression of a polynucleotide encoding the US10 protein is under the control of its native promoter. 4. An oncolytic herpes simplex virus type 1 (HSV-1), A cassette integrated into one or both of the γ34.5 loci, the cassette 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 and another cassette integrated into the US10-12 locus, the cassette comprising, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding a native US11 protein, a further polynucleotide encoding a native US11 protein, a polynucleotide encoding a US10 protein, a polyadenylation signal operably linked to the polynucleotide encoding the US10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV1 promoter controlling expression of the CD40 agonist, an MMLV promoter controlling expression of IL-12, a polynucleotide encoding IL-12, and a polyadenylation signal operably linked to the polynucleotide encoding IL-12. Including, the polynucleotide of hFLT3L encodes the amino acid sequence set forth in SEQ ID NO:71, the polynucleotide of UL49.5 encodes the amino acid sequence set forth in SEQ ID NO:82, the polynucleotide of IL-12 encodes the amino acid sequence set forth in SEQ ID NO:8, the polynucleotide of CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO:28, the polynucleotide of CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO:56, the polynucleotide of the variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO:204, a further polynucleotide of US11 encodes the amino acid sequence set forth in SEQ ID NO:80, and the polynucleotide of US10 encodes the amino acid sequence set forth in SEQ ID NO:90; Oncolytic herpes simplex virus type 1 (HSV-1). 5. The oncolytic HSV-1 of embodiment 4, wherein the bGH polyadenylation signal is SEQ ID NO: 102, the hGH polyadenylation signal is SEQ ID NO: 209, the GAPDH synthetic polyadenylation signal is SEQ ID NO: 213, the C2 transcriptional pause site is SEQ ID NO: 214, the hGT transcriptional pause site is SEQ ID NO: 215, and the hBG polyadenylation signal is SEQ ID NO: 216. 6. An oncolytic virus comprising one or more expression cassettes comprising a polynucleotide encoding IL-12, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding a CTLA-4 binding protein. 7. The oncolytic virus of embodiment 6, wherein the virus comprises a backbone nucleic acid encoding one or more native viral proteins associated with viral replication and / or packaging. 8. The oncolytic virus of embodiment 6, wherein one or both of the native γ34.5 genes are inactivated by deletion, substitution, or insertion of the backbone nucleic acid. 9. An oncolytic virus according to any one of embodiments 6 to 8, wherein the native US12 gene of the virus is inactivated by a deletion, substitution or insertion in the backbone nucleic acid. 10. The oncolytic virus of any one of embodiments 6 to 9, wherein IL-12 is a heterodimer comprising a p35 subunit and a p40 subunit. 11. The oncolytic virus of embodiment 10, wherein the p35 subunit and / or the p40 subunit is human. 12. The oncolytic virus of embodiment 10 or 11, wherein the p35 subunit comprises the amino acid sequence of SEQ ID NO: 1. 13. An oncolytic virus according to any one of embodiments 10 to 12, wherein the p40 subunit comprises the amino acid sequence of SEQ ID NO:2. 14. The oncolytic virus of embodiment 10, wherein the p35 subunit and / or the p40 subunit is murine. 15. The oncolytic virus of embodiment 14, wherein the p35 subunit comprises the amino acid sequence of SEQ ID NO:5. 16. The oncolytic virus of embodiment 14 or 15, wherein the p40 subunit comprises the amino acid sequence of SEQ ID NO:6. 17. An oncolytic virus according to any one of embodiments 10 to 16, wherein the p35 subunit and the p40 subunit are linked by a peptide linker. 18. The oncolytic virus of embodiment 14, wherein the peptide linker comprises an amino acid sequence comprising a glycine residue and a serine residue. 19. The oncolytic virus of embodiment 18, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7. 20. The oncolytic virus of any one of embodiments 6 to 19, wherein the CD40 agonist is a CD40 ligand. 21. The oncolytic virus of any one of embodiments 6 to 20, wherein the CD40 agonist comprises a CD40 ligand ectodomain. 22. The oncolytic virus of embodiment 21, wherein the CD40 agonist is a trimer of three single-chain trimeric CD40 ligand ectodomains. 23. The oncolytic virus of embodiment 21 or 22, wherein the CD40 ligand ectodomain is human. 24. The oncolytic virus of embodiment 23, wherein the CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 20. 25. The oncolytic virus of embodiment 21 or 22, wherein the CD40 ligand ectodomain is murine. 26. The oncolytic virus of embodiment 24, wherein the CD40 ligand ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 26. 27. The oncolytic virus of any one of embodiments 22 to 26, wherein the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. 28. The oncolytic virus of embodiment 27, wherein the trimerization motif is a T4 fibritin trimerization motif. 29. The oncolytic virus of embodiment 27 or 28, wherein the trimerization motif is linked to each of the three single-chain trimeric ectodomains of the CD40 agonist by a linker comprising a glycine residue and a serine residue. 30. The oncolytic virus of any one of embodiments 6 to 29, wherein the CTLA-4 binding protein is a CTLA-4 antibody or an antigen-binding fragment thereof. 31. The oncolytic virus of embodiment 30, wherein the CTLA-4 antibody or antigen-binding fragment thereof is an scFv. 32. The oncolytic virus of embodiment 30 or 31, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof specifically binds to human CTLA-4. 33. The oncolytic virus of any one of embodiments 30-32, wherein the anti-CTLA-4 antibody or antigen-binding fragment is bivalent. 34. The oncolytic virus of any one of embodiments 31 to 33, wherein the anti-CTLA-4 scFv is fused to the N-terminus of the IgG1 constant domain. 35. The oncolytic virus of embodiment 34, wherein the human IgG1 is a variant human IgG1 comprising a C220S substitution, numbering according to the EU index numbering. 36. The anti-CTLA-4 antibody or antigen-binding fragment thereof is CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 40; CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 41; CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 42; CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 43; CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 44; and CDRL3 comprising the amino acid sequence set forth in SEQ ID NO:45 36. An oncolytic virus according to any one of embodiments 30 to 35, comprising: 37. The oncolytic virus of any one of embodiments 30-35, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 47. 38. The oncolytic virus of embodiment 36 or 37, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 50. 39. The oncolytic virus of embodiment 30, wherein the CTLA-4 antibody is a camelid antibody comprising an anti-CTLA-4 VHH. 40. The oncolytic virus of embodiment 39, wherein the VHH is fused to the heavy chain of mouse IgG2a Fc. 41. The oncolytic virus of embodiment 40, wherein the anti-CTLA-4 VHH comprises the amino acid sequence set forth in SEQ ID NO: 54. 42. The oncolytic virus of any one of embodiments 6 to 41, wherein the one or more expression cassettes comprise a nucleic acid encoding a FLT3 ligand (FLT3L). 43. The oncolytic virus of embodiment 42, wherein FLT3L is human. 44. The oncolytic virus of embodiment 42 or 43, wherein FLT3L is a homodimer. 45. An oncolytic virus according to any one of embodiments 42 to 44, wherein FLT3L comprises a signal peptide that directs secretion to the plasma membrane. 46. ​​The oncolytic virus of any one of embodiments 42-44, wherein FLT3L comprises the amino acid sequence set forth in SEQ ID NO: 71. 47. The oncolytic virus of any one of embodiments 6 to 46, wherein one or more of the one or more expression cassettes further comprises a polynucleotide comprising a variant US11 gene. 48. The oncolytic virus of embodiment 47, wherein the polynucleotide comprising the variant US11 gene is human-codon optimized compared to the native gene encoding US11. 49. The oncolytic virus of embodiment 47 or 48, wherein the variant US11 gene comprises the polynucleotide sequence of SEQ ID NO: 204. 50. An oncolytic virus according to any one of embodiments 47 to 49, wherein the variant US11 gene is operably associated with an immediate-early promoter. 51. The oncolytic virus of embodiment 50, wherein the variant US11 gene is operably associated with the native US12 immediate-early promoter to express the immediate-early US11 protein. 52. An oncolytic virus according to any one of embodiments 6 to 51, wherein the oncolytic virus does not express granulocyte-macrophage colony-stimulating factor (GM-CSF). 53. An oncolytic virus according to any one of embodiments 6 to 52, comprising a native late US11 gene. 54. An oncolytic virus according to any one of embodiments 6 to 53, further comprising a transporter associated with antigen processing (TAP) inhibitor. 55. An oncolytic virus according to embodiment 54, wherein the TAP inhibitor is derived from herpesvirus 1 or herpesvirus 2. 56. The oncolytic virus according to embodiment 55, wherein the TAP inhibitor is derived from Bovine Herpesvirus 1. 57. An oncolytic virus according to embodiment 54 or 55, wherein the TAP inhibitor is UL49.5, US6 or ICP47. 58. The oncolytic virus of embodiment 57, wherein the TAP inhibitor is UL49.5. 59. An oncolytic virus according to any one of embodiments 54 to 58, wherein the TAP inhibitor is expressed as an immediate early gene. 60. The oncolytic virus of any one of embodiments 42 to 59, wherein the expression cassette comprises a polynucleotide encoding FLT3L. 61. The oncolytic virus of embodiment 60, wherein the expression cassette comprises a polynucleotide encoding a TAP inhibitor. 62. The oncolytic virus of embodiment 61, wherein the expression cassette comprises a polynucleotide encoding a self-cleaving peptide. 63. The oncolytic virus of embodiment 62, wherein the polynucleotide encoding the self-cleaving peptide is located between the polynucleotide encoding FLT3L and the polynucleotide encoding the TAP inhibitor. 64. The oncolytic virus of embodiment 62 or 63, wherein the self-cleaving peptide is P2A. 65. The oncolytic virus of embodiment 64, wherein P2A comprises the amino acid sequence of SEQ ID NO: 91. 66. An oncolytic virus according to any one of embodiments 6 to 65, wherein at least one of the native γ34.5 loci is functionally inactivated by insertion of an expression cassette. 67. The oncolytic virus of embodiment 66, wherein both native γ34.5 loci are replaced or substantially replaced by copies of the expression cassette. 68. An oncolytic virus according to any one of embodiments 61 to 67, wherein the one or more expression cassettes comprise a CMV promoter regulating the expression of a polynucleotide encoding FLT3L and a polynucleotide encoding a TAP inhibitor. 69. The oncolytic virus of embodiment 68, wherein the expression cassette comprises a polyadenylation signal. 70. The oncolytic virus of embodiment 69, wherein the polyadenylation signal is the bovine growth hormone polyadenylation signal (BGHpA). 71. The expression cassette contains a long terminal repeat (TR L 71. The oncolytic virus of any one of embodiments 63-70, wherein the cassette replaces all or substantially all of one or both of the γ34.5 loci of the γ34.5 gene in the γ34.5 locus ... 72. Expression cassette is TR L 72. The oncolytic virus of embodiment 71, wherein the oncolytic virus replaces all or substantially all of both of the γ34.5 loci of the α-terminal nucleotide sequence of the γ34.5 gene. 73. The oncolytic virus of any one of embodiments 56-63, wherein the expression cassette replaces all or substantially all of the γ34.5 locus of internal repeats (IRLs) and comprises (i) a nucleic acid encoding a TAP inhibitor, (ii) a nucleic acid encoding a self-cleaving peptide, and (iii) a nucleic acid encoding FLT3L, and wherein the cassette is oriented relative to the UL region of the genome as follows: UL-(iii)-(ii)-(i). 74. The oncolytic virus of embodiment 73, wherein the expression cassette replaces all or substantially all of the IRL. 75. An oncolytic virus according to any one of embodiments 6 to 74, wherein one of the expression cassettes is inserted into the native US10-US12 locus. 76. The oncolytic virus of embodiment 75, wherein the expression cassette comprises a polynucleotide encoding IL-12. 77. The oncolytic virus of embodiment 75 or 76, wherein the expression cassette comprises a polynucleotide encoding a CD40 agonist. 78. An oncolytic virus described in any one of embodiments 75 to 77, wherein the expression cassette comprises a polynucleotide encoding a CTLA-4 binding protein. 79. An oncolytic virus according to any one of embodiments 75 to 78, wherein the expression cassette comprises a polynucleotide encoding a US10 protein. 80. The oncolytic virus of embodiment 79, wherein the expression cassette comprises a polyadenylation signal located after the polynucleotide encoding the US10 protein. 81. The oncolytic virus of embodiment 80, wherein the polyadenylation signal is the human growth hormone polyadenylation signal (hGHpA). 82. An oncolytic virus according to any one of embodiments 75 to 81, wherein the expression cassette comprises the native late US11 gene. 83. An oncolytic virus according to any one of embodiments 75 to 82, wherein the expression cassette comprises a polynucleotide comprising a variant US11 gene. 84. An oncolytic virus according to any one of embodiments 75 to 83, wherein the expression cassette does not express a US12 protein. 85. An oncolytic virus described in any one of embodiments 78 to 84, wherein the expression cassette comprises a CMV promoter positioned upstream of the polynucleotide encoding the CTLA-4 binding protein. 86. An oncolytic virus described in any one of embodiments 78 to 85, wherein the expression cassette comprises a polyadenylation signal located after the polynucleotide encoding the CTLA-4 binding protein. 87. The oncolytic virus of embodiment 86, wherein the polyadenylation signal is a polyadenylation signal derived from the human GAPDH gene. 88. The oncolytic virus of any one of embodiments 77 to 87, further comprising a polyadenylation signal located after the polynucleotide encoding the CD40 agonist. 89. The oncolytic virus of embodiment 88, wherein the polyadenylation signal located after the polynucleotide encoding the CD40 agonist is hBGpA. 90. An oncolytic virus according to any one of embodiments 77 to 89, wherein the expression cassette comprises an AoHV1 promoter operably linked to a polynucleotide encoding a CD40 agonist. 91. An oncolytic virus described in any one of embodiments 76 to 90, wherein the expression cassette comprises an MMLV promoter operably linked to a polynucleotide encoding IL-12. 92. An oncolytic virus according to any one of embodiments 76 to 91, wherein the expression cassette comprises a polyadenylation signal following the polynucleotide encoding IL-12. 93. The oncolytic virus of embodiment 92, wherein the polyadenylation signal following the polynucleotide encoding IL-12 is US9-10pA. 94. An oncolytic virus described in any one of embodiments 78 to 87, wherein the expression cassette comprises (i) a polynucleotide encoding an IL-12 protein, (ii) a nucleic acid encoding a CD40 agonist, and (iii) a polynucleotide encoding a CTLA-4 binding protein, and the expression cassette is oriented relative to the internal short repeat (IRS) region of the genome of IRS-(i)-(ii)-(iii). 95. The oncolytic virus of embodiment 94, wherein the expression cassette comprises a polynucleotide encoding (iv) the US10 protein, and the expression cassette is oriented relative to the IRS region of the genome of IRS-(i)-(ii)-(iii)-(iv). 96. An oncolytic virus according to embodiment 95, wherein the cassette comprises a polynucleotide encoding (v) the US11 protein, and expression is oriented relative to the IRS region of the genome of IRS-(i)-(ii)-(iii)-(iv)-(v). 97. The oncolytic virus of embodiment 96, wherein the cassette comprises a nucleic acid comprising (vi) a variant US11 gene, and the expression cassette is oriented relative to the IRS region of the genome as follows: IRS-(i)-(ii)-(iii)-(iv)-(v)-(vi). 98. An oncolytic virus according to any one of embodiments 95 to 97, wherein the polynucleotide encoding the CD40 agonist is in a reverse orientation within the cassette relative to the polynucleotide encoding IL-12 and the polynucleotide encoding the CTLA-4 binding protein. 99. An oncolytic virus described in any one of embodiments 6 to 98, which exhibits increased T cell activation as assessed by an in vitro IL-2 secretion assay compared to an oncolytic virus lacking any one, any two, or all of the genes encoding IL-12 protein, CD40 agonist, and CTLA-4 binding protein. 100. An oncolytic virus according to any one of embodiments 1 to 100, wherein the virus is attenuated compared to the wild-type virus. 101. An oncolytic virus according to any one of embodiments 42 to 99, wherein the oncolytic virus has increased abscopal efficacy compared to an oncolytic virus lacking any one, any two, or any three of FLT3L, IL-12, CD40 agonist, and CTLA-4 binding protein. 102. An oncolytic virus according to any one of embodiments 1 to 100, wherein the virus is attenuated compared to the wild-type virus. 102. The oncolytic virus of any one of embodiments 1 to 101, wherein the virus is capable of evading the human immune system. 103. A pharmaceutical composition comprising an oncolytic virus according to any one of embodiments 1 to 102 and a pharmaceutically acceptable excipient. 104. a polynucleotide encoding a TAP inhibitor, and Polynucleotide encoding FLT3 ligand an expression cassette comprising: 105. An expression cassette according to embodiment 104, comprising a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide encoding the self-cleaving peptide is located between the TAP inhibitor and the FLT3 ligand. 106. The expression cassette of embodiment 105, wherein the self-cleaving peptide is P2A. 107. The expression cassette of embodiment 106, wherein P2A comprises the amino acid sequence of SEQ ID NO: 91. 108. An expression cassette according to any one of embodiments 104 to 107, wherein the TAP inhibitor is derived from herpesvirus 1 or herpesvirus 2. 109. An expression cassette according to any one of embodiments 104 to 108, wherein the TAP inhibitor is derived from Bovine Herpesvirus 1. 110. An expression cassette according to any one of embodiments 104 to 107, wherein the TAP inhibitor is UL49.5, US6 or ICP47. 111. An expression cassette according to embodiment 110, wherein the TAP inhibitor is UL49.5. 112. An expression cassette according to embodiment 111, wherein the TAP inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 82. 113. An expression cassette according to any one of embodiments 104 to 112, wherein the TAP inhibitor is expressed as an immediate early gene. 114. The expression cassette of any one of embodiments 104-113, wherein FLT3L comprises the amino acid sequence set forth in SEQ ID NO: 71. 115. An expression cassette according to any one of embodiments 104 to 114, comprising a CMV promoter regulating the expression of a polynucleotide encoding FLT3L and a polynucleotide encoding a TAP inhibitor. 116. An expression cassette according to any one of embodiments 104 to 115, comprising a polyadenylation signal located after the polynucleotide encoding FLT3L and the polynucleotide encoding a TAP inhibitor. 117. The expression cassette of embodiment 116, wherein the polyadenylation signal is the bovine growth hormone polyadenylation signal (BGHpA). 118. An expression cassette according to embodiment 117, comprising, from upstream to downstream, a CMV promoter, a polynucleotide encoding FLT3L, a polynucleotide encoding a P2A peptide, a polynucleotide encoding a UL49.5 protein, and a BGHpA polyadenylation signal. 119. An expression cassette according to embodiment 118, wherein the polynucleotide for the CMV promoter comprises the polynucleotide sequence set forth in SEQ ID NO: 107, the polynucleotide for hFLT3L encodes the amino acid sequence set forth in SEQ ID NO: 71, the polynucleotide for P2A encodes the amino acid sequence set forth in SEQ ID NO: 91, the polynucleotide for UL49.5 encodes the amino acid sequence set forth in SEQ ID NO: 82, and the polynucleotide for the BGHpA polyadenylation signal comprises the polynucleotide sequence set forth in SEQ ID NO: 102. 120. A modified HSV genome comprising an expression cassette according to any one of embodiments 104 to 119. 121. The modified HSV genome of embodiment 120, wherein one or both of the γ34.5 loci of the modified HSV genome have been replaced with a cassette. 122. An oncolytic virus comprising an expression cassette according to any one of embodiments 104 to 119. 123. An oncolytic virus according to embodiment 122, wherein one or both of the γ34.5 loci of the oncolytic virus have been replaced with an expression cassette. 124. An oncolytic virus according to embodiment 122 or 123, wherein both γ34.5 loci of the oncolytic virus have been replaced with expression cassettes. 125. a polynucleotide encoding IL-12; a polynucleotide encoding a CD40 agonist, and Polynucleotides encoding CTLA-4 binding proteins an expression cassette comprising: 126. The expression cassette of embodiment 125, wherein IL-12 is a heterodimer comprising a p35 subunit and a p40 subunit. 127. The expression cassette of embodiment 126, wherein the p35 subunit and / or the p40 subunit is human. 128. The expression cassette of embodiment 126 or 127, wherein the p35 subunit comprises the sequence of amino acids of SEQ ID NO:1. 129. An expression cassette according to any one of embodiments 126 to 128, wherein the p40 subunit comprises the sequence of amino acids of SEQ ID NO:2. 130. The expression cassette of embodiment 126, wherein the p35 subunit and / or the p40 subunit is murine. 131. The expression cassette of embodiment 130, wherein the p35 subunit comprises the amino acid sequence of SEQ ID NO:5. 132. The expression cassette according to embodiment 130 or 131, wherein the p40 subunit comprises the sequence of amino acids of SEQ ID NO:6. 133. The expression cassette according to any one of embodiments 126 to 132, wherein the p35 subunit and the p40 subunit are linked by a peptide linker. 134. The expression cassette of embodiment 133, wherein the linker comprises an amino acid sequence comprising a glycine residue and a serine residue. 135. The expression cassette of embodiment 134, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 3 or 7. 136. The expression cassette according to any one of embodiments 125 to 135, wherein the CD40 agonist is a CD40 ligand. 137. The expression cassette according to any one of embodiments 125-136, wherein the CD40 agonist comprises a CD40 ligand ectodomain. 138. The expression cassette of embodiment 137, wherein the CD40 ligand is a trimer of three single-chain trimeric CD40 ligand ectodomains. 139. The expression cassette of embodiment 137 or 138, wherein the CD40 ligand ectodomain is human. 140. The expression cassette of embodiment 139, wherein the CD40 ligand ectodomain comprises the sequence of amino acids of SEQ ID NO: 20. 141. The expression cassette of embodiment 137 or 138, wherein the CD40 ligand ectodomain is murine. 142. The expression cassette of embodiment 141, wherein the CD40 ligand ectodomain comprises the sequence of amino acids of SEQ ID NO: 26. 143. The expression cassette according to any one of embodiments 136-142, wherein the CD40 agonist comprises a trimerization motif operably linked to each of the three single-chain trimeric CD40 ligand ectodomains. 144. The expression cassette of embodiment 143, wherein the trimerization motif is a T4 fibritin trimerization motif. 145. The expression cassette according to embodiment 143 or 144, wherein the trimerization motif is linked to each of the three single-chain trimeric ectodomains of the CD40 agonist by a linker comprising a glycine residue and a serine residue. 146. The expression cassette of any one of embodiments 125-145, wherein the CTLA-4 binding protein is a CTLA-4 antibody or an antigen-binding fragment thereof. 147. The expression cassette of embodiment 146, wherein the CTLA-4 antibody or antigen-binding fragment thereof is an scFv. 148. The expression cassette of embodiment 146 or 147, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof specifically binds to human CTLA-4. 149. The expression cassette of any one of embodiments 146-148, wherein the anti-CTLA-4 antibody or antigen-binding fragment is bivalent. 150. The expression cassette of any one of embodiments 147-149, wherein the anti-CTLA-4 scFv is fused to the N-terminus of the IgG1 constant domain. 151. The expression cassette of embodiment 150, wherein the human IgG1 is a variant human IgG1 comprising a C220S substitution according to EU numbering. 152. An anti-CTLA-4 antibody or antigen-binding fragment thereof, CDRH1 comprising the amino acid sequence of SEQ ID NO: 40; CDRH2 comprising the amino acid sequence of SEQ ID NO: 41; CDRH3 comprising the amino acid sequence of SEQ ID NO: 42; CDRL1 comprising the amino acid sequence of SEQ ID NO: 43; CDRL2 comprising the amino acid sequence of SEQ ID NO: 44; and CDRL3 comprising the amino acid sequence of SEQ ID NO: 45 152. The expression cassette of any one of embodiments 146 to 151, comprising: 153. The expression cassette of any one of embodiments 146 to 152, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 47. 154. The expression cassette of any one of embodiments 146 to 153, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises the sequence of amino acids of SEQ ID NO: 50. 155. The expression cassette of embodiment 146, wherein the CTLA-4 antibody is a camelid antibody comprising an anti-CTLA-4 VHH. 156. The expression cassette according to embodiment 155, wherein the VHH is fused to the heavy chain of mouse IgG2a Fc. 157. The expression cassette according to any one of embodiments 125 to 156, further comprising a polynucleotide encoding a US10 protein. 158. An expression cassette according to any one of embodiments 125 to 157, wherein the expression cassette comprises the native late US11 gene. 159. An expression cassette according to any one of embodiments 125 to 158, wherein the expression cassette comprises a variant polynucleotide encoding a US11 protein. 160. The expression cassette of any one of embodiments 125-159, wherein the expression cassette does not express a US12 protein. 161. An expression cassette according to any one of embodiments 125 to 160, wherein the expression cassette comprises an mCMV promoter positioned upstream of the polynucleotide encoding the CTLA-4 binding protein. 162. The expression cassette of any one of embodiments 125 to 161, wherein the expression cassette comprises a polyadenylation signal located after the polynucleotide encoding the CTLA-4 binding protein. 163. The expression cassette of embodiment 162, wherein the polyadenylation signal is a polyadenylation signal derived from the human GAPDH gene. 164. The expression cassette according to any one of embodiments 125 to 163, further comprising a polyadenylation signal located after the polynucleotide encoding the CD40 agonist. 165. The expression cassette according to embodiment 164, wherein the polyadenylation signal located after the polynucleotide encoding the CD40 agonist is hBGpA. 166. The expression cassette of any one of embodiments 125-165, wherein the expression cassette comprises an AoHV1 promoter operably linked to a polynucleotide encoding a CD40 agonist. 167. The expression cassette of any one of claims 125-166, wherein the expression cassette comprises an MMLV promoter operably linked to a polynucleotide encoding IL-12. 168. The expression cassette of any one of embodiments 125 to 167, wherein the expression cassette comprises a polyadenylation signal following the polynucleotide encoding IL-12. 169. The expression cassette of embodiment 168, wherein the polyadenylation signal following the polynucleotide encoding IL-12 is US9-10pA. 170. The expression cassette according to any one of embodiments 157 to 169, wherein the expression cassette comprises a polyadenylation signal located after the polynucleotide encoding the US10 protein. 171. The expression cassette of embodiment 170, wherein the polyadenylation signal is the human growth hormone polyadenylation signal (hGHpA). 172. An expression cassette according to any one of embodiments 160 to 171, comprising, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene, a polynucleotide encoding a naturally occurring late US11 protein, a polynucleotide encoding a US10 protein, a polynucleotide encoding a CTLA-4 binding protein, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding IL-12. 173. The expression cassette of embodiment 168, wherein the expression cassette comprises, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene, a polynucleotide encoding a naturally occurring late US11 protein, a polynucleotide encoding a US10 protein, an hGHpA polyadenylation sequence, an mCMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal derived from the human GAPDH gene, an hBGpA polyadenylation sequence, a polynucleotide encoding a CD40 agonist, an AoHV1 promoter, an MMLV promoter, a polynucleotide encoding IL-12, and a US9-10pA polyadenylation sequence. 174. A polynucleotide for a variant US11 gene comprises the polynucleotide sequence set forth in SEQ ID NO: 204, a polynucleotide encoding a US11 protein encodes the amino acid sequence set forth in SEQ ID NO: 80, a polynucleotide encoding a US10 protein encodes the amino acid sequence set forth in SEQ ID NO: 90, a polynucleotide corresponding to an mCMV promoter comprises the polynucleotide sequence set forth in SEQ ID NO: 107, a polynucleotide encoding a CTLA-4 binding protein encodes the amino acid sequence set forth in SEQ ID NO: 50, a polynucleotide for a polyadenylation signal derived from the human GAPDH gene comprises SEQ ID NO: 213, 174. The expression cassette of embodiment 173, wherein the polynucleotide corresponding to the BGp polyadenylation signal comprises the polynucleotide sequence set forth in SEQ ID NO: 102, the polynucleotide for the CD40 agonist encodes the amino acid sequence set forth in SEQ ID NO: 25, the polynucleotide for the AoHV1 promoter comprises the polynucleotide sequence set forth in SEQ ID NO: 310, the polynucleotide for the MMLV promoter comprises the polynucleotide sequence set forth in SEQ ID NO: 311, the polynucleotide for IL-12 encodes the amino acid sequence set forth in SEQ ID NO: 4, and the polynucleotide for the US9-10pA polyadenylation sequence comprises SEQ ID NO: 314. 175. A modified HSV genome comprising the expression cassette of any one of embodiments 125 to 174. 176. The modified HSV genome of embodiment 175, wherein the expression cassette is integrated into the US10-12 locus of the modified HSV genome. 177. An oncolytic virus comprising an expression cassette according to any one of embodiments 104 to 119 and an expression cassette according to any one of embodiments 125 to 174. 178. The oncolytic virus of embodiment 177, wherein the virus is a herpes simplex virus (HSV). 179. The oncolytic virus of embodiment 178, wherein the virus is HSV-1 or HSV-2. 180. The oncolytic virus according to any one of embodiments 177 to 179, wherein the virus is attenuated compared to the wild-type virus. 181. An oncolytic virus according to any one of embodiments 177 to 180, wherein the virus is capable of evading the human immune system. 182. A method for treating cancer in an individual, comprising administering to the individual an effective amount of an oncolytic virus according to any one of embodiments 1 to 102, 122 to 124, 177 to 181 or a pharmaceutical composition according to embodiment 102. 183. The method of embodiment 121, wherein the cancer is locally advanced cancer, metastatic cancer, or recurrent cancer. 184. The method of embodiment 182 or 183, wherein the cancer comprises a solid tumor. 185. A method for killing tumor cells in an individual, comprising administering to the individual an oncolytic virus according to any one of embodiments 1 to 102, 122 to 124, 177 to 181 or a pharmaceutical composition according to embodiment 103. 186. The method of embodiment 185, wherein the individual has tumor cells at a first and a second site, an oncolytic virus is administered to the first site, and the oncolytic virus induces an immune response at the first site that results in cell death of the tumor cells at the second site. 187. The method of any one of embodiments 182-186, wherein administering the oncolytic virus to the individual results in an immune response in the individual. 188. The method of any one of embodiments 182 to 187, wherein the oncolytic virus causes tumor growth inhibition. 189. The method of any one of embodiments 182 to 188, wherein the oncolytic virus generates a sustained anti-tumor immune response. 190. The method of any one of embodiments 182-189, wherein the oncolytic virus preferentially lyses tumor cells. 191. The method of any one of embodiments 182-190, wherein the oncolytic virus enhances T cell effector function and / or depletes Tregs in the tumor microenvironment. 192. The method of any one of embodiments 182-191, wherein the oncolytic virus recruits dendritic cells to the tumor microenvironment. 193. The method of any one of embodiments 182-192, wherein the oncolytic virus matures dendritic cells. 194. A method for producing an oncolytic substance, comprising culturing cells containing an oncolytic virus according to any one of embodiments 1-102, 122-124, 177-181, lysing the cells to produce a cell lysate, and purifying the oncolytic virus from the cell lysate. 195. A CD40 agonist protein comprising three single-chain trimeric CD40 ligand ectodomains each fused to a trimerization motif, wherein the CD40 agonist protein is a trimer. 196. The CD40 agonist protein according to embodiment 195, wherein the CD40 agonist is a CD40 ligand. 197. The CD40 agonist protein according to embodiment 195 or 196, wherein the CD40 ligand ectodomain is human. 198. The CD40 agonist protein according to any one of embodiments 195 to 197, wherein the CD40 ligand ectodomain comprises the sequence of amino acids according to SEQ ID NO: 20. 199. The CD40 agonist protein according to any one of embodiments 195-198, wherein the CD40 ligand ectodomain is murine. 200. The CD40 agonist protein according to embodiment 199, wherein the CD40 ligand ectodomain comprises the sequence of amino acids set forth in SEQ ID NO: 26. 201. The CD40 agonist protein according to any one of embodiments 195-200, wherein the trimerization motif is a T4 fibritin trimerization motif. 202. The CD40 agonist protein of any one of embodiments 195-201, wherein the trimerization motif comprises the amino acid sequence set forth in SEQ ID NO: 21. 203. The CD40 agonist protein of any one of embodiments 195-202, wherein the trimerization motif is linked to each of the three single-chain trimer ectodomains of the CD40 agonist by a peptide linker. 204. The CD40 agonist protein according to embodiment 203, wherein the peptide linker comprises glycine and serine residues. 205. The CD40 agonist protein according to embodiment 203 or 204, wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 23 or 27. 206. The CD40 agonist protein of any one of embodiments 195-205, wherein the CD40 agonist protein activates dendritic cells. 207. A polynucleotide encoding the CD40 agonist protein of any one of embodiments 195-206. 208. A vector comprising a polynucleotide according to embodiment 207. 209. A host cell comprising the vector according to embodiment 208. 210. A method for inhibiting tumor growth in an individual, comprising administering to the individual an effective amount of the CD40 agonist protein of any one of embodiments 195-206, or a polynucleotide encoding the CD40 agonist protein of any one of embodiments 195-206. 211. The method of any one of embodiments 182-193 and 210, wherein the individual is a human. 212. Oncolytic virus corresponding to JP-OV-2. 213. Oncolytic virus corresponding to the virus shown in Figure 15E (Step 3 virus (JP-OV-2)).

[0148] Any embodiment or aspect of the present disclosure that refers in the specification or claims to a method of treatment is applicable, mutatis mutandis, to the manufacture of a medicament for treatment. [Example]

[0149] The subject matter of the present disclosure will be better understood by reference to the following examples, which are provided by way of illustration of the invention and not by way of limitation.

[0150] Example 1: Engineering an HSV-1 oncolytic virus with innate and adaptive immune stealth functions. This example describes the engineering of a herpes simplex virus type 1 (HSV-1) oncolytic virus (OV) with stealth features that enhance viral evasion of innate and adaptive antiviral host responses.

[0151] Manipulation of enhanced innate immune stealth function Previously developed HSV-1 OVs have employed several strategies to achieve partial attenuation of the virus while maintaining sufficient viral replication in tumor cells. For example, Talimogene laherparepvec (a previously reported genetically engineered oncolytic herpesvirus) is an HSV-1 OV that harbors an inactive γ34.5 gene (Δγ34.5) encoding a neurovirulence factor and possesses immediate-early (IE) expression of the US11 gene to improve viral replication. Deletion of the γ34.5 gene results in a highly attenuated virus that is safe but replicates poorly. IE-US11 expression is achieved by deleting the US12 gene, which causes the US11 gene to be driven by the US12 promoter, resulting in IE-US11 expression instead of the late (L) expression typically seen with the endogenous US11 promoter.IE-US11 expression has been shown to partially compensate for the deletion of the γ34.5 gene, resulting in a virus that replicates better than the highly attenuated Δγ34.5 single mutant without re-establishing neurovirulence (Cassady et al. The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2. J. Virol. 1998;72(11):8620-8626; Mulvey et al. Regulation of eIF2alpha phosphorylation by different functions that act during discrete phases in the herpes simplex virus type 1 life cycle. J. Virol. 2003;77(20):10917-10928; Mohr et al. A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells are severely attenuated in animals.J.Virol.2001;75(11):5189-5196; and Taneja et al.Enhanced anti-tumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells.Proc Natl Acad Sci US A.2001;98(15):8804-8808).However, IE-US11 expression by deletion of US12 eliminates L-US11 expression from its native promoter and results in reduced levels of US11 compared to wild-type (WT) virus at later time points of infection, which may make the virus vulnerable to inhibition by the innate antiviral response as the infection cycle progresses.

[0152] To circumvent the innate antiviral response throughout the infection cycle, HSV-1 OV was engineered to contain two copies of the US11 gene. The first copy of the US11 gene was a codon-optimized IE-US11 under the control of the US12 promoter, which partially compensates for the deletion of the γ34.5 gene, as described above, allowing efficient viral replication in tumor cells. The second copy of the US11 gene was an endogenous L-US11 gene under the endogenous US11 promoter, which was intended to protect the virus from protein kinase R (PKR)-mediated translational arrest throughout the temporal viral gene expression cycle.

[0153] Manipulation of enhanced adaptive immune stealth function As mentioned above, previously developed HSV-1 OVs, such as previously reported genetically engineered oncolytic herpesviruses, contain deletions of the US12 gene to drive IE-US11 expression from the US12 promoter. US12 encodes ICP47, a transporter associated with inhibitors of antigen processing (TAP), which normally prevents viral antigen presentation on the cell surface. Removal of ICP47 increases viral antigen presentation and leads to rapid elimination of virus-infected cells by antiviral T cells, potentially limiting OV efficacy. Therefore, deletion of US12 may affect in vivo viral replication and susceptibility to the host adaptive immune system (Pourchet A, Fuhrmann SR, Pilones KA, et al. CD8(+) T-cell immune evasion enables oncolytic v...

Claims

1. an oncolytic herpes simplex virus type 1 (HSV-1), a. a cassette integrated into one or both of the γ34.5 loci, the cassette 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 into the US10-12 locus, the cassette comprising, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding a native US11 protein, an additional polynucleotide encoding a native US11 protein, a polynucleotide encoding a US10 protein, a polyadenylation signal operably linked to the polynucleotide encoding the US10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV1 promoter controlling expression of the CD40 agonist, an MMLV promoter controlling expression of IL-12, a polynucleotide encoding IL-12, and a polyadenylation signal operably linked to the polynucleotide encoding IL-12. Including, the hFLT3L polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:71, the UL49.5 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:82, the IL-12 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:4, the CD40 agonist polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:25, the CTLA-4 binding protein polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:50, the variant US11 gene polynucleotide comprises the polynucleotide sequence set forth in SEQ ID NO:204, the US11 further polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:80, and the US10 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:

90. Oncolytic herpes simplex virus type 1 (HSV-1).

2. an oncolytic herpes simplex virus type 1 (HSV-1), a. a cassette integrated into one or both of the γ34.5 loci, the cassette comprising, in order from upstream to downstream, a CMV promoter, a polynucleotide encoding a hFLT3 protein, a P2A cleavage sequence, a polynucleotide encoding UL49.5, and a polyadenylation signal; and b. Another cassette integrated into the US10-12 locus, the cassette comprising, in order from upstream to downstream, a polynucleotide comprising a variant US11 gene encoding a native US11 protein, an additional polynucleotide encoding a native US11 protein, a polynucleotide encoding a US10 protein, a polyadenylation signal operably linked to the polynucleotide encoding the US10 protein, a CMV promoter, a polynucleotide encoding a CTLA-4 binding protein, a polyadenylation signal operably linked to the polynucleotide encoding the CTLA-4 binding protein, a polyadenylation signal operably linked to a polynucleotide encoding a CD40 agonist, a polynucleotide encoding a CD40 agonist, an AoHV1 promoter controlling expression of the CD40 agonist, an MMLV promoter controlling expression of IL-12, a polynucleotide encoding IL-12, and a polyadenylation signal operably linked to the polynucleotide encoding IL-12. Including, the hFLT3L polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:71, the UL49.5 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:82, the IL-12 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:8, the CD40 agonist polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:28, the CTLA-4 binding protein polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:56, the variant US11 gene polynucleotide comprises the polynucleotide sequence set forth in SEQ ID NO:204, the US11 further polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:80, and the US10 polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:

90. Oncolytic herpes simplex virus type 1 (HSV-1).

3. An oncolytic virus comprising one or more expression cassettes comprising a polynucleotide encoding IL-12, a polynucleotide encoding a CD40 agonist, and a polynucleotide encoding a CTLA-4 binding protein.

4. 4. The oncolytic virus of claim 3, wherein one or both of the native γ34.5 genes are inactivated by deletion, substitution, or insertion of backbone nucleic acid.

5. 5. The oncolytic virus of claim 3 or claim 4, wherein the native US12 gene of the virus is inactivated by a deletion, substitution, or insertion in the backbone nucleic acid.

6. The oncolytic virus of any one of claims 3 to 5, wherein the one or more expression cassettes comprise a polynucleotide encoding a FLT3 ligand (FLT3L).

7. The oncolytic virus of any one of claims 3 to 6, wherein the one or more expression cassettes further comprise a polynucleotide comprising a variant US11 gene.

8. 8. The oncolytic virus of claim 7, wherein the variant US11 gene is operably associated with an immediate-early promoter.

9. 9. The oncolytic virus of claim 3, comprising a native late US11 gene.

10. The oncolytic virus of any one of claims 3 to 9, further comprising a transporter associated with antigen processing (TAP) inhibitor.

11. The oncolytic virus of any one of claims 3 to 10, wherein one of the expression cassettes is inserted into the natural US10-US12 locus.

12. A pharmaceutical composition comprising the oncolytic virus according to any one of claims 3 to 11 and a pharmaceutically acceptable excipient.

13. a. a polynucleotide encoding a TAP inhibitor, and b. Polynucleotides encoding FLT3 ligands an expression cassette comprising:

14. A modified HSV genome comprising the expression cassette of claim 13.

15. 15. The modified HSV genome of claim 14, wherein one or both of the γ34.5 loci of the modified HSV genome are replaced with the cassette.

16. An oncolytic virus comprising the expression cassette of claim 13.

17. 17. The oncolytic virus of claim 16, wherein one or both of the γ34.5 loci of the oncolytic virus are replaced with the expression cassette.

18. a. a polynucleotide encoding IL-12; b. a polynucleotide encoding a CD40 agonist, and c. Polynucleotides encoding CTLA-4 binding proteins an expression cassette comprising:

19. 19. The expression cassette of claim 18, further comprising a polynucleotide encoding a US10 protein.

20. 20. The expression cassette of claim 18 or claim 19, wherein the expression cassette comprises a native late US11 gene.

21. 21. The expression cassette of claim 20, wherein the expression cassette comprises a variant polynucleotide encoding a US11 protein.

22. A modified HSV genome comprising an expression cassette according to any one of claims 18 to 21.

23. 23. The modified HSV genome of claim 22, wherein the expression cassette is integrated into the US10-12 locus of the modified HSV genome.

24. An oncolytic virus comprising an expression cassette according to any one of claims 16 and 18 to 21.

25. 25. The oncolytic virus of claim 24, wherein the virus is a herpes simplex virus (HSV).

26. 12. A method for treating cancer in an individual, comprising administering to said individual an effective amount of an oncolytic virus according to any one of claims 3 to 11.

27. A method for killing tumor cells in an individual, comprising administering an oncolytic virus according to any one of claims 3 to 11.

28. 12. A method for producing an oncolytic substance, comprising culturing cells containing the oncolytic virus of any one of claims 3 to 11, lysing the cells to produce a cell lysate, and purifying the oncolytic virus from the cell lysate.

29. A CD40 agonist protein comprising three single-chain trimeric CD40 ligand ectodomains, each fused to a trimerization motif, wherein said CD40 agonist protein is a trimer.

30. 30. The CD40 agonist protein of claim 29, wherein the trimerization motif is a T4 fibritin trimerization motif.

31. A polynucleotide encoding the CD40 agonist protein of claim 29 or claim 30.

32. A vector comprising the polynucleotide of claim 31.

33. A host cell comprising the vector of claim 32.

34. A method for inhibiting tumor growth in an individual, comprising administering an effective amount of a CD40 agonist protein of claim 29 or claim 30.

Images