Modified viruses

Abstract

To provide a virus that can be used as an oncolytic therapy.SOLUTION: The present invention provides an oncolytic virus encoding a CTLA-4 inhibitor such as an anti-CTLA-4 antibody or an antigen binding fragment thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to oncolytic immunotherapeutic agents and the use of oncolytic immunotherapeutic agents in the treatment of cancer. [Background technology]

[0002] Viruses have the inherent ability to invade cells with high efficiency. After cell entry, viral genes are expressed and the virus replicates. This usually results in the death of the infected cell and the release of antigenic components of the cell as the cell ruptures upon death. As a result, virus-mediated cell death tends to result in an immune response by the host against these cellular components, which is amplified due to the recognition of so-called damage-associated molecular patterns (DAMPs), both derived from the host cell and encoded or incorporated by the virus itself, which aid in the activation of the immune response.

[0003] Viruses also engage various mediators of the innate immune response as part of the host response to recognition of viral infection, for example, through Toll-like receptors and cGAS / STING signaling, which result in inflammation and activation of the interferon response, which are also immunogenic signals to the host, and recognition of pathogen-associated molecular patterns (PAMPs). These immune responses can provide immunogenic benefits to cancer patients, such that immune responses to tumor antigens provide a systemic benefit resulting in the treatment of non-virally infected tumors, including micrometastatic disease, and provide vaccination against recurrence.

[0004] The combined direct ("oncolytic") effect of the virus and the immune response to tumor antigens (including non-self "neo-antigens," i.e., derived from specific mutated genes in individual tumors) is called "oncolytic immunotherapy."

[0005] Viruses can also be used as delivery vehicles ("vectors") to express heterologous genes inserted into the viral genome in infected cells. These properties make viruses useful in a variety of biotechnological and medical applications. For example, viruses expressing heterologous therapeutic genes can be used in gene therapy. In the context of oncolytic immunotherapy, the delivered genes may include genes encoding specific tumor antigens, genes intended to induce an immune response or increase the immunogenicity of released antigens after viral replication and cell death, genes intended to shape the immune response generated, genes that increase the general immune activation state of the tumor, or genes that increase the direct oncolytic properties (i.e., cytotoxic effect) of the virus. Importantly, viruses have the ability to deliver encoded molecules intended to help initiate, augment, or shape a systemic anti-tumor immune response directly and selectively to tumors. This may have the benefit of, for example, reduced toxicity or concentrating beneficial effects in tumors (including those not infected with the virus) rather than off-target effects in normal (i.e., non-cancerous) tissues compared to systemic administration of these same molecules or other molecules targeting the same pathway.

[0006] Numerous viruses, such as herpes simplex virus (HSV), have demonstrated utility in oncolytic cancer therapy. For use in oncolytic cancer therapy, HSV must be rendered ineffective so that it is no longer pathogenic but can invade and kill tumor cells. Numerous null mutations in HSV have been identified, including disruption of the genes encoding ICP34.5, ICP6, and / or thymidine kinase, which do not prevent the virus from replicating in tumor tissue in culture or in vivo, but do prevent significant replication in normal tissues. HSV with only the ICP34.5 gene disrupted replicates in many tumor cell types in vitro and selectively replicates in tumor tissue but not in surrounding tissues in mouse tumor models. Clinical trials of HSV with deleted ICP34.5, or deleted ICP34.5 and ICP6, have also demonstrated safety and selective replication in human tumor tissue.

[0007] As discussed above, oncolytic viruses such as HSV can also be used to deliver therapeutic genes in cancer treatment. This type of ICP34.5-deleted virus, further deleted for ICP47 and encoding a heterologous gene for GM-CSF, has also been tested in clinical trials, including a phase 3 trial in melanoma, where safety and efficacy in humans have been demonstrated. GM-CSF is a proinflammatory cytokine with multiple functions, including stimulating monocytes to escape the circulation and migrate to tissues where they proliferate and mature into macrophages and dendritic cells. GM-CSF is important for the proliferation and maturation of antigen-presenting cells, and its activity is necessary for the activation of antitumor immune responses. Study data demonstrated tumor responses in injected tumors and, to a lesser extent, in uninjected tumors. Responses tended to be durable (lasting months to years), and responding patients appeared to achieve a survival benefit. Each of these demonstrated the involvement of the immune system in cancer treatment in addition to direct oncolytic effects. However, this and other data regarding oncolytic viruses have generally shown that not all tumors respond to treatment and not all patients achieve a survival benefit. Thus, improvements to the art of oncolytic therapy are clearly needed.

[0008] Recently, it has been shown that oncolytic immunotherapy can be used in combination with immune co-inhibitory pathway blockade (i.e., inhibition or "antagonism" of immune checkpoint pathways, also known as immune co-inhibitory pathways), to produce additive or synergistic therapeutic effects. Immune co-inhibitory pathway blockade is intended to block host immune inhibitory mechanisms that typically serve to prevent the development of autoimmunity. However, in cancer patients, these mechanisms may also serve to inhibit or block the induction of potentially beneficial effects of any tumor-induced immune response.

[0009] Systemic blockade of these pathways with agents targeting cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4), PD-1, or PD-L1 has shown efficacy in many tumor types, including melanoma and lung cancer. However, based on their mechanism of action, off-target toxicity can occur due to the induction of autoimmunity. Nevertheless, these agents are well tolerated to provide significant clinical benefit. Other immune co-inhibitory pathways and associated targets for which agents (primarily antibodies) have been developed include LAG-3, TIM-3, VISTA, CSF1R, IDO, CEACAM1, and CD47. Optimal clinical activity of these agents, such as PD1, PDL1, LAG-3, TIM-3, VISTA, CSF1R, IDO, CD47, and CEACAM1, may require systemic administration or presence in all tumors depending on their mechanism of action (i.e., targeting the interface between immune effector cells and tumors or other immune inhibitory mechanisms in / at tumors). In some cases, agents that target a more localized presence, such as CTLA-4, in only some tumors or in only some lymph nodes may also be optimally effective.

[0010] An alternative approach to increasing anti-tumor immune responses in cancer patients is to target (activate) immune costimulatory pathways, as opposed to inhibiting immune co-inhibitory pathways. These pathways send activation signals to T cells and other immune cells, typically resulting from the interaction of relevant ligands on antigen-presenting cells (APCs) with relevant receptors on the surface of T cells and other immune cells. Depending on the ligand / receptor, these signals can result in increased activation of T cells and / or APCs and / or NK cells and / or B cells, including specific subtypes; increased differentiation and proliferation of T cells and / or APCs and / or NK cells and / or B cells, including specific subtypes; or suppression of the immune inhibitory activity of T cells, such as regulatory T cells. Thus, while activation of these pathways is predicted to result in an increased anti-tumor immune response, systemic activation of these pathways, i.e., activation of a global immune response rather than a specific or selective anti-tumor immune response, can be predicted to result in significant off-target toxicity in non-tumor tissues, the extent of which depends on the specific immune co-inhibitory pathway being targeted. Nevertheless, drugs targeting immune co-inhibitory pathways (mainly agonist antibodies or, less frequently, soluble ligands for the receptors in question), such as drugs targeting GITR, 4-1-BB, OX40, CD40 or ICOS, that are intended for systemic use (i.e., intravenous delivery) are in clinical development or have been proposed for clinical development.

[0011] For many of these approaches targeting immune co-inhibitory pathways or co-inhibitory pathways to be successful, a pre-existing immune response against the tumor is required, i.e., it can enhance the pre-existing immune response or alleviate the blockade of the anti-tumor immune response. The presence of an inflamed tumor microenvironment, which indicates such an ongoing response, is also required. A pre-existing immune response against tumor neoantigens appears to be particularly important for the activity of immune co-inhibitory pathway blockade and related drugs. Only some patients may have an ongoing immune response against tumor antigens, including neoantigens and / or an inflamed tumor microenvironment, both of which are required for optimal activity of these drugs. Therefore, oncolytic agents that can induce immune responses against tumor antigens, including neoantigens, and / or induce an inflamed tumor microenvironment are attractive for use in combination with immune co-inhibitory pathway blockade and immune-enhancing agents. This may explain the promising combined anti-tumor effects of oncolytic agents and immune co-inhibitory pathway blockade observed to date in mice and humans.

[0012] The above discussion demonstrates that there remains significant room for improvement in cancer treatment using oncolytic agents and agents that target oncolytics, anti-tumor immune responses, and immune co-inhibitory or co-stimulatory pathways. Summary of the Invention

[0013] The present invention provides oncolytic viruses expressing CTLA-4 inhibitors. The viruses may further comprise other immunomodulatory agents. In particular, the viruses may comprise GM-CSF and / or at least one molecule targeting the immune costimulatory pathway. CTLA-4 inhibitors act to block the costimulatory pathway, i.e., prevent the interaction between CTLA-4 and B7. GM-CSF helps induce an inflammatory tumor microenvironment and stimulates the proliferation and maturation of antigen-presenting cells, such as dendritic cells, which help induce anti-tumor immune responses. These immune responses can be amplified by activating immune costimulatory pathways or pathways using immune costimulatory pathway-activating molecules or molecules also delivered by the oncolytic viruses.

[0014] Oncolytic viruses replicate within tumors, causing tumor cell lysis and the release of tumor antigens, combined with the activation of local inflammation and innate immune responses, all of which are beneficial to the activation of antitumor immune responses and the activity of inhibitors of CTLA-4 / B7 interactions.

[0015] Direct delivery of molecules that inhibit CTLA-4 / B7 interaction to immune response-initiating tumors, including potential transport to draining lymph nodes, focuses the inhibitor's immune-enhancing effects on tumors—i.e., tumor antigens present within the tumor—reducing systemic toxicity and preventing the activation of regulatory T cells (Tregs), which would otherwise block T cell activation at the site of immune response initiation. Using oncolytic viruses to deliver molecules targeting CTLA-4 and, optionally, immune costimulatory pathways to tumors, aims to enhance the immune effect of antitumor immune responses and reduce the enhancement of immune responses to nontumor antigens. Thus, immune cells in tumors and tumor-draining lymph nodes are selectively affected by virus-expressed molecules rather than general immune cells. This results in enhanced immune cell stimulation and reduced off-target toxicity. This is also important for focusing the combined effects of systemic immune costimulatory pathway blockade and immune costimulatory pathway activation on tumors, i.e., the immune response from which the costimulatory blockade is released is an antitumor immune response, rather than a response to nontumor antigens.

[0016] The present invention takes advantage of the fact that, when delivered by oncolytic viruses, the site of action of CTLA-4 blockade and optional immune costimulatory pathway activation and GM-CSF expression is the tumor and / or tumor-draining lymph nodes, but the result of such activation (an amplified systemic anti-tumor immune response) is systemic. This targets the tumor globally, not just the tumor to which the oncolytic virus delivered the immunomodulatory molecule. Thus, the oncolytic viruses of the present invention result in improved cancer treatment through the generation of improved tumor-focused immune responses. The oncolytic viruses of the present invention provide improved anti-tumor immunostimulatory effects, such as increased immune-mediated effects against tumors not destroyed by oncolysis, including micrometastatic disease, leading to more effective destruction of these tumors, preventing future recurrence and improving overall survival, and more effective long-term anti-tumor vaccination.

[0017] Anti-tumor efficacy is improved when the oncolytic viruses of the invention are used as single agents and when the viruses are used in combination with other anti-cancer modalities, such as chemotherapy, targeted drug therapy, radiation, and in preferred embodiments, immune checkpoint blockade (i.e., antagonists of immune co-inhibitory pathways, e.g., antibodies against PD1 or PD-L1) and / or agonists of immune co-stimulatory pathways.

[0018] Thus, the present invention provides an oncolytic virus encoding a CTLA-4 inhibitor, which is preferably an anti-CTLA-4 antibody or antibody-like molecule, or an antigen-binding fragment thereof.

[0019] The virus further provides an oncolytic virus comprising (i) a gene encoding GM-CSF, and / or (ii) an immune costimulatory pathway activating molecule or a gene encoding an immune costimulatory pathway activating molecule. The virus may encode more than one immune costimulatory pathway activating molecule / gene.

[0020] The immune costimulatory pathway activating molecule is preferably GITRL, 4-1-BBL, OX40L, ICOSL, or CD40L, or a modified version of any of them. Examples of modified versions include costimulatory pathway agonists that are secreted rather than membrane bound, and / or agonists that have been modified to form protein multimers.

[0021] The virus can be a modified clinical isolate, such as a modified clinical isolate of a virus, which kills two or more tumor cell lines more quickly and / or at lower doses in vitro than one or more reference clinical isolates of the same virus.

[0022] The virus is preferably a herpes simplex virus (HSV), such as HSV 1. HSV typically does not express functional ICP34.5 and / or functional ICP47, and / or expresses the US11 gene as an immediate early gene.

[0023] The present invention also provides: - a pharmaceutical composition comprising the virus of the invention and a pharmaceutically acceptable carrier or diluent, - a virus of the invention for use in a method for treating the human or animal body by therapy, - a virus of the invention for use in a method for treating cancer, the method optionally comprising administering a further anti-cancer agent. - a product containing the virus of the invention in a sterile vial, ampoule or syringe, - a method for treating cancer, comprising administering a therapeutically effective amount of the virus or pharmaceutical composition of the present invention to a patient in need thereof, the method optionally comprising administering a further anti-cancer agent; - Use of a virus of the invention in the manufacture of a medicament for use in a method of treating cancer, the method optionally comprising administering a further anti-cancer agent. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1 shows the structure of a virus used to construct an exemplary virus of the invention, comprising an anti-mouse or anti-human CTLA-4 construct, which is a secreted scFv molecule linked to the Fc region of a codon-optimized human or mouse IgG1. The scFv contains light and heavy chain variable regions derived from 9D9 (the first mouse antibody used to validate CTLA-4; WO 2007 / 123737: mouse version) or ipilimumab (WO 2014 / 066532: human version), linked by a 15-mer [G4S]3 (GGGGSGGGGSGGGGS). This virus is a modified version of the HSV1 RH018A strain (clinical strain 18). The ICP34.5 and ICP47 genes are inactivated in the virus. The US11 gene is placed under the control of the ICP47 immediate-early gene promoter by deletion of the ICP47 promoter. An expression cassette is inserted into the ICP34.5 locus. In virus 17, the expression cassette contains the human GM-CSF gene under the control of a CMV promoter and the GALV gene under the control of an RSV promoter. Virus 16 is the same as virus 17, except that it contains human GM-CSF instead of mouse GM-CSF. Viruses 25 and 29 are the same as viruses 16 and 17, respectively, except that each additionally contains a GFP gene under the control of an MMLV promoter in the expression cassette. Viruses 27 and 31 are the same as viruses 25 and 29, respectively, except that the GFP gene has been replaced with mouse anti-CTLA4 and human anti-CTLA4. [Figure 1B] This is a continuation of Figure 1A. [Figure 2A] FIG. 2 shows the structure of the plasmids used to construct exemplary viruses of the invention. [Figure 2B] Continuation of Figure 2A. [Figure 2C] Continuation of Figure 2A. [Figure 2D] Continuation of Figure 2A. [Figure 3]Figure 3 shows the structures of codon-optimized secreted scFv molecule anti-mouse or anti-human CTLA-4 constructs linked to the Fc region of human or mouse IgG1. The scFvs contain linked ([G4S]3) light and heavy variable chains derived from 9D9 (the first mouse antibody used to validate CTLA-4; US2011044953: mouse version) or ipilimumab (US20150283234: human version). The structures of the resulting CTLA-4 inhibitors are also shown. [Figure 4] Figure 4 shows a Western blot demonstrating the expression of anti-mouse CTLA-4 from virus 27. The gel used was a reduced, denatured PVDF membrane Tris-glycine gel. Anti-CTLA-4 was detected using an alkaline phosphatase-conjugated anti-mouse IgG1 antibody. Lane 1: Spectra broad-range ladder; Lane 2: Virus 27, undiluted supernatant; Lane 3: Virus 27, 2-fold dilution of supernatant; Lane 4: Virus 27, 4-fold dilution of supernatant; Lane 5: Virus 27, 8-fold dilution of supernatant; Lane 6: Virus 27, 16-fold dilution of supernatant; Lane 7: Virus 27, 32-fold dilution of supernatant; Lane 8: Negative control virus, undiluted supernatant; The expected size of anti-CTLA-4 (reduced) is 57 kDa. [Figure 5] Figure 5 shows the superior tumor control and tumor regression in uninjected tumors with a virus expressing anti-mCTLA-4 (virus 27) compared to an identical virus (virus 16) that does not express CTLA-4. The virus dose used was 5x104 pfu (50 ul of 1x106 pfu / ml in each case), administered three times per week. This viral dose level is subtherapeutic for uninjected tumors compared to virus 16, allowing the benefit of delivery of the additional molecule encoded by virus 27 to be clearly seen. [Figure 6]Figure 6 shows superior tumor control and tumor regression in tumors injected and uninjected with a virus expressing anti-mCTLA-4 (virus 27) compared to an identical virus (virus 16) that does not express CTLA-4. The viral dose used was 5x10 pfu per week for the right tumor with anti-mCTLA-4 expressing virus (virus 27) compared to an identical virus (virus 16) that does not express CTLA-4. Each line represents a different mouse. [Figure 7-1] Figure 7 shows the effect of combined treatment of bilateral murine A20 tumors with anti-PD1 and virus 27 expressing mGM-CSF, GALVR, and anti-mCTLA-4. The top panel shows the effect of anti-PD1 alone on both injected (right) and uninjected (left) tumors. The middle panel shows the effect of virus 27 alone on both injected (right) and uninjected (left) tumors. The bottom panel shows the excellent tumor control and tumor regression achieved when anti-PD1 and virus 27 were both injected into the right tumor. The enhanced anti-tumor effect of combined treatment is observed in both injected (right) and uninjected (left) tumors. Each line represents a different mouse. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8]Figure 8 shows the superior tumor control and tumor regression effects of virus 31 expressing hGM-CSF, GALVR, and anti-human CTLA-4 compared with virus 17 expressing only hGM-CSF and GALVR in mouse MC38 tumors in knock-in mice expressing human CTLA-4. The anti-tumor effect of virus 31 is observed when the virus is administered alone or in combination with anti-PD1. Superior tumor control and tumor regression in injected tumors is achieved with virus 31 expressing anti-human CTLA-4 compared with an identical virus that does not express anti-human CTLA-4 (left panel). This effect is further enhanced when treatment with the virus is combined with anti-PD1 treatment. When treatment with either virus is combined with anti-PD1 treatment, superior tumor control and tumor regression are also observed in uninjected tumors (right panel). This improvement is significantly greater with virus 31 expressing anti-CTLA-4 than with virus 17 that does not express anti-CTLA-4. Each line represents a different mouse.

[0025] Brief description of the sequence listing SEQ ID NO: 1 is the amino acid sequence of the light chain variable region of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 2 is the complete light chain amino acid sequence, including the light chain variable region amino acid sequence, of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 3 is the heavy chain variable region amino acid sequence of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 4 is the heavy chain CH1 amino acid sequence of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 5 is the heavy chain CH2 / 3 amino acid sequence of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 6 is the complete heavy chain amino acid sequence of the human CTLA-4 antibody used in the Examples. SEQ ID NO: 7 is the amino acid sequence of the signal peptide present in the CTLA-4 antibody of the example. SEQ ID NO: 8 is the amino acid sequence of the linker present between the light chain variable region and the heavy chain variable region in the CTLA-4 antibody of the example. SEQ ID NO: 9 is the amino acid sequence of the human scFv CTLA-4 antibody of the example. SEQ ID NO: 10 is the nucleotide sequence of an example human scFv CTLA-4 antibody. SEQ ID NO: 11 is the amino acid sequence of the light chain variable region of the murine CTLA-4 antibody used in the Examples. SEQ ID NO: 12 is the heavy chain variable region amino acid sequence of the murine CTLA-4 antibody used in the Examples. SEQ ID NO: 13 is the complete heavy chain amino acid sequence of the murine CTLA-4 antibody used in the Examples. SEQ ID NO: 14 is the amino acid sequence of the murine scFv CTLA-4 antibody of the example. SEQ ID NO: 15 is the nucleotide sequence of an example murine scFv CTLA-4 antibody. SEQ ID NO: 16 is the nucleotide sequence of an example murine scFv CTLA-4 antibody present in an exemplary virus with restriction sites inserted at the N- and C-termini for cloning purposes. The restriction sites are the first 6 and last 8 nucleotides of the sequence. SEQ ID NO: 17 is the nucleotide sequence of an example human scFv CTLA-4 antibody present in an exemplary virus with restriction sites inserted at the N- and C-termini for cloning purposes. The restriction sites are the first 6 and last 8 nucleotides of the sequence. SEQ ID NO: 18 is the nucleotide sequence of mouse GM-CSF. SEQ ID NO: 19 is the nucleotide sequence of a codon-optimized version of mouse GM-CSF. SEQ ID NO: 20 is the nucleotide sequence of human GM-CSF. SEQ ID NO: 21 is the nucleotide sequence of a codon-optimized version of human GM-CSF. SEQ ID NO: 22 is the amino acid sequence of mouse GM-CSF. SEQ ID NO: 23 is the amino acid sequence of human GM-CSF. SEQ ID NO: 24 is the nucleotide sequence of GALV-R-. SEQ ID NO: 25 is the nucleotide sequence of the codon-optimized version of GALV-R-. SEQ ID NO: 26 is the amino acid sequence of GALV-R-. SEQ ID NO:27 is the nucleotide sequence of a codon-optimized version of the human / mouse hybrid membrane-bound version of CD40L. SEQ ID NO: 28 is the amino acid sequence of the human / mouse hybrid membrane-bound version of CD40L. SEQ ID NO:29 is the nucleotide sequence of a codon-optimized version of the multimeric secreted version of human CD40L. SEQ ID NO: 30 is the amino acid sequence of the multimeric secreted version of human CD40L. SEQ ID NO: 31 is the nucleotide sequence of a codon-optimized version of the multimeric secreted version of mouse CD40L. SEQ ID NO: 32 is the amino acid sequence of the multimeric secreted version of mouse CD40L. SEQ ID NO: 33 is the nucleotide sequence of wild-type human CD40L. SEQ ID NO: 34 is the amino acid sequence of wild-type human CD40L. SEQ ID NO: 35 is the nucleotide sequence of wild-type mouse CD40L. SEQ ID NO: 36 is the amino acid sequence of wild-type mouse CD40L. SEQ ID NO: 37 is the nucleotide sequence of the CMV promoter. SEQ ID NO:38 is the nucleotide sequence of the RSV promoter. SEQ ID NO: 39 is the nucleotide sequence of BGH polyA. SEQ ID NO:40 is the nucleotide sequence of SV40 late polyA. SEQ ID NO: 41 is the nucleotide sequence of rabbit β-globulin poly A. SEQ ID NO: 42 is the nucleotide sequence of GFP. SEQ ID NO: 43 is the nucleotide sequence of the retroviral LTR from MMLV. SEQ ID NO: 44 is the nucleotide sequence of the EF1a promoter. SEQ ID NO:45 is the nucleotide sequence of the SV40 promoter. SEQ ID NO: 46 is the nucleotide sequence of HGH polyA.

[0026] Detailed Description of the Invention Oncolytic viruses The virus of the present invention is oncolytic. An oncolytic virus is a virus that infects and replicates in tumor cells, thereby killing the tumor cells. Therefore, the virus of the present invention is replicable. Preferably, the virus is selectively replicable in tumor tissue. A virus is selectively replicable in tumor tissue if it replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus to replicate in different tissue types can be determined using standard techniques in the art.

[0027] The virus of the present invention can be any virus with these properties, such as herpesvirus, poxvirus, adenovirus, retrovirus, rhabdovirus, paramyxovirus, or reovirus, or any species or strain within these larger groups.The virus of the present invention can be wild-type (i.e., not modified from the parent virus species), or can have gene disruption or gene addition.Which of these depends on the virus species used.Preferably, the virus is a herpesvirus species, more preferably a HSV strain including HSV1 and HSV2 strains, and most preferably a HSV1 strain.In a particularly preferred embodiment, the virus of the present invention is based on a clinical isolate of the virus species used.Clinical isolates may be selected based on those with particularly advantageous properties for cancer treatment.

[0028] The virus can be a modified clinical isolate, where the clinical isolate kills two or more tumor cell lines in vitro more rapidly and / or at lower doses than one or more reference clinical isolates of the same virus. Typically, the clinical isolate kills two or more tumor cell lines within 48 hours, preferably 24 hours, of infection at a multiplicity of infection (MOI) of 0.1 or less. Preferably, the clinical isolate kills a broad range of tumor cell lines, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or for example all, of the following human tumor cell lines: U87MG (glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreatic), CAPAN-1 (pancreatic), HT1080 (fibrosarcoma).

[0029] In a preferred embodiment, the virus of the invention is a strain selected from: strain RH018A, which has accession number ECCAC16121904; strain RH004A, which has accession number ECCAC16121902; strain RH031A, which has accession number ECCAC16121907; strain RH040B, which has accession number ECCAC16121908; strain RH015A, which has accession number ECCAC16121903; strain RH021A, which has accession number ECCAC16121905; strain RH023A, having accession number ECCAC16121906; and strain RH047A with accession number ECCAC16121909.

[0030] More preferably, the virus of the invention is a strain selected from: strain RH018A, which has accession number ECCAC16121904; strain RH004A, which has accession number ECCAC16121902; strain RH031A, which has accession number ECCAC16121907; strain RH040B, having accession number ECCAC16121908; and strain RH015A with accession number ECCAC16121903.

[0031] Most preferably, the virus of the invention is strain RH018A, which has accession number EACC16121904. Any one of the deposited strains may be modified as provided herein.

[0032] The HSV of the present invention can selectively replicate in tumors, such as human tumors. Typically, HSV replicates efficiently in target tumors but not in non-tumor tissues. The HSV may contain one or more mutations in one or more viral genes that inhibit replication in normal tissues but still allow replication in tumors. The mutations may be, for example, mutations that prevent the expression of functional ICP34.5, ICP6, and / or thymidine kinase by HSV.

[0033] In one preferred embodiment, the gene encoding ICP34.5 is mutated to confer selective oncolytic activity to HSV. Mutations in the gene encoding ICP34.5 that prevent the expression of functional ICP34.5 are described in Chou et al. (1990) Science 250:1262-1266, Maclean et al. (1991) J. Gen. Virol. 72:631-639, and Liu et al. (2003) Gene Therapy 10:292-303, which are incorporated herein by reference. Additionally, the gene encoding ICP6 and / or the gene encoding thymidine kinase can be inactivated, as can other genes, as long as such inactivation does not prevent viral infection or replication within tumors.

[0034] HSV may contain additional mutations that enhance HSV replication in tumors. The resulting enhancement of viral replication in tumors not only leads to direct "oncolytic" tumor cell killing by the virus, but also enhances the level of heterologous (i.e., genes inserted into the virus, in the case of the viruses of the present invention, genes encoding CTLA-4 inhibitors, GM-CSF, and / or immune costimulatory pathway-activating molecules) gene expression, increasing the amount of tumor antigens released when tumor cells die. Both of these may also improve the immunogenic properties of therapies for cancer treatment. For example, in a preferred embodiment of the present invention, deletion of the gene encoding ICP47 in a manner that places the US11 gene under the control of the immediate-early promoter that normally regulates the expression of the gene encoding ICP47 results in enhanced replication in tumors (see Liu et al., 2003, incorporated herein by reference).

[0035] Other mutations can also be introduced into the virus of the present invention, placing the sequence encoding HSV late gene US11 under the control of a promoter that is not dependent on viral replication.This mutation allows US11 to be expressed before HSV replication occurs, enhancing viral replication in tumors.In particular, this mutation enhances the replication of HSV that lacks the gene encoding functional ICP34.5.

[0036] Thus, in one embodiment, the HSV of the present invention comprises a US11 gene operably linked to a promoter, the activity of which is independent of viral replication. The promoter can be an immediate-early (IE) promoter or a non-HSV promoter active in mammalian, preferably human, tumor cells. The promoter can be, for example, a eukaryotic promoter, such as a promoter derived from a mammalian, preferably human, genome. The promoter can be a ubiquitous promoter (such as a β-actin or tubulin promoter) or a cell-specific promoter, such as a tumor-specific promoter. The promoter can be a viral promoter, such as the Moloney murine leukemia virus long terminal repeat (MMLV LTR) promoter or a human or murine cytomegalovirus (CMV) IE promoter. HSV immediate-early (IE) promoters are well known in the art. The HSV IE promoter can be a promoter driving expression of ICP0, ICP4, ICP22, ICP27, or ICP47.

[0037] The above-mentioned genes, whose functional inactivation confers tumor-selective properties to the virus, can be rendered functionally inactive by any suitable method, for example, by deleting or replacing all or part of the gene and / or its regulatory sequences, or by inserting one or more nucleic acids into or in place of the gene and / or its regulatory sequences. For example, the viruses of the invention can be generated using homologous recombination methods, which are standard in the art. Alternatively, bacterial artificial chromosome (BAC)-based approaches can be used.

[0038] As used herein, the term "gene" is intended to mean a nucleotide sequence that encodes a protein, i.e., the coding sequence of a gene. The various genes mentioned above can be rendered non-functional by mutating the gene itself or the regulatory sequences adjacent to the gene, such as promoter sequences. Deletions can remove one or more portions of a gene, the entire gene, or the entire gene and all or part of the regulatory sequences. For example, deletion of only a single nucleotide within a gene resulting in a frameshift can occur. However, larger deletions can occur, such as deletion of at least about 25%, more preferably at least about 50%, of the entire coding and / or non-coding sequence. In one preferred embodiment, a gene that is functionally inactive is deleted. For example, the entire gene and, optionally, part of the flanking sequences can be removed from the virus. If two or more copies of a gene are present in the viral genome, both copies of the gene are rendered functionally inactive.

[0039] A gene can be inactivated by replacing it with another sequence, for example, by replacing all or part of the endogenous gene with a heterologous gene and, optionally, a promoter sequence. If the promoter sequence is not replaced, the heterologous gene can be inserted so that it is regulated by the promoter of the non-functionalized gene. In the HSV of the present invention, the gene encoding ICP34.5 is preferably rendered non-functional by inserting a heterologous gene, and a promoter sequence operably linked thereto, and optionally other control elements, such as a polyadenylation sequence, into each of the loci encoding ICP34.5.

[0040] The viruses of the present invention are used to express a CTLA-4 inhibitor and, optionally, GM-CSF and / or an immune costimulatory pathway activating molecule in tumors. This is typically achieved by inserting a heterologous gene encoding a CTLA-4 inhibitor and, optionally, a heterologous gene encoding GM-CSF and / or a heterologous gene encoding an immune costimulatory pathway activating molecule into the genome of a selectively replicating virus, with each gene under the control of a promoter sequence. Because replication of such viruses occurs selectively in tumor tissue, viral expression of the CTLA-4 inhibitor and, if present, GM-CSF and / or an immune costimulatory pathway activating molecule is also enhanced in tumor tissue compared to non-tumor tissues of the body. Enhanced expression occurs when expression is greater in tumors compared to other tissues of the body. Proteins expressed by oncolytic viruses are also expected to be present in tumor-draining lymph nodes infected by the oncolytic viruses, including due to trafficking of the expressed proteins and viruses in or on tumor-derived antigen-presenting cells. Thus, the present invention provides the benefit of expressing a CTLA-4 inhibitor, and optionally co-expressed GM-CSF and / or immune co-stimulatory pathway activating molecules, selectively in tumors and tumor-draining lymph nodes in combination with the anti-tumor effects provided by oncolytic viral replication.

[0041] The viruses of the present invention include a CTLA-4 inhibitor. A CTLA-4 inhibitor is a molecule, typically a peptide or protein molecule, that binds to CTLA-4 and reduces or blocks signaling by CTLA-4. By reducing CTLA-4 signaling, the inhibitor reduces or eliminates blockage of the immune stimulatory pathway by CTLA-4.

[0042] The CTLA-4 inhibitor is preferably an antibody or an antigen-binding fragment thereof.

[0043] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An antibody refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (kappa) (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The constant regions of antibodies may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0044] The antibody is typically a monoclonal antibody. The antibody may be a chimeric antibody. The antibody is preferably a humanized antibody, more preferably a human antibody.

[0045] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to CTLA-4. Antigen-binding fragments also retain the ability to inhibit CTLA-4, thus reducing or eliminating CTLA-4 blockade of a stimulatory immune response. Examples of suitable fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementarity-determining regions (CDRs). Single-chain antibodies such as scFvs and heavy-chain antibodies such as VHHs and camelid antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. In a preferred embodiment, the antibody is an scFv. Examples of suitable scFv molecules are disclosed, for example, in WO2007 / 123737 and WO2014 / 066532, which are incorporated herein by reference.

[0046] The antibody-encoding sequence typically encodes an antibody or antibody fragment having an N-terminal signal sequence. The signal sequence may have the amino acid sequence set forth in SEQ ID NO: 7. For example, this signal sequence is contained in the scFv having the amino acid sequence set forth in SEQ ID NO: 9 and encoded by the nucleotide sequence set forth in SEQ ID NO: 10, and the scFv having the amino acid sequence set forth in SEQ ID NO: 14 and encoded by the nucleotide sequence set forth in SEQ ID NO: 15.

[0047] In an antibody or antibody fragment, the light and heavy chain sequences can be linked by an amino acid linker. The linker typically contains about 15 to about 25 amino acids, for example, about 18 or 20 amino acids. Any suitable linker can be used, such as a linker containing glycine and serine residues, for example, the amino acid sequence set forth in SEQ ID NO: 8. For example, this linker is contained in an scFv having the amino acid sequence set forth in SEQ ID NO: 9 and encoded by the nucleotide sequence set forth in SEQ ID NO: 10, and an scFv having the amino acid sequence set forth in SEQ ID NO: 14 and encoded by the nucleotide sequence set forth in SEQ ID NO: 15. Both are preferred antibody fragments for use in the present invention.

[0048] Other antibody fragments having similar structures are also preferred. Thus, the viruses of the present invention can encode antibodies or fragments that comprise or consist essentially of a light chain variable region, a linker, a heavy chain variable region, a heavy chain CH1 domain, a heavy chain CH2 domain, and a heavy chain CH3 domain. The viruses can further encode a signal sequence at the N-terminus of the antibody.

[0049] The antibody or antibody fragment of the present invention may comprise an Fc region that is preferably an IgG1, IgG2, IgG3, or IgG4 region, more preferably an IgG1 region. The antibody is preferably an scFv antibody in which the scFv is linked to the CH2 and CH3 domains of an IgG heavy chain.

[0050] A preferred CTLA-4 antibody or fragment comprises a heavy chain variable region set forth in SEQ ID NO:3 and / or a light chain variable region set forth in SEQ ID NO:1, or a heavy chain variable region set forth in SEQ ID NO:11 and / or a light chain variable region set forth in SEQ ID NO:12. The antibody may comprise a heavy chain CH1 domain having the amino acid sequence set forth in SEQ ID NO:4 and / or a CH2 / CH3 domain set forth in SEQ ID NO:5. The antibody may comprise a light chain amino acid sequence set forth in SEQ ID NO:2. The antibody of the invention may alternatively comprise a variant of one of these heavy or light chain variable regions or CDR sequences. For example, the variant may be a substitution, deletion, or addition variant of any of the above amino acid sequences.

[0051] Variant antibodies may contain 1, 2, 3, 4, 5, 10, 20, 30, or more amino acid substitutions and / or deletions from the specific sequences and fragments described above while retaining the activity of the antibodies described herein. "Deletion" variants may include deletion of, for example, 1, 2, 3, 4, or 5 individual amino acids, or one or more deletions of small groups of amino acids, such as 2, 3, 4, or 5 amino acids. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids, making conservative amino acid substitutions. For example, amino acids may be substituted with alternative amino acids having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0052] The viruses of the present invention comprise one or more polynucleotide sequences encoding a CTLA-4 inhibitor. The polynucleotide sequences are under the control of a suitable promoter. The virus may comprise a first polynucleotide sequence encoding an antibody heavy chain variable region and a second polynucleotide encoding an antibody light chain variable region. The first polynucleotide can encode a full-length heavy chain, and / or the second polynucleotide can encode a full-length light chain. The first and second polynucleotides, optionally with an IRES, may be under the control of a single promoter or two separate promoters. The separate promoters may be the same or different.

[0053] The first polynucleotide may comprise, consist essentially of, or consist of the coding sequence for the heavy chain variable region set forth in SEQ ID NO:9, and / or the second polynucleotide may comprise, consist essentially of, or consist of the coding sequence for the heavy chain variable region set forth in SEQ ID NO:10. The first polynucleotide may comprise, consist essentially of, or consist of the coding sequence for the heavy chain variable region set forth in SEQ ID NO:19, and / or the second polynucleotide may comprise, consist essentially of, or consist of the coding sequence for the heavy chain variable region set forth in SEQ ID NO:20.

[0054] The first and / or second polynucleotide sequences may be variants of SEQ ID NO: 9, 10, 19 or 20. For example, variants may be substitution, deletion or addition variants of any of these nucleic acid sequences. Variant polynucleotides may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions of SEQ ID NO: 9, 10, 19 or 20.

[0055] Suitable variants may be at least 70% homologous to the polynucleotide of any one of the nucleic acid sequences disclosed herein, preferably at least 80 or 90%, and more preferably at least 95%, 97%, or 99% homologous. These levels of homology and identity preferably exist at least over the coding regions of the polynucleotides. Methods for measuring homology are well known in the art, and those skilled in the art will understand that in the present context, homology is calculated based on nucleic acid identity. Such homology may exist over a region of at least 15, preferably at least 30, e.g., at least 40, 60, 100, 200, or more contiguous nucleotides. Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0056] Methods for measuring polynucleotide homology or identity are known in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (e.g., used with default settings) (Devereux et al. (1984) Nucleic Acids Research 12, p387-395).

[0057] For example, as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10, the PILEUP and BLAST algorithms can also be used to calculate homology and align sequences (typically using default settings).

[0058] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short strings of length W within the query sequence that match or satisfy some positive threshold score T when aligned with a string of the same length in a database sequence. T is referred to as the neighboring character score threshold (Altschul et al., supra). These initial neighboring character hits act as seeds to initiate searches for HSPs containing them. The character hits are extended in both directions along each sequence for as long as the combined alignment score increases. Extension of the string search in both directions stops when the sum of the alignment scores falls below zero due to an increase in the alignment of one or more negative-scoring residues, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program defaults to using a word length (W) of 11 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), an alignment of 50 (B), an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0059] The BLAST algorithm performs a statistical analysis of the similarity between two sequences. See, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that two nucleotide or amino acid sequences will be similar by chance. For example, a sequence is considered to be similar to another sequence if the minimum sum probability when comparing the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0060] In one embodiment, a variant sequence may differ from a specific sequence in a sequence listing due to redundancy in the genetic code. The DNA code has four primary nucleic acid residues (A, T, C, and G) that are used to "spell" three-letter codons, which represent amino acids in proteins encoded by an organism's genes. The linear sequence of codons along a DNA molecule is translated into the linear sequence of amino acids in the proteins encoded by those genes. The code is highly degenerate, with 61 codons encoding the 20 natural amino acids and three codons representing "stop" signals. Thus, most amino acids are encoded by more than one codon, and in fact, some are encoded by four or more different codons. Thus, a variant polynucleotide of the invention may encode the same polypeptide sequence as another polynucleotide of the invention, but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids. Codons may also be optimized to increase the level of expression of the encoded protein in target cells compared to the use of the unmodified sequence.

[0061] Preferably, the virus of the present invention comprises GM-CSF, and the sequence of the gene encoding GM-CSF may be codon-optimized to increase the expression level of the respective protein in target cells compared to the use of the unmodified sequence.

[0062] The viruses of the present invention preferably comprise one or more immune costimulatory pathway-activating molecules and / or one or more genes encoding immune costimulatory pathway-activating molecules. Immune costimulatory pathway-activating molecules include proteins and nucleic acid molecules (e.g., aptamer sequences). Examples of immune costimulatory pathway-activating molecules include CD40 ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, ICOS ligand, flt3 ligand, TL1A, CD30 ligand, CD70, and single-chain antibodies targeting the receptors for these molecules (CD40, GITR, 4-1-BB, OX40, ICOS, flt3, DR3, CD30, and CD27).

[0063] Activators of immune costimulatory pathways include mutant or wild-type, soluble, secreted and / or membrane-bound ligands, and agonistic antibodies, including single-chain antibodies. Preferably, the viruses of the invention encode one or more of CD40L, ICOSL, 4-1-BBL, GITRL, or OX40L.

[0064] The viruses of the invention can encode one or more immune costimulatory pathway activating molecules, preferably 1, 2, 3 or 4 immune costimulatory pathway activating molecules, more preferably 1 or 2 immune costimulatory pathway activating molecules.

[0065] The sequence of the gene encoding the immune costimulatory pathway activating molecule can be codon-optimized to increase the expression level of the respective protein in the target cell compared to when the unmodified sequence is used.

[0066] The viruses of the present invention may contain one or more additional heterologous genes in addition to the CTLA-4 inhibitor and GM-CSF and / or immune costimulatory pathway activating molecule. In a preferred embodiment, the viruses may further contain a fusogenic protein such as GALVR.

[0067] The fusogenic protein can be any heterologous protein capable of promoting the fusion of a cell infected with a virus of the present invention to another cell. A fusogenic protein, preferably a wild-type or modified viral glycoprotein (i.e., modified to enhance its fusogenic properties), is a protein capable of inducing cell-cell fusion (syncitia formation) of cells in which it is expressed. Examples of fusogenic glycoproteins include VSV-G, syncytin-1 (from human endogenous retrovirus-W (HERV-W)), or syncytin-2 (from HERVFRDE1), paramyxovirus SV5-F, measles virus-H, measles virus-F, RSV-F, and glycoproteins from retroviruses or lentiviruses, such as gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), and equine infectious anemia virus (EIAV), in which the R transmembrane peptide has been removed (R-version). In a preferred embodiment, the fusogenic protein is derived from GALV and the R peptide has been removed (GALV-R-).

[0068] Viruses of the invention can optionally contain multiple copies of a gene encoding a fusogenic protein, preferably 1 or 2 copies. A virus can contain two or more different fusogenic proteins, including any of the fusogenic proteins listed above.

[0069] The fusogenic proteins optionally expressed by the viruses of the invention may be identical to naturally occurring proteins or may be modified proteins.

[0070] The gene encoding the fusogenic protein (fusogenic gene) can have a naturally occurring nucleic acid sequence or a modified sequence. The sequence of the fusogenic gene can be modified, for example, to enhance the fusogenic properties of the encoded protein or to provide codon optimization and thus increase the expression efficiency of the encoded protein.

[0071] The present invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, expressing at least three heterologous genes, each driven by a different promoter selected from the group consisting of a CMV promoter, a RSV promoter, an EF1a promoter, an SV40 promoter, and a retroviral LTR promoter. The virus may express, for example, four heterologous genes, each driven by a different promoter selected from the group consisting of a CMV promoter, a RSV promoter, an EF1a promoter, an SV40 promoter, and a retroviral LTR promoter. The retroviral LTR is preferably derived from MMLV. The heterologous genes may be terminated by a polyadenylation sequence, which may be the same or different. Preferably, each heterologous gene is terminated by a different polyadenylation sequence, preferably selected from the group consisting of BGH, SV40, HGH, and RBG polyadenylation sequences. The present invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, that expresses at least three heterologous genes, each terminated by a different polyadenylation sequence selected from BGH, SV40, HGH, and RBG polyadenylation sequences. The virus can, for example, express four heterologous genes, each terminated by a BGH, SV40, HGH, and RBG polyadenylation sequence, respectively.

[0072] The at least three heterologous genes may be selected from, for example, a gene encoding a CTLA-4 inhibitor, GM-CSF, a gene encoding an immune costimulatory pathway activating molecule, and a fusogenic gene. Examples of the three heterologous genes include a gene encoding a CTLA-4 inhibitor, GM-CSF, and a gene encoding an immune costimulatory pathway activating molecule; a gene encoding a CTLA-4 inhibitor, GM-CSF, and a fusogenic gene; and a CTLA-4 inhibitor, a gene encoding an immune costimulatory pathway activating molecule, and a fusogenic gene. Four heterologous genes may be, for example, a gene encoding a CTLA-4 inhibitor, GM-CSF, a gene encoding an immune costimulatory pathway activating molecule, and a fusogenic gene. The three or four heterologous genes may include, for example, two or more genes encoding immune costimulatory pathway activating molecules and / or two or more fusogenic genes.

[0073] In one embodiment, the promoters controlling the expression of the three heterologous genes are the CMV, RSV, and MMLV promoters. For example, a preferred virus may contain a GM-CSF gene under the control of a CMV promoter, a GALV gene under the control of an RSV promoter, and a CTLA-4 inhibitor under the control of an MMLV promoter.

[0074] In one embodiment, the polyadenylation sequences terminating the at least three heterologous genes are SV40, BGH, and RBG polyadenylation sequences. Expression of the three heterologous genes is controlled by CMV, RSV, and MMLV promoters. For example, a preferred virus may contain a GM-CSF gene terminated by the BGH polyadenylation sequence, a GALV gene terminated by the SV40 polyadenylation sequence, and a CTLA-4 inhibitor terminated by the RGB polyadenylation sequence.

[0075] Any combination of various promoters and polyadenylation sequences may be used with any of the heterologous genes. For example, a preferred virus may contain a GM-CSF gene under the control of a CMV promoter terminated by a BGH polyadenylation sequence, a GALV gene under the control of an RSV promoter terminated by an SV40 polyadenylation sequence, and a CTLA-4 inhibitor under the control of an MMLV promoter terminated by an RGB polyadenylation sequence.

[0076] Virus production Viruses of the invention are constructed using methods well known in the art. For example, a plasmid (for smaller viruses and single- and multi-genome component RNA viruses) or BAC (for larger DNA viruses, including herpesviruses) encoding the viral genome to be packaged, containing genes encoding fusogenic and immunostimulatory molecules under appropriate regulatory control, can be constructed by standard molecular biology techniques and transfected into permissive cells from which recombinant virus can be recovered.

[0077] Alternatively, in a preferred embodiment, a plasmid containing DNA regions flanking the intended insertion site can be constructed and then co-transfected with viral genomic DNA into permissive cells, resulting in homologous recombination between the flanking regions of the target insertion site in the plasmid and the same regions in the parent virus. Recombinant viruses can then be selected and purified through the loss or addition of functions inserted or deleted by the plasmid used for modification, such as by inserting or deleting a marker gene, such as GFP or lacZ, from the parent virus at the intended insertion site. In a most preferred embodiment, the insertion site is the HSV ICP34.5 locus, and thus the plasmid used for engineering contains HSV sequences flanking this insertion site, with expression cassettes encoding GM-CSF and immune costimulatory pathway activating molecules between them. In this case, the parent virus can contain a cassette encoding GFP in place of ICP34.5, and recombinant virus plaques are selected by the loss of GFP expression. In a most preferred embodiment, the HSV US11 gene is also expressed as an IE gene. This can be achieved through deletion of the ICP47 coding region or by other means.

[0078] Additional protein-coding sequences, such as a CTLA-4 inhibitor and, optionally, a GM-CSF-encoding sequence and a costimulatory pathway activating molecule-encoding sequence, and / or a fusogenic protein such as GALVR-encoding sequence, are inserted into the viral genome under appropriate regulatory control. Depending on the species and insertion site, this may be under the regulatory control of the native promoter of the viral species used in the present invention, or, preferably, under the regulation of a heterologous promoter. Suitable heterologous promoters include mammalian promoters such as the IEF2a promoter or the actin promoter. Strong viral promoters such as the CMV IE promoter, RSV LTR, MMLV LTR, other retroviral LTR promoters, or promoters derived from SV40 are more preferred. Preferably, each foreign gene (i.e., encoding GM-CSF and a costimulatory pathway activating molecule) is under the regulation of a separate promoter, but they can also be expressed from a single RNA transcript, for example, by inserting an internal ribosome entry site (IRES) between the protein-coding sequences. The RNA from each promoter is typically terminated with a polyadenylation sequence (e.g., a mammalian sequence such as the bovine or human growth hormone (BGH) polyA sequence, a synthetic polyadenylation sequence, a rabbit beta globin polyadenylation sequence, or a viral sequence such as the SV40 early or late polyadenylation sequence).

[0079] Each heterologous gene in the virus is typically under the control of a promoter. The promoters controlling the expression of the heterologous genes may be the same or different. For example, one or more of the genes encoding anti-CTLA-4, GM-CSF, a fusogenic gene, and an immune costimulatory pathway activating molecule may be under the control of a CMV promoter, an RSV promoter, an EF1a promoter, an SV40 promoter, or a retroviral LTR promoter. Alternatively, for example, anti-CTLA-4 may be under the control of a retroviral LTR promoter such as an MMLV promoter, GM-CSF may be under the control of a CMV promoter, and / or a fusogenic gene such as GALVR may be under the control of an RSV promoter.

[0080] Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline, such as phosphate-buffered saline. The composition may further comprise other ingredients, such as sugars or proteins, to improve properties such as product stability. Alternatively, a lyophilized formulation may be used that is reconstituted in a pharmaceutically acceptable carrier or diluent before use.

[0081] If necessary, the choice of carrier is often a function of the delivery route of the composition. Within the scope of the present invention, the composition can be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents are those used in compositions suitable for intratumoral administration, intravenous / intraarterial administration, administration to the brain, or administration to a body cavity (e.g., bladder, pleural cavity, or intraperitoneal administration). The composition can be administered in any suitable form, preferably as a liquid.

[0082] The present invention also provides an article of manufacture comprising a virus of the invention in a sterile vial, ampoule, or syringe.

[0083] Medical Uses / Treatment Methods The present invention provides a virus of the present invention for use in the treatment of the human or animal body by therapy, particularly for use in a method of treating cancer. Cancer typically occurs in mammals, preferably humans. The virus kills infected tumor cells by lysis and by causing infected tumor cells to fuse with each other. The virus of the present invention also induces a systemic anti-tumor immune response enhanced by the expression of a CTLA-4 inhibitor, which also kills cancer cells, and optionally GM-CSF and costimulatory pathway activating molecules.

[0084] The present invention also provides a method of treating cancer, comprising administering a therapeutically effective amount of a virus of the present invention to an individual in need thereof.

[0085] The invention further provides the use of a virus of the invention in the manufacture of a medicament for treating cancer.

[0086] The viruses of the invention are particularly useful for treating any solid tumor, including any adenocarcinoma, carcinoma, melanoma, or sarcoma. For example, the viruses of the invention are useful for treating cancer of the head and neck, prostate, breast, ovary, lung, liver, endometrium, bladder, gallbladder, pancreas, colon, kidney, stomach / gastric, esophagus, or cervix, mesothelioma, melanoma, or other skin cancers, lymphoma, glioma, or other cancers of the nervous system, or sarcomas, such as soft tissue sarcomas.

[0087] The viruses of the invention can be used to treat malignant tumors, including tumors that have metastasized from the site of the original tumor. In this embodiment, the virus can be administered to the primary tumor or to one or more secondary tumors.

[0088] The viruses of the present invention can be administered in combination with other therapeutic agents, including chemotherapy, targeted therapy, immunotherapy (including immune checkpoint blockade, i.e., administration of one or more antagonists of immune co-inhibitory pathways and / or one or more agonists of immune co-stimulatory pathways), and / or in combination with radiation therapy, and / or in combination with any combination thereof. The therapeutic agent is preferably an anti-cancer agent.

[0089] The viruses of the invention can be administered in combination with a second virus, such as a second oncolytic virus.

[0090] For example, therapeutic agents may include immunogens (including recombinant or naturally occurring antigens, including combinations of such antigens or antigens delivered as DNA or RNA encoding them) to further stimulate an immune response, e.g., a cellular or humoral immune response, against tumor cells, particularly neoantigens. Therapeutic agents may be agents intended to increase or enhance the immune response, such as cytokines, agents intended to inhibit immune checkpoint pathways or stimulate immune enhancement pathways, or agents that inhibit the activity of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs).

[0091] The therapeutic agent may be a drug known for use in existing cancer treatments. The therapeutic agent may be a radiotherapeutic agent or a chemotherapeutic agent. The therapeutic agent may be selected from cyclophosphamide, alkylating agents such as cisplatin or melphalan, plant alkaloids and terpenoids such as vincristine or paclitaxel (taxol), antimetabolites such as 5-fluorouracil, topoisomerase inhibitors type I or II such as camptothecin or doxorubicin, cytotoxic antibiotics such as actinomycin, anthracyclines such as epirubicin, glucocorticoids such as triamcinolone, inhibitors of protein, DNA, and / or RNA synthesis such as methotrexate and dacarbaxin, histone deacetylase (HDAC) inhibitors, or any other chemotherapeutic agent.

[0092] The therapeutic agent may be one or a combination of the following: an immunotherapeutic or immunomodulatory agent, such as a TLR agonist; an agent that downregulates T regulatory cells, such as cyclophosphamide; or an agent designed to block immune checkpoints or stimulate immune enhancing pathways, including, but not limited to, a monoclonal antibody, such as a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a CEACAM1 inhibitor, a GITR agonist, a 4-1-BB agonist, a KIR inhibitor, an SLAMF7 inhibitor, an OX40 agonist, a CD40 agonist, an ICOS agonist, or a CD47 inhibitor. In a preferred embodiment, the therapeutic agent is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody, a PD1 inhibitor, such as an anti-PD-1 antibody, or a PD-L1 inhibitor, such as an anti-PD-L1 antibody. Such inhibitors, agonists and antibodies can be generated and tested by standard methods known in the art.

[0093] Immunotherapeutic agents may also include bispecific antibodies, dendritic cell-based cell-based therapies, NK cells, or engineered T cells, e.g., CAR-T cells or T cells expressing engineered T cell receptors. Immunotherapeutic agents also include agents that target specific genetic mutations occurring in tumors, agents intended to elicit an immune response against specific tumor antigens or combinations of tumor antigens, e.g., neoantigens, and / or agents intended to activate the STING / cGAS pathway, TLRs, or other innate immune responses and / or inflammatory pathways, e.g., intratumoral agents.

[0094] For example, the viruses of the invention may be used in combination with dacarbazine, a BRAF inhibitor and / or a PD1 or PD-L1 blocker to treat melanoma; in combination with taxol, doxorubicin, vinorelbine, cyclophosphamide and / or gemcitabine to treat breast cancer; in combination with 5-fluorouracil, and optionally in combination with leucovorin, irinotecan and / or oxaliplatin to treat colorectal cancer; in combination with taxol, carboplatin, vinorelbine and / or gemcitabine, a PD-1 or PD-L1 blocker to treat lung cancer; and in combination with cisplatin and / or radiation therapy to treat head and neck cancer.

[0095] The therapeutic agent can be an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway. Examples of IDO inhibitors include epcadostat (INCB024360), 1-methyl-tryptophan, indoximod (1-methyl-D-tryptophan), GDC-0919, or F001287.

[0096] The mechanism of action of IDO in suppressing anti-tumor immune responses may also suppress immune responses that occur after oncolytic virotherapy. IDO expression is induced by Toll-like receptor (TLR) activation and interferon-γ, both of which may result from oncolytic virus infection. One embodiment of the use of oncolytic virotherapy for cancer treatment involves the combination of an oncolytic virus, including a CTLA-4 inhibitor and, optionally, a virus expressing GM-CSF and / or an immune costimulatory pathway-activating molecule, with an inhibitor of the IDO pathway and, optionally, one or more antagonists of immune co-inhibitory pathways, including those targeting PD-1 and / or PD-L1, and / or an additional agonist of an immune costimulatory pathway.

[0097] When a therapeutic agent and / or radiation therapy is used together with the virus of the present invention, the administration of the virus and the therapeutic agent and / or radiation therapy may be simultaneous or separated by time. The composition of the present invention may be administered before, simultaneously with, or after the therapeutic agent or radiation therapy. The method of treating cancer may include multiple administrations of the virus of the present invention and / or the therapeutic agent and / or radiation therapy. In a preferred embodiment, in combination with an immune checkpoint blocker or other immune enhancing agent, the virus of the present invention is administered once or multiple times before the subsequent simultaneous administration of the immune checkpoint blocker or other immune enhancing agent, or is administered simultaneously with the administration of the immune checkpoint blocker or other immune enhancing agent without prior administration of the virus of the present invention.

[0098] The virus of the present invention can be administered to a subject by any suitable route. Typically, the virus of the present invention is administered by direct intratumoral injection. Intratumoral injection includes direct injection into epidermal, subcutaneous, or nodular tumors, as well as image-guided (such as CT, MRI, or ultrasound) injection to localize deeper or more firmly deposits, for example, in visceral organs and other locations. The virus can be administered into a body cavity, for example, into the pleural cavity, bladder, or by intraperitoneal administration. The virus can be injected into a blood vessel, preferably into a blood vessel supplying a tumor.

[0099] Therapeutic agents that can be combined with the viruses of the present invention can be administered to human or animal subjects in vivo using a variety of known routes and techniques. For example, the compositions can be provided as injectable solutions, suspensions, or emulsions and administered via parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe or a liquid jet injection system. The compositions can be administered topically to the skin or mucosal tissue, such as nasal, intratracheal, intestinal, sublingual, rectal, or vaginal, or can be provided as a finely divided spray suitable for respiratory or pulmonary administration. In a preferred embodiment, the compositions are administered by intravenous infusion, orally, or directly to a tumor.

[0100] The virus and / or therapeutic agent can be administered to a subject in an amount compatible with a therapeutically effective dosage composition. The administration of the virus of the present invention is for "therapeutic" purposes. As used herein, the term "therapeutic" or "treatment" includes any one or more of the following purposes: preventing any metastasis or further metastasis that occurs, reducing or eliminating symptoms, reducing or completely eliminating tumors or cancer, increasing the time to cancer progression in a patient, increasing the time to recurrence after treatment, or increasing survival time.

[0101] Therapeutic treatment is given for stage I, II, III or IV cancer, preferably stage II, III or IV, more preferably stage III or IV, before or after surgical intervention (i.e., after recurrence or incomplete removal of tumor after surgery), preferably before any surgical intervention (either for resection of primary or recurrent / metastatic disease) or after recurrence after surgery or incomplete surgical removal of disease, i.e., while residual tumor remains.

[0102] Therapeutic treatment can be carried out after injecting the viral composition into the target tissue, which may be a tumor, into a body cavity, or directly into a blood vessel. As a guideline, the amount of virus administered can range from 10 to 100 mg / kg for HSV. 4 ~10 10 pfu, preferably 10 5 ~10 9 In the case of HSV, the initial low dose (e.g., 10 4 ~10 7 pfu) to seroconvert patients who are seronegative for HSV and to boost immunity in seropositive patients, followed by higher doses (e.g., 10 6 ~10 9pfu) can then be given. Typically, up to 20 ml of a pharmaceutical composition consisting essentially of virus and a suitable pharmaceutically acceptable carrier or diluent can be used for direct injection into a tumor, or up to 50 ml (which may be subjected to further dilution in a suitable diluent before administration) can be used for administration into a body cavity or into the bloodstream. However, for some oncolytic therapy applications, larger or smaller volumes can also be used, depending on the tumor and the route and site of administration.

[0103] The described routes of administration and dosages are intended as guidelines only, as a skilled physician can easily determine the optimal route of administration and dosage. The dosage can be determined according to various parameters, particularly the location of the tumor, tumor size, the age, weight, and condition of the patient being treated, and the route of administration. Preferably, the virus is administered by direct injection into the tumor or body cavity. The virus can also be administered by injection into a blood vessel. The optimal route of administration depends on the location and size of the tumor. Multiple doses may be required to achieve an immunological or clinical effect, typically administered at intervals of 2 days to 12 weeks, preferably 3 days to 3 weeks, as needed. Repeated administrations can be given for up to 5 years or more, preferably up to 1 month to 2 years, depending on the response rate of the type of tumor being treated and the response of the particular patient, as well as any concomitant therapy that may be given. The present invention encompasses, for example, the following embodiments: [Embodiment 1] An oncolytic virus encoding a CTLA-4 inhibitor. [Embodiment 2] The virus described in embodiment 1, wherein the CTLA-4 inhibitor is a CTLA-4 antibody or an antigen-binding fragment thereof. [Embodiment 3] The virus described in embodiment 2, wherein the fragment comprises an scFv molecule. [Embodiment 4] The virus described in embodiment 2, wherein the fragment is an scFv molecule linked to one or more IgG1 constant regions. [Embodiment 5] The virus of any one of embodiments 2 to 4, wherein the antibody or fragment comprises a light chain variable region linked to an IgG heavy chain. [Embodiment 6] A virus described in any of embodiments 2 to 5, wherein the antibody or fragment comprises (a) a light chain variable region sequence shown in SEQ ID NO: 1 and a heavy chain variable region sequence shown in SEQ ID NO: 3, or (b) a light chain variable region sequence shown in SEQ ID NO: 11 and a heavy chain variable region sequence shown in SEQ ID NO: 12. [Embodiment 7] The virus described in embodiment 6, wherein the antibody or fragment comprises (a) the amino acid sequence of SEQ ID NO: 9, or (b) the amino acid sequence of SEQ ID NO: 14. [Embodiment 8] The virus described in embodiment 7, wherein the antibody or fragment is encoded by (a) the nucleotide sequence of SEQ ID NO: 10, or (b) the nucleotide sequence of SEQ ID NO: 15. [Embodiment 9] The virus described in any one of embodiments 1 to 8, further comprising a gene encoding GM-CSF. [Embodiment 10] A virus described in any one of embodiments 1 to 9, further comprising an immune costimulatory pathway activating molecule or a gene encoding an immune costimulatory pathway activating molecule. [Embodiment 11] The virus described in embodiment 10, wherein the gene encoding an immune costimulatory pathway activating molecule encodes CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27, or flt3 ligand, or a modified version of any of these. [Embodiment 12] The virus described in embodiment 10 or 11, wherein the gene encoding an immune costimulatory pathway activating molecule encodes a CD40 ligand, a GITR ligand, a 4-1-BB ligand, an OX40 ligand, an ICOS ligand, or a modified version of any of these. [Embodiment 13] A virus described in any one of embodiments 1 to 12, further comprising a gene encoding a fusogenic protein. [Embodiment 14] The virus described in embodiment 13, wherein the fusogenic protein is selected from the group consisting of vesicular stomatitis virus (VSV) G protein, syncytin-1, syncytin-2, simian virus 5 (SV5) F protein, measles virus (MV) H protein, MV F protein, respiratory syncytial virus (RSV) F protein, and glycoproteins derived from R peptide-deleted gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer simian virus (MPMV), or equine infectious anemia virus (EIAV). [Embodiment 15] A virus described in embodiment 13 or 14, wherein the fusogenic protein is a glycoprotein derived from gibbon ape leukemia virus (GALV) and the R transmembrane peptide has been mutated or deleted (GALV-R-). [Embodiment 16] A virus described in any one of embodiments 1 to 15, encoding one or more immune costimulatory pathway activating molecules. [Embodiment 17] A virus described in any one of embodiments 1 to 16, which is derived from a clinical isolate of the virus. [Embodiment 18] A virus described in any of embodiments 1 to 17, which is a modified clinical isolate of a virus that kills two or more tumor cell lines in vitro more quickly and / or at lower doses than one or more reference clinical isolates of the same virus. [Embodiment 19] A virus described in any one of embodiments 1 to 18, selected from the group consisting of herpesviruses, poxviruses, adenoviruses, retroviruses, rhabdoviruses, paramyxoviruses, and reoviruses. [Embodiment 20] A virus described in any one of embodiments 1 to 19, which is a herpes simplex virus (HSV). [Embodiment 21] The virus described in embodiment 20, which is HSV1. [Embodiment 22] HSV is (a) do not express functional ICP34.5; (b) does not express functional ICP47, and / or (c) expressing the US11 gene as an immediate early gene; 22. The virus of embodiment 21. [Embodiment 23] A virus described in any of embodiments 20 to 22, in which a gene encoding an anti-CTLA-4 inhibitor protein is inserted into the gene locus encoding ICP34.5 by insertion, partial deletion, or complete deletion. [Embodiment 24] The virus described in embodiment 23, wherein the gene encoding the anti-CTLA-4 inhibitor protein is contained in a cassette that also contains one or more immune stimulatory genes such as GM-CSF and / or genes encoding immune costimulatory pathway activating molecules and / or genes encoding fusogenic proteins. [Embodiment 25] A virus described in any of embodiments 1 to 24, wherein the sequence encoding a CTLA-4 inhibitor, the sequence encoding GM-CSF, the sequence encoding an immune costimulatory pathway activating molecule, and / or the gene encoding a fusogenic protein are codon-optimized to increase their expression levels in target cells. [Embodiment 26] A virus described in any one of embodiments 1 to 25, which expresses three heterologous genes, each of which is driven by a different promoter selected from a CMV promoter, an RSV promoter, an SV40 promoter (sequence number), and a retroviral LTR promoter. [Embodiment 27] A virus described in embodiment 26, which expresses four heterologous genes driven by a CMV promoter, an RSV promoter, an SV40 promoter, and a retroviral LTR promoter, respectively. [Embodiment 28] A virus described in embodiment 26 or 27, wherein the retroviral LTR is derived from MMLV (sequence number). [Embodiment 29] A virus described in any of embodiments 1 to 28, which expresses three heterologous genes, each of which is terminated by a different polyadenylation sequence selected from BGH, SV40, HGH and RBG polyadenylation sequences. [Embodiment 30] A virus described in embodiment 29, which expresses four heterologous genes, each terminated by a BGH, SV40, HGH, and RBG polyadenylation sequence. [Embodiment 31] A virus described in any one of embodiments 26 to 30, which is a poxvirus. [Embodiment 32] A pharmaceutical composition comprising the virus according to any one of embodiments 1 to 31 and a pharmaceutically acceptable carrier or diluent. [Embodiment 33] A virus described in any one of embodiments 1 to 31 for use in a method for treating the human or animal body by therapy. [Embodiment 34] A virus described in any one of embodiments 1 to 31 for use in a method for treating cancer. [Embodiment 35] The virus for use according to embodiment 34, wherein the method comprises administering an additional anticancer agent. [Embodiment 36] A virus for use according to embodiment 35, wherein the additional anticancer agent is selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiation therapy and / or chemotherapy, agents targeting specific gene mutations occurring in tumors, agents intended to induce an immune response against one or more tumor antigens or neo-antigens, cellular products derived from T cells or NK cells, agents intended to stimulate STING, cGAS, TLR or other innate immune responses and / or inflammatory pathways, a second virus, optionally an oncolytic virus, and combinations thereof. [Embodiment 37] The virus for use according to embodiment 36, wherein the agent targeting the immune co-inhibitory pathway is a PD-1 inhibitor, PD-L1 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, CSF1R inhibitor, IDO inhibitor, KIR inhibitor, SLAMF7 inhibitor, CEACAM1 inhibitor or CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, 4-1-BB agonist, OX40 agonist, CD40 agonist or ICOS agonist. [Embodiment 38] A virus for use according to any one of embodiments 35 to 37, wherein the additional anticancer agent is an antibody. [Embodiment 39] A virus for use according to any of embodiments 35 to 38, wherein the method comprises administering an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and an additional antagonist of an immune co-inhibitory pathway or an additional agonist of an immune co-stimulatory pathway. [Embodiment 40] A virus for use described in any of embodiments 34 to 39, wherein the virus and the additional anticancer drug are administered separately. [Embodiment 41] A virus for use described in any of embodiments 34 to 39, wherein the virus and a further anticancer drug are administered simultaneously. [Embodiment 42] A virus for use according to any one of embodiments 34 to 41, wherein the cancer is a solid tumor. [Embodiment 43] A product containing the virus described in any one of embodiments 1 to 31 in a sterile vial, ampoule, or syringe. [Embodiment 44] A method for treating cancer, comprising administering a therapeutically effective amount of a virus described in any one of embodiments 1 to 31 or a pharmaceutical composition described in embodiment 32 to a patient in need thereof. [Embodiment 45] The method of embodiment 44, further comprising administering a therapeutically effective amount of an additional anticancer agent to a patient in need thereof. [Embodiment 46] The method of embodiment 45, wherein the additional anticancer agent is selected from the group consisting of agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiation therapy and / or chemotherapy, agents targeting specific gene mutations occurring in tumors, agents intended to induce an immune response against one or more tumor antigens or neo-antigens, cellular products derived from T cells or NK cells, agents intended to stimulate STING, cGAS, TLR or other innate immune response and / or inflammatory pathways, a second virus, optionally an oncolytic virus, and combinations thereof. [Embodiment 47] The method of embodiment 46, wherein the agent targeting the immune co-inhibitory pathway is a PD-1 inhibitor, PD-L1 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, CSF1R inhibitor, IDO inhibitor, KIR inhibitor, SLAMF7 inhibitor, CEACAM1 inhibitor or CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, 4-1-BB agonist, OX40 agonist, CD40 agonist or ICOS agonist. [Embodiment 48] The method of any one of embodiments 45 to 47, wherein the additional anticancer agent comprises an antibody. [Embodiment 49] A method according to any one of embodiments 45 to 48, wherein the virus and the additional anticancer agent are administered separately. [Embodiment 50] A method according to any one of embodiments 45 to 48, wherein the virus and the additional anticancer agent are administered simultaneously. [Embodiment 51] The method described in any one of embodiments 44 to 50, wherein the cancer is a solid tumor. [Embodiment 52] Use of a virus described in any one of embodiments 1 to 31 in the manufacture of a medicament for use in a method for treating cancer. [Embodiment 53] The use described in embodiment 52, wherein the method includes administering an additional anticancer agent.

[0104] The following examples illustrate the present invention. [Example]

[0105] [Example 1] Construction of the viruses of the invention The viral species used to illustrate the present invention is HSV, specifically HSV1.

[0106] A schematic diagram of the plasmids used is shown in Figure 2. A schematic diagram of the viruses is shown in Figure 1. All viruses were constructed using HSV1 strain RH018A. Plasmids used in virus construction were generated by a combination of gene synthesis and subcloning performed by Genscript Inc.

[0107] A virus expressing anti-mouse CTLA4 together with mouse GM-CSF and GALV was constructed as follows: Plasmid 77 was cotransfected with virus 16 DNA, and GFP-expressing plaques were selected. GFP was then inserted into virus 16 to yield virus 25. Subsequently, GFP was knocked out from virus 25 by cotransfection with plasmid 119 and virus 25 DNA, yielding virus 27.

[0108] A virus expressing anti-human CTLA4 together with human GM-CSF and GALV was constructed as follows: Plasmid 78 was cotransfected with virus 17 DNA, and GFP-expressing plaques were selected. GFP was then inserted into virus 17 to yield virus 29. Subsequently, virus 29 DNA was cotransfected with plasmid 122 to knock out GPF from virus 29, yielding virus 31.

[0109] Viruses expressing anti-mouse CTLA-4 and costimulatory ligands along with mouse GM-CSF and GALV were constructed by cotransfecting virus 27 with a plasmid encoding GFP driven by the SV40 promoter located between the mouse GM-CSF and anti-mouse CTLA-4 coding sequences. GFP was then knocked out from the resulting virus by substituting plasmids encoding each of the individual mouse costimulatory ligands for GFP.

[0110] Viruses expressing anti-human CTLA4 and costimulatory ligands along with human GM-CSF and GALV were constructed by cotransfecting virus 31 with a plasmid encoding GFP driven by the SV40 promoter located between the human GM-CSF and anti-human CTLA-4 coding sequences. GFP was then knocked out from the resulting virus with plasmids encoding each of the individual human costimulatory ligands in place of GFP.

[0111] FIG. 4 shows a Western blot demonstrating the expression of anti-mouse CTLA-4 from virus 27.

[0112] [Example 2] Effect of combined expression of oncolytic virus-derived GALV, GM-CSF, and anti-CTLA4

[0113] The utility of the present invention is demonstrated by the following method: A20 cells are injected into both flanks of Balb / c mice, and A20 tumors are allowed to grow to a diameter of approximately 0.5 cm.

[0114] Each mouse in the group then received the following treatments three times a week on only one flank (right tumor). 50 μl of solvent (group 1); Mouse GM-CSF and GALVR-only inserted HSV (virus 16) 10 6 50 μl of pfu / ml; GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody-injected HSV (virus 27) 6 50 μl of pfu / ml

[0115] The effect on tumor growth is then monitored for up to one month. The virus dose used is 5x10 4 pfu (1x10 in each case) 6 The doses were administered at a concentration of 50 μl (pfu / ml) three times a week. This viral dose level was subtherapeutic for virus 16 in uninjected tumors, allowing the benefit of delivery of the additional molecule encoded by virus 27 to be clearly seen. Figures 5 and 6 show superior tumor control and tumor regression in uninjected tumors for virus expressing anti-CTLA-4 compared to virus 16, which does not express CTLA-4.

[0116] [Example 3] Combined expression of oncolytic virus-derived GALV, GM-CSF, and anti-CTLA4 and its effect with anti-PD-1

[0117] A20 cells are injected into both flanks of Balb / c mice, and A20 tumors are allowed to grow to approximately 0.5 cm in diameter.

[0118] Groups of mice (10 per group) are then treated three times per week for one week on only one flank of each mouse with the following treatment: 50 μl of solvent; intraperitoneal anti-mouse PD1 (Bioxcell, RMP-1-14, 10 mg / kg every 3 days); GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody inserted HSV (virus 27) 10 7 50 μl of pfu / ml; GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody inserted HSV (virus 27) 10 7 pfu / ml 50 μl; intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (Group 3)

[0119] The effect on tumor growth is then monitored for up to 80 days. When viral treatment was combined with anti-PD1 treatment, there was excellent tumor control and regression in both injected and uninjected tumors. This data is shown in Figure 7.

[0120] [Example 4] The combined effects of oncolytic virus-derived GALV, GM-CSF, and anti-human CTLA4 expression alone and in combination with anti-PD-1

[0121] MC38 cells are injected into both flanks of C57BL / 6 mice that have been gene-edited to express human CTLA-4 rather than mouse CTLA-4, rendering them susceptible to anti-human CTLA-4 antibodies such as ipilimumab. MC38 tumors are allowed to grow to approximately 0.5 cm in diameter.

[0122] Groups of mice (10 per group) then received the following treatments on only one flank, three times per week for two weeks: 50 μl of solvent; Virus 17 (i.e., hGM-CSF and GALV expression) 10 850 μl of pfu / ml; Virus 31 (i.e., hGM-CSF, GALV, and anti-human CTLA-4 expression) 10 8 50 μl of pfu / ml; Virus 10 of 17 8 50 μl pfu / ml and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days); Virus 10 of 31 8 pfu / ml at 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days)

[0123] The effect on tumor growth is then monitored for up to 35 days. Superior tumor control and regression are observed in tumors injected with anti-human CTLA-4-expressing viruses, and this effect is further enhanced by combined treatment with anti-PD1. When treatment with either virus is combined with anti-PD1, superior tumor control and regression are observed in uninjected tumors. This improvement is even more pronounced with the anti-CTLA4-expressing viruses. The data are shown in Figure 8.

[0124] [Example 5] Combined expression of oncolytic virus-derived GALV, GM-CSF, and anti-CTLA4 and its effect with anti-PD-1

[0125] A20 cells are injected into both flanks of Balb / c mice and A20 tumors are allowed to grow to approximately 0.5 cm in diameter.

[0126] Groups of mice (10 per group) then received the following treatments three times a week for two weeks on only one flank of each mouse: 50 μl of solvent (group 1); intraperitoneal anti-mouse PD1 (Bioxcell, RMP-1-14, 10 mg / kg every 3 days); Mouse GM-CSF and GALVR-only inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 50 μl of pfu / ml (group 3); GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody-inserted HSV105 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml at 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (group 3); Mouse GM-CSF, GALVR-only inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml at 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (group 3); GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody-inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (Group 3)

[0127] The effect on tumor growth was then monitored for up to one month. Anti-CTLA-4 expressing viruses induced superior tumor control and regression in both injected and uninjected tumors, and this effect was further enhanced by combined treatment with anti-PD1, as evidenced by an improved dose-response curve compared to other groups.

[0128] [Example 6] The combined effects of oncolytic virus-derived GALV, GM-CSF, and anti-human CTLA4 expression alone and in combination with anti-PD-1

[0129] MC38 cells are injected into both flanks of C57BL / 6 mice that have been gene-edited to express human CTLA-4 rather than mouse CTLA-4, rendering them susceptible to anti-human CTLA-4 antibodies such as ipilimumab. MC38 tumors are allowed to grow to approximately 0.5 cm in diameter.

[0130] Groups of mice (10 per group) are then treated three times per week for two weeks on one flank only with the following treatments: 50 μl of solvent (group 1); intraperitoneal anti-mouse PD1 (Bioxcell, RMP-1-14, 10 mg / kg every 3 days); Mouse GM-CSF and GALVR-only inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 50 μl of pfu / ml (group 3); GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody-inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml at 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (group 3); Mouse GM-CSF, GALVR-only inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml at 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (group 3); GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody-inserted HSV10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml 50 μl and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) (Group 3)

[0131] The effect on tumor growth was then monitored for up to one month. The anti-human CTLA-4 virus induced excellent tumor control and tumor shrinkage in both injected and uninjected tumors, and this effect was further enhanced by combined treatment with anti-PD1, as evidenced by an improved dose-response curve compared to other groups.

[0132] [Example 7] Effect of combined expression of oncolytic virus-derived GALV, GM-CSF, anti-CTLA4, and immune costimulatory pathway activating molecules

[0133] The experiment of Example 3 above is repeated, except that mice are administered a virus that further expresses an immune co-stimulatory pathway ligand in addition to GALV, mGM-CSF, and anti-CTLA4.

[0134] More specifically, groups of mice are administered: (1) solvent; (2) intraperitoneal anti-mouse PD1; (3) mGM-CSF, GALVR-, and anti-CTLA4 inserted HSV of Example 2; (4) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse CD40L; (5) mGM-CSF, GALVR-, anti-CTLA4, and murine 4-1BBL-inserted HSV; (6) HSV with mGM-CSF, GALVR-, anti-CTLA4, and mouse GITRL inserts; (7) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse OX40L; (8) HSV with mGM-CSF, GALVR-, anti-CTLA4, and mouse ICOSL inserts; (9) mGM-CSF, GALVR-, and anti-CTLA4-inserted HSV of Example 2 with intraperitoneal anti-PD1; (10) mGM-CSF, GALVR-, anti-CTLA4, and mouse CD40L-injected HSV with intraperitoneal anti-PD1; (11) mGM-CSF, GALVR-, anti-CTLA4, and mouse 4-1BBL-injected HSV and intraperitoneal anti-PD1; (12) mGM-CSF, GALVR-, anti-CTLA4, and mouse GITRL-inserted HSV with intraperitoneal anti-PD1; (13) mGM-CSF, GALVR-, anti-CTLA4, and mouse OX40L-injected HSV with intraperitoneal anti-PD1; or (14) mGM-CSF, GALVR-, anti-CTLA4, and mouse ICOSL-injected HSV and intraperitoneal anti-PD1

[0135] Superior tumor control has been observed with viruses expressing immune costimulatory ligands.

[0136] Deposit information The following HSV1 strains were deposited by Replimune Limited at the ECACC, Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK on 19 December 2016 and assigned the indicated accession numbers: RH004A-Accession No. 16121902 RH015A - Accession number 16121903 RH018A - Accession number 16121904 RH021A - Accession number 16121905 RH023A - Accession number 16121906 RH031A - Accession number 16121907 RH040B-Accession No. 16121908 RH047A - Accession number 16121909

Claims

1. An oncolytic herpes simplex virus (HSV) comprising a gene encoding a CTLA-4 inhibitor, the oncolytic HSV being modified HSV1 strain RH018A having accession number ECACC16121904.

2. The oncolytic HSV of claim 1, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

3. The oncolytic HSV of claim 1 or 2, wherein the CTLA-4 inhibitor is an scFv molecule.

4. The oncolytic HSV of claim 3, wherein the scFv molecule is linked to one or more IgG1 constant regions.

5. The oncolytic HSV of any one of claims 2 to 4, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region sequence linked to an IgG heavy chain.

6. The oncolytic HSV according to any one of claims 1 to 5, further comprising: (a) one or more genes encoding fusogenic proteins; (b) one or more genes encoding immune costimulatory pathway activating molecules; and / or (c) The gene encoding GM-CSF.

7. 7. The oncolytic HSV of any one of claims 1 to 6, further comprising a glycoprotein from Gibbon Ape Leukemia Virus (GALV), in which the R transmembrane peptide has been mutated or deleted (GALV-R-).

8. The oncolytic HSV according to claim 6 or 7, wherein the one or more genes encoding immune costimulatory pathway activating molecules encode any one of CD40 ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, and ICOS ligand.

9. 9. An oncolytic HSV according to any one of claims 1 to 8, comprising one or more mutations in one or more viral genes that inhibit replication in normal tissues but still allow replication in tumors.

10. 10. The oncolytic HSV of claim 9, wherein the one or more mutations prevent expression by the HSV of functional ICP34.5, functional ICP6, functional thymidine kinase, or a combination thereof.

11. One or more mutations (a) expression of functional ICP34.5 is blocked; (b) expression of functional ICP47 is prevented, and / or (c) US11 gene is expressed as an immediate early gene.

11. An oncolytic HSV according to claim 9 or 10.

12. 12. The oncolytic HSV of any one of claims 1 to 11, wherein a gene encoding a CTLA-4 inhibitor is inserted into the gene locus encoding ICP34.5 by insertion, partial deletion, or complete deletion.

13. 13. The oncolytic HSV of any one of claims 1 to 12, wherein the gene encoding the CTLA-4 inhibitor is contained in a cassette that also comprises one or more immune stimulatory genes and / or genes encoding immune costimulatory pathway activating molecules and / or genes encoding fusogenic proteins, and the cassette is inserted into the ICP34.5-encoding gene locus by insertion, partial deletion or complete deletion.

14. 14. The oncolytic HSV of any one of claims 1 to 13, wherein the sequence encoding the CTLA-4 inhibitor is codon-optimized to increase expression levels in target cells.

15. An oncolytic HSV according to any one of claims 1 to 14, which expresses: (a) three heterologous genes, each driven by a different promoter selected from the group consisting of a CMV promoter, an RSV promoter, an SV40 promoter, and a retroviral LTR promoter, and / or each terminated by a different polyadenylation sequence selected from the group consisting of BGH, SV40, HGH, and RBG polyadenylation sequences; or (b) Four heterologous genes driven by a CMV promoter, an RSV promoter, an SV40 promoter and a retroviral LTR promoter, respectively, and / or terminated by a BGH, an SV40, an HGH and an RBG polyadenylation sequence, respectively.

16. The oncolytic HSV of claim 15, wherein the retroviral LTR promoter is derived from MMLV.

17. A pharmaceutical composition comprising an oncolytic HSV according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier or diluent.

18. 17. A composition for use in a method of treating cancer, comprising an oncolytic HSV according to any one of claims 1 to 16.

19. 19. The composition of claim 18, wherein the cancer is a solid tumor.

20. 20. The composition of claim 18 or 19, wherein the method comprises administering, simultaneously with or separately from the virus, an additional anti-cancer agent.

21. Further anti-cancer drugs (a) an anti-cancer agent selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiation therapy and / or chemotherapy, agents targeting specific genetic mutations occurring in tumors, agents intended to induce an immune response against one or more tumor antigens or neo-antigens, cellular products derived from T cells or NK cells, agents intended to stimulate STING, cGAS, TLR or other innate immune response and / or inflammatory pathways, a second virus, optionally an oncolytic virus, and combinations thereof; (b) an agent that targets an immune co-inhibitory pathway selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a KIR inhibitor, an SLAMF7 inhibitor, a CEACAM1 inhibitor, and / or a CD47 inhibitor; (c) an agent that targets an immune costimulatory pathway selected from a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40 agonist, and / or an ICOS agonist; (d) is an antibody; and / or (e) an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and a further antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway, 21. The composition of claim 20.

22. 20. Use of an oncolytic HSV according to any one of claims 1 to 16 in the manufacture of a medicament for use in a method of treating cancer.

23. 23. The use according to claim 22, wherein the medicament comprises a further anti-cancer agent.

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