Use of oncolytic viruses, alone or in combination with checkpoint inhibitor, for treatment of cancer

Abstract

To provide an effective cancer therapy with reduced side effects (e.g., compared to chemotherapy).SOLUTION: Provided is use of oncolytic viruses (e.g., Talimogene laherparepvec) either alone or in combination with an immune checkpoint inhibitor (e.g., anti-CTLA-4, anti-PD-1 and anti-PD-L1 compounds such as antibodies). In addition, the present invention provides compositions and kits related to such use of oncolytic viruses either alone or in combination with an immune checkpoint inhibitor.SELECTED DRAWING: Figure 11a

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 471,875, filed on Mar. 15, 2017, which is hereby incorporated by reference herein in its entirety under 35 U.S.C. § 119(e).

Background Art

[0002] In view of the progress of cancer treatment over the past few decades, the cancer - related mortality rates for the most common types of cancer, such as lung cancer, colon cancer, breast cancer, and prostate cancer, have been continuously decreasing for both men and women. This improvement in survival rates is likely due to the progress of diagnosis at the early stages of certain cancers, the improvement of treatment methods, and the results of public health efforts to encourage preventive measures and screening.

[0003] Nevertheless, cancer remains a major public health problem, with over 16 million people being diagnosed each year. In addition, a cancer diagnosis has a profound impact on the patient and their family and friends. In fact, in the United States, cancer remains the second - leading cause of death (behind only heart disease), accounting for nearly one in four deaths. See progressreport.cancer.gov / introduction, accessed on Mar. 8, 2017.

[0004] A desirable goal of cancer therapy is to preferentially kill cancer cells without causing harmful effects on normal cells. To achieve this goal, several methods have been used, including surgery, radiation therapy, chemotherapy, and therapy with oncolytic viruses.

[0005] Local treatments such as radiotherapy and surgery provide methods for reducing tumor burden within regions of the body accessible through surgical techniques or high-dose radiotherapy. However, the main approach to cancer treatment is chemotherapy. However, chemotherapeutic agents have limited effectiveness in treating many cancer types, including many common solid tumors. This lack of success is due in part to drug resistance (whether acquired or intrinsic) in many tumor cells. A significant obstacle to the use of chemotherapeutic agents is their severe side effects. These include myelosuppression, nausea, vomiting, hair loss, and mouth ulcers.

[0006] Proposed alternative therapies include the administration of oncolytic viruses and the use of viral vectors to deliver transgenes with anti-cancer activity. The genetic engineering of viruses for use as oncolytic agents initially focused on the use of replication-deficient viruses in an attempt to prevent virus-induced damage to non-tumor cells. The main limitation of this approach was that these replication-deficient viruses required helper viruses to enable integration and / or replication in host cells. These viruses have limited effectiveness because each replication-defective retroviral particle can enter only a single cell and thereafter cannot undergo productive infection in other cells. Therefore, they cannot spread away from producer cells and cannot fully penetrate many tumor cells in vivo. More recently, the genetic engineering of oncolytic viruses has focused on the generation of "replication-restricted" viruses that can spread to other tumor cells while avoiding systemic infection.

[0007] Currently, the only approved oncolytic virus-based agent in the United States and Europe is talimogene laherparepvec (IMLYGIC®). Talimogene laherparepvec is HSV-1 derived from clinical strain JS1 (deposited with the European collection of cell cultures (ECAAC) under accession number 01010209). In talimogene laherparepvec, the HSV-1 viral genes encoding ICP34.5 and ICP47 are functionally deleted. The functional deletion of ICP47 results in the early expression of US11, a gene that promotes viral growth in tumor cells without reducing tumor selectivity. In addition, the coding sequence for human GM-CSF is inserted into the viral genome at the former ICP34.5 gene site. See Lie et al., Gene Ther., 10:292-303, 2003.

[0008] The therapeutic combination of oncolytic viruses and checkpoint inhibitors is being investigated. For example, the combination of talimogene laherparepvec and immunotherapy (e.g., ipilimumab and pembrolizumab) is currently being investigated in clinical trials for melanoma (NCT01740297 and NCT02263508) and head and neck squamous cell carcinoma (NCT02626000). Checkpoint inhibitors such as ipilimumab (a CTLA-4 antibody), pembrolizumab, nivolumab (an anti-PD-1 antibody), and atezolizumab (an anti-PD-L1 antibody) have demonstrated efficacy in various tumor types. See Grosso et al., Cancer Immun., 13:5 (2013); Pardoll, Nat Rev Cancer, 12:252-264 (2012); and Chen et al., Immunity, 39:1-10 (2013).

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, there remains a need to further develop effective cancer therapies with reduced side effects (e.g., compared to chemotherapy). There also remains a need to further develop cancer therapies that are effective against metastatic cancer. The present invention addresses these and other needs.

Means for Solving the Problems

[0011] In one embodiment, the present invention relates to a method of treating Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer (i.e., colon cancer), melanoma, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma by administering a therapeutically effective amount of an oncolytic virus. In some embodiments, the cancer is metastatic cancer. In some embodiments, the oncolytic virus is a herpes simplex virus. This herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. Herpes simplex virus type 1 can also be modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0012] The present invention also relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of an oncolytic virus and (ii) a therapeutically effective amount of a checkpoint inhibitor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the checkpoint inhibitor is a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody). In certain embodiments, the anti-CTLA-4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor is a PD-L1 blocker (e.g., an anti-PD-L1 antibody). In certain embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the checkpoint inhibitor is a PD-1 blocker (e.g., an anti-PD-1 antibody). In certain embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab. In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. Herpes simplex virus type 1 can also be modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0013] In a specific embodiment, the present invention also relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of a oncolytic virus (e.g., talimogene laherparepvec) and (ii) a therapeutically effective amount of a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody, such as ipilimumab). In some embodiments, the cancer is metastatic cancer. In another embodiment, the present invention relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of a oncolytic virus (e.g., talimogene laherparepvec) and (ii) a therapeutically effective amount of a PD-L1 blocker (e.g., an anti-PD-L1 antibody, such as atezolizumab). In other embodiments, the present invention relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of a oncolytic virus (e.g., talimogene laherparepvec) and (ii) a therapeutically effective amount of a PD-1 blocker (e.g., an anti-PD-1 antibody, such as nivolumab or pembrolizumab).

[0014] The present invention also relates to a method of treating B cell lymphoma by administering (i) a therapeutically effective amount of an oncolytic virus and (ii) a therapeutically effective amount of a GITR agonist. In some embodiments, the cancer is metastatic B cell lymphoma. In certain embodiments, the GITR agonist is AMG228 (also referred to as 9H6v3), TRX518, MEDI1873, or MK-4166. See PCT Publication No. WO 2015 / 031667 and U.S. Patent No. 9,464,139 (both of which are incorporated herein by reference in their entirety). In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is (i) modified such that it does not contain an intact ICP34.5 gene. Herpes simplex virus type 1 can also be modified (i) such that it does not contain an intact ICP34.5 gene and (ii) such that it does not contain an intact ICP47 gene. In yet other embodiments, herpes simplex virus type 1 is (i) modified such that it does not contain an intact ICP34.5 gene, (ii) modified such that it does not contain an intact ICP47 gene, and (iii) modified such that it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0015] The present invention further relates to a therapeutically effective amount of an oncolytic virus for use in the treatment of Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma. In some embodiments, the cancer is a metastatic cancer. In yet another aspect, the present invention relates to a pharmaceutical composition for use in a method of treating Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma, the pharmaceutical composition comprising an oncolytic virus. In such embodiments, the oncolytic virus can be a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it also does not contain an intact ICP47 gene. In yet other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0016] In another aspect, the present invention relates to a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer). In some embodiments, the cancer is metastatic cancer. In another embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer), the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor. In such embodiments, the checkpoint inhibitor is a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody). In certain embodiments, the anti-CTLA-4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor is a PD-L1 blocker (e.g., an anti-PD-L1 antibody). In certain embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the checkpoint inhibitor is a PD-1 blocker (e.g., an anti-PD-1 antibody). In certain embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab. In some embodiments, the oncolytic virus is herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it also does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0017] In addition, the present invention relates to a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody, such as ipilimumab, etc.) for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer). In some embodiments, the cancer is metastatic cancer. In another embodiment, the present invention relates to a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a PD-L1 blocker (e.g., an anti-PD-L1 antibody, such as atezolizumab, etc.) for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer). In another embodiment, the present invention relates to a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a PD-1 blocker (e.g., an anti-PD-1 antibody, such as nivolumab, pembrolizumab, etc.). In some embodiments, the oncolytic virus is a herpes simplex virus. This herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it also does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0018] In another embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer), the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody, such as ipilimumab). In some embodiments, the cancer is metastatic cancer. In another embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer), the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a PD-L1 blocker (e.g., an anti-PD-L1 antibody, such as atezolizumab). In another embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer), the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus (e.g., talimogene laherparepvec) and a PD-1 blocker (e.g., an anti-PD-1 antibody, such as nivolumab or pembrolizumab). In some embodiments, the oncolytic virus is herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it also does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF).In certain embodiments, the oncolytic virus is talimogene laherparepvec.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] The headings of the sections used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited in the text of this specification are hereby expressly incorporated by reference in their entirety.

[0021] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzyme reactions and purification techniques can be performed according to the manufacturer's specifications or as commonly achieved in the art or as described herein. The following methods and techniques can generally be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. For example, for any purpose, see Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is hereby incorporated by reference in this specification. Unless specific definitions are provided, the nomenclature, laboratory methods, and techniques used in connection with analytical chemistry, organic chemistry, pharmaceuticals, and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and patient delivery and treatment.

[0022] Oncolytic virus As discussed herein, the present invention has demonstrated that oncolytic viruses can provide an anti-tumor effect in various tumor types, either alone or in combination with checkpoint inhibitors. The significant benefit of the oncolytic viruses of the present invention is, for example, that the anti-tumor effect is accompanied by side effects that are not as severe / negative as those of chemotherapy. For example, in one embodiment, the present invention relates to the use of oncolytic viruses in the treatment of cancer. In another embodiment, the present invention relates to the use of oncolytic viruses for the treatment of Ewing sarcoma, neuroblastoma, rhabdomyosarcoma-like tumors, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma. In another embodiment, the present invention relates to the use of a combination of an oncolytic virus and a checkpoint inhibitor for the treatment of B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma or breast cancer (e.g., triple-negative breast cancer).

[0023] In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus can be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene and (ii) it does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene, (ii) it does not contain an intact ICP47 gene, and (iii) it contains a gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laherparepvec.

[0024] Talimogene laherparepvec, HSV-1 [JS1 strain] ICP34.5- / ICP47- / hGM-CSF (formerly OncoVex GM-CSFTalimogene laherparepvec (previously known as) is an intratumoral delivered oncolytic immunotherapy that includes an immune-enhancing HSV-1 that selectively replicates within solid tumors (Lui et al., Gene Therapy, 10:292-303, 2003; U.S. Patent No. 7,223,593 and U.S. Patent No. 7,537,924). HSV-1 is derived from the JS1 strain deposited with the European Collection of Authenticated Cell Cultures (ECAAC) under accession number 01010209. In talimogene laherparepvec, the HSV-1 viral gene encoding ICP34.5 is functionally deleted. The functional deletion of ICP34.5, which acts as a pathogenicity factor during HSV infection, restricts replication in non-dividing cells and renders the virus non-pathogenic. The safety of HSV with a functionally deleted ICP34.5 has been shown in multiple clinical trials (MacKie et al, Lancet 357:525-526, 2001; Markert et al, Gene Ther 7:867-874, 2000; Rampling et al, Gene Ther 7:859-866, 2000; Sundaresan et al, J.Virol 74:3822-3841, 2000; Hunter et al, J Virol Aug;73(8):6319-6326, 1999). In addition, ICP47 (which blocks viral antigen presentation to major histocompatibility complex class I and II molecules) is functionally deleted from talimogene laherparepvec. The functional deletion of ICP47 also results in the early expression of US11, a gene that promotes viral growth in tumor cells without reducing tumor selectivity. As used herein, "functionally virus gene lacking" means that the gene is partially or completely deleted, replaced, rearranged, or otherwise altered in the herpes simplex virus genome such that a functional virus protein can no longer be expressed from that gene by herpes simplex virus. The coding sequence for human GM-CSF, a cytokine involved in the stimulation of the immune response, is inserted into the viral genome of talimogene laherparepvec (at two previous sites of the ICP34.5 gene).The insertion of the gene encoding human GM-CSF is performed such that it replaces substantially all of the ICP34.5 gene, ensuring that any potential recombination events between talimogene laherparepvec and the wild-type virus can only result in non-pathogenic viruses rendered non-functional, and that the generation of wild-type viruses carrying the gene for human GM-CSF cannot occur. The HSV thymidine kinase (TK) gene remains intact in talimogene laherparepvec, which renders the virus sensitive to antiviral agents such as acyclovir. Thus, acyclovir can be used to block talimogene laherparepvec replication, if necessary.

[0025] Examples of additional HSV genes that can be modified include ICP6, the large subunit of ribonucleotide reductase, which is involved in nucleotide metabolism and viral DNA synthesis in non-dividing cells but not in dividing cells. Thymidine kinase, which is responsible for phosphorylating acyclovir to acyclovir monophosphate, the virion trans-activating factor protein vmw65, glycoprotein H, vhs, ICP43 and ICP4, ICP27, ICP22 and / or the early gene encoding ICP0 can also be modified.

[0026] Modifications can also be made to alter the timing of expression of herpes simplex virus genes. For example, US11 can be expressed as an early gene by placing the US11 gene under the control of the Us12 promoter (Mulvey et al. (1999) J Virology, 73:4, 3375-3385, U.S. Patent No. 5,824,318, Mohr & Gluzman (1996) EMBO 15:4759-4766).

[0027] As would be understood by those skilled in the art, heterologous genes such as those encoding human GM-CSF can be inserted into the HSV viral genome, and viral genes such as ICP34.5 and ICP47 can be functionally deleted using homologous recombination with plasmid DNA.

[0028] Talimogene laherparepvec creates an anti-tumor immune response that is enhanced by the direct oncolytic effect of virus replication in tumors and the local expression of GM-CSF and the release of tumor-derived antigens through lysis. Since many cancers exist as primary and secondary (i.e., metastatic) tumors in patients, this dual activity is beneficial as a therapeutic treatment. The intended clinical effects include the destruction of injected tumors, the destruction of non-injected tumors in the local area where they are located, the reduction of the formation of new metastases, the overall rate of progression and the reduction of the recurrence rate after treatment of the initially existing disease, and the extension of the overall survival period.

[0029] As used herein, the terms "patient" or "subject" are used interchangeably and mean a mammal, including but not limited to humans, non-human mammals such as cows, horses, dogs, sheep, or cats. Preferably, the patient is a human.

[0030] Talimogene laherparepvec and OncoVex mGM-CSF (HSV-1 virus having the same genetic modification as talimogene laherparepvec except that human GM-CSF is replaced by murine GM-CSF) has been tested for efficacy in various in vitro (cell line) and in vivo murine tumor models and has been shown to eradicate tumors or substantially inhibit their growth at doses comparable to those used in clinical trials. Non-clinical evaluations have also confirmed that GM-CSF enhances the resulting immune response, enhances both the response of injected tumors and the response of non-injected tumors, and that the increased surface level of MHC class I molecules results from the deletion of ICP47. Talimogene laherparepvec has been injected into normal and tumor-bearing mice to evaluate its safety. Generally, this virus has shown good tolerance, and doses of up to 1×10 8 PFU / dose showed no signs of concern regarding safety. (See, for example, Liu et al., Gene Ther 10:292-303, 2003).

[0031] Clinical trials have been or are currently being conducted in several progressive tumor types, and over 400 subjects have been treated with talimogene laherparepvec (see, e.g., Hu et al., Clin Can Res 12:6737-6747, 2006; Harrington et al., J Clin Oncol. 27(15a):abstract 6018, 2009; Kaufman et al., Ann Surgic Oncol. 17:718-730, 2010; Kaufman and Bines, Future Oncol. 6(6):941-949, 2010). Clinical data indicate that talimogene laherparepvec may provide overall clinical utility for patients with progressive melanoma. In particular, high rates of complete response have been achieved in stage IIIc-IV melanoma (Scenzer et al., J.Clin.Oncol. 271(12):907-913, 2009). In addition, responses were observed in both injected and non-injected sites, including the site of injection.

[0032] The virus of the present invention may also be derived from a herpes simplex virus type 2 (HSV-2) strain or a derivative thereof. Derivatives include intertype recombinants containing DNA from HSV-1 and HSV-2 strains. Such intertype recombinants are described in the art, for example, by Thompson et al., (1998) Virus Genes 1(3);275 286 and Meignier et al., (1998) J.Infect.Dis. 159;602 614.

[0033] Herpes simplex virus strains can be derived from clinical isolates. Such strains are isolated from infected individuals such as those having recurrent herpes simplex. As described in U.S. Patent Nos. 7,063,835 and 7,223,593 (each of which is incorporated by reference in its entirety), clinical isolates can be screened for desired capabilities or properties such as enhanced replication in tumors and / or other cells in vitro and / or in vivo as compared to standard laboratory strains. In one embodiment, the herpes simplex virus is a clinical isolate from recurrent herpes simplex.

[0034] Examples of herpes simplex virus type 1 virus strains include, but are not limited to, the JS1 strain, the 17+ strain, the F strain, the KOS strain, and the Patton strain.

[0035] Further examples of modified herpes simplex viruses include, but are not limited to, the Seprehvir™ (HSV1716) 17+ strain of herpes simplex virus type 1, which has a 759 bp deletion located within each copy of the BamHI fragment (0 - 0.02 and 0.81 - 0.83 map units) of the long repeat region of the HSV genome, removes one complete copy from the 18 bp DR to the "a" sequence element, and terminates 1105 bp upstream of the 5' end of the immediate early (IE) gene 1 (see MacLean et al., (1991) Journal of General Virology 79:631 - 639).

[0036] G207 is an oncolytic HSV-1 derived from the wild-type HSV-1 F strain and has deletions of both copies of the ICP34.5 gene, a major determinant of HSV neurovirulence, and an inactivating insertion of the Escherichia coli (E. coli) lacZ gene in UL39, which encodes infected cell protein 6 (ICP6) (see Mineta et al. (1995) Nat Med. 1:938 - 943).

[0037] OrienX010 is a herpes simplex virus that has deletions of both copies of the gamma 34.5 and ICP47 genes, as well as an interruption of the ICP6 gene and an insertion of the human GM-CSF gene (see Liu et al., (2013) World Journal of Gastroenterology 19(31):5138-5143).

[0038] NV1020 is a herpes simplex virus in which the junction region of the long (L) and short (S) regions, which contains one copy of ICP34.5, UL24, and UL56.34, 35, is deleted. The deleted region has been replaced with a fragment of HSV-2 US DNA (US2, US3(PK), gJ, and gG) (see Todo, et al. (2001) Proc Natl Acad Sci USA. 98:6396-6401).

[0039] M032 is a herpes simplex virus that has deletions of both copies of the ICP34.5 gene and an insertion of interleukin 12 (see Cassady and Ness Parker, (2010) The Open Virology Journal 4:103-108).

[0040] ImmunoVEX HSV-2 is a herpes simplex virus (HSV-2) that has functional deletions of the genes encoding vhs, ICP47, ICP34.5, UL43, and US5.

[0041] OncoVex GALV / CD is a state in which the genes encoding ICP34.5 and ICP47 are functionally deleted, and the genes encoding cytosine deaminase and the gibbon ape leukemia fusion glycoprotein are inserted into the viral genome in place of the ICP34.5 gene. This also is derived from the JS1 strain of HSV-1.

[0042] Examples of modified herpes simplex viruses include G47 delta, G47 delta IL-12, ONCR-001, OrienX-010, NSC 733972, HF-10, BV-2711, JX-594, Myb34.5, AE-618, Brainwel™, and Heapwel™.

[0043] Herpes virus strains and methods of making such strains are also described in U.S. Patent Nos. 5,824,318; 6,764,675; 6,770,274; 7,063,835; 7,223,593; 7,749,745; 7,744,899; 8,273,568; 8,420,071; 8,470,577; WIPO Publication Nos. WO 96 / 00007; WO 96 / 39841; WO 99 / 07394; WO 00 / 54795; WO 06 / 002394; WO 13 / 06795; Chinese Patent Nos. 128,303; 10,230,334; and 10,230,335; Varghese and Rabkin, (2002) Cancer Gene Therapy 9:967-97; and Cassady and Ness Parker, (2010) The Open Virology Journal 4:103-108, each of which is incorporated herein by reference.

[0044] Checkpoint inhibitors Immune checkpoints are proteins that regulate certain types of immune system cells, such as T cells, which play a central role in cell-mediated immunity. Immune checkpoints assist in suppressing the immune response, but they can also prevent T cells from killing cancer cells. Checkpoint inhibitors (or simply "checkpoint inhibitors") can block immune checkpoint protein activity, release the "brakes" of the immune system, and enable T cells to better kill cancer cells.

[0045] As used herein, the terms "immune checkpoint inhibitor" or "checkpoint inhibitor" refer to a molecule that fully or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins control T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer 12:252-264, 2012). These proteins are responsible for co-stimulatory or inhibitory interactions of the T cell response. Immune checkpoint proteins control and maintain self-tolerance and the duration and amplitude of the physiological immune response. Immune checkpoint inhibitors can include or be derived from antibodies.

[0046] Checkpoint inhibitors can include small molecule inhibitors, or antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+ (αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. B-7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antibody-binding fragments thereof that bind to one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049 and block or inhibit the activity of one or more of these, other binding proteins, biotherapeutics, or small molecules.

[0047] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint molecule that downregulates the pathway of T cell activation. CTLA-4 is a negative regulator of T cell activation. Blockade of CTLA-4 has been shown to enhance T cell activation and proliferation. The combination of herpes simplex virus and anti-CTLA-4 antibody is intended to improve T cell activation through two different mechanisms in order to enhance the anti-tumor immune response against tumor antigens released after the cytolytic replication of the virus within the tumor. Therefore, the combination of herpes simplex virus and anti-CTLA-4 antibody increases the destruction of the injected tumor and the uninjected / distant tumors, improves the overall tumor response, and in particular, can extend the overall survival period when compared to that obtained by using anti-CTLA-4 antibody alone.

[0048] Programmed cell death protein 1 (PD-1) is a 288-amino acid cell surface protein molecule that is expressed on T cells and pro-B cells and plays a role in their fate / differentiation. The two ligands of PD-1, PD-L1 and PD-L2, are members of the B7 family. PD-1 restricts the activity of T cells in peripheral tissues during the inflammatory response to infection, and blockade of autoimmune PD-1 in vitro restricts T cell proliferation and cytokine production in response to challenge by specific antigen targets or allogeneic cells during the mixed lymphocyte reaction. A strong correlation between the expression and response of PD-1 has been shown by blockade of PD-1 (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). PD-1 blockade can be achieved by various mechanisms including antibodies that bind to PD-1 or PD-L1.

[0049] Programmed cell death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene. See Entrez Gene: CD274 for the CD274 molecule. PD-L1 is a 40 kDa type I transmembrane protein that plays a role in the suppression of the immune system and binds to its receptor (PD-1), which is found on activated T cells, B cells, and myeloid cells, to regulate cell activation or inhibition. See Chemnitz et al., Journal of Immunology, 173(2):945-54(2004).

[0050] Other immune checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Also included are B7 inhibitors, such as B7-H3 and B7-H4 inhibitors (e.g., anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15(18)3834)). Another checkpoint inhibitor is TIM3 (T cell immunoglobulin domain and mucin domain 3) (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94).

[0051] As further described herein, in one aspect, the present invention relates to the use of a combination of an oncolytic virus and a checkpoint inhibitor for the treatment of cancer. In another aspect, the present invention relates to a pharmaceutical composition comprising a combination of an oncolytic virus and a checkpoint inhibitor.

[0052] Accordingly, in one aspect of the present invention, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4, PD-1, PD-L1 or PD-L2. In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4 such as tremelimumab, ipilimumab (also known as 10D1, MDX-D010), BMS-986249, AGEN-1884, and anti-CTLA-4 antibodies (described in U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720, and 7,605,238, each incorporated herein by reference).In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of PD-L1 or PD-1 (e.g., a molecule that inhibits the interaction of PD-1 with PD-L1 and / or a PD-L2 inhibitor), such as pembrolizumab (an anti-PD-1 antibody), nivolumab (an anti-PD-1 antibody), CT-011 (an anti-PD-1 antibody), CX-072 (an anti-PD-L1 antibody), IO-103 (anti-PD-L1), BGB―A333 (anti-PD-L1), WBP-3155 (anti-PD-L1), MDX-1105 (anti-PD-L1), LY-3300054 (anti-PD-L1), KN-035 (anti-PD-L1), FAZ-053 (anti-PD-L1), CK-301 (anti-PD-L1), AK-106 (anti-PD-L1), M-7824 (anti-PD-L1), CA-170 (anti-PD-L1), CS-1001 (an anti-PD-L1 antibody), SHR-1316 (an anti-PD-L1 antibody), BMS 936558 (an anti-PD-1 antibody), BMS-936559 (an anti-PD-1 antibody), atezolizumab (an anti-PD-L1 antibody), AMP 224 (a fusion protein of the extracellular domain of PD-L2 and an IgG1 antibody designed to block the PD-L2 / PD-1 interaction), MEDI4736 (durvalumab; an anti-PD-L1 antibody), MSB0010718C (an anti-PD-L1 antibody), and those described in U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Published Patent Application International Publications Nos. 03042402, 2008156712, 2010089411, 2010036959, 2011066342, 2011159877, 2011082400, and 2011161699 (each of which is incorporated herein by reference).Examples of additional anti-PD-1 antibodies include PDR-001, SHR-1210, BGB-A317, BCD-100, JNJ-63723283, PF-06801591, BI-754091, JS-001, AGEN-2034, MGD-013, LZM-009, GLS-010, MGA-012, AK-103, genolimzumab, dostarlimab, semiprimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

[0053] BMS 936558 is a fully human IgG4 monoclonal antibody that targets PD-1. In a phase 1 clinical trial, bi-weekly administration of BMS-936558 in subjects with advanced refractory malignancies demonstrated sustained partial or complete regression. The most prominent response rates were observed in subjects with melanoma (28%) and renal cell carcinoma (27%), although substantial clinical activity was also observed in subjects with non-small cell lung cancer (NSCLC), and some responses persisted for over a year.

[0054] BMS 936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand, PD-L1. Phase 1 trial results indicated that bi-weekly administration of this agent resulted in durable responses, particularly in subjects with melanoma. Objective response rates ranged from 6% to 17% depending on cancer type in subjects with advanced NSCLC, melanoma, RCC, or ovarian cancer, and some subjects experienced responses that persisted for over a year.

[0055] AMP 224 is a fusion protein of the extracellular domain of the second PD-1 ligand, PD-L2, and IgG1, and has the ability to block the PD-L2 / PD-1 interaction. AMP-224 is currently in a phase 1 clinical trial as monotherapy in subjects with advanced cancer.

[0056] MEDI4736 is an anti-PD-L1 antibody that demonstrated an acceptable safety profile and durable clinical activity in this dose escalation trial. Development of MEDI4736 in multiple cancers and as monotherapy as well as in combination is ongoing.

[0057] GITR agonist Glucocorticoid-induced TNFR-related gene (GITR: TNFRSF 18), also sometimes called activation-induced TNFR family member (AITR), is a receptor belonging to the TNF receptor superfamily (TNFRSF). It is activated by its cognate ligand, GITR ligand (GITRL, TNFSF18). GITR is a type I transmembrane protein containing a cysteine-rich extracellular domain characteristic of TNFR family members. For example, the cytoplasmic domain of GITR shares close homology with certain other TNFR family members such as 4-1BB and CD27 (Nocentini, et al., Proc. Natl. Acad. Sci., 94:6216-6221 (1997)). GITR agonist antibodies are currently being validated as a means to expand the CD8+ T effector memory cell population and, on the other hand, as a means to promote the depletion or inhibition of Tregs.

[0058] Costimulation of responder T cells and abrogation of the inhibitory activity of regulatory T cells implies that GITR activation results in enhanced immune responses. Such activation has the potential to restore immune responses to infection and tumors. Thus, molecules capable of activating GITR would be valuable as immunostimulants in situations where it is desirable to induce enhanced immune responses.

[0059] As further described herein, in one aspect, the invention relates to the use of a combination of an oncolytic virus and a GITR agonist in the treatment of cancer. In another aspect, the invention relates to a pharmaceutical composition comprising a combination of an oncolytic virus and a GITR agonist.

[0060] In some embodiments, the GITR agonist is AMG 228 (also referred to as 9H6v3), TRX518, MEDI1873, MK-4166, BMS-986156, MK-1248, INCAGN01876 or GWN323.

[0061] TRX518 is a humanized Fc-inactivated anti-GITR monoclonal antibody that has been shown to block the interaction of GITR and act synergistically with chemotherapeutic agents in cancer models. TRX518 is currently under investigation in clinical trials including NCT01239134 (Phase III or IV malignant melanoma or other solid tumors) and NCT02628574 (advanced solid tumors).

[0062] MEDI1873 is a GITR agonist (GITR ligand (GITRL) IgG1 fusion protein) with potential immunomodulatory and anti-neoplastic activities. MEDI1873 is currently under investigation in clinical trials including NCT02583165 (advanced solid tumors).

[0063] MK-4166 is an anti-GITR agonistic monoclonal antibody that has been shown to act synergistically with chemotherapeutic agents in cancer models. MK-4166 is currently under investigation in clinical trials including NCT02132754 (in combination with pembrolizumab in advanced solid tumors).

[0064] BMS-986156 is an anti-GITR agonistic monoclonal antibody. BMS-986156 is currently under investigation in clinical trials including NCT02598960 (as monotherapy and in combination with nivolumab in subjects with advanced solid tumors).

[0065] MK-1248 is an anti-GITR agonistic monoclonal antibody. MK-1248 is currently under investigation in clinical trials including NCT02553499 (as monotherapy and in combination with pembrolizumab in subjects with advanced solid tumors).

[0066] INCAGN01876 is an anti-GITR agonistic monoclonal antibody. INCAGN01876 is currently under investigation in clinical trials including NCT02697591 (in subjects with progressive or metastatic solid tumors).

[0067] GWN323 is an anti-GITR agonistic monoclonal antibody. GWN323 is currently under investigation in clinical trials including NCT02697591 (in subjects with progressive cancer or lymphoma, as monotherapy and in combination with PDR001).

[0068] Method for treating a disease or disorder The present invention also relates to a method for treating a disease or disorder such as cancer. In some embodiments, the cancer is Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma. In other embodiments, the cancer is B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma or breast cancer (e.g., triple-negative breast cancer). In some embodiments, the cancer is metastatic cancer.

[0069] The term "metastatic cancer" refers to cancer that has spread from the part of the body where it originated (i.e., the primary site) to other parts of the body. When cancer spreads to a new area (i.e., metastasizes), it is still named according to the part of the body where it originated. For example, colon cancer that has metastasized to the pancreas is not pancreatic cancer but is referred to as "metastatic colon cancer to the pancreas". Treatment is also based on where the cancer occurred. Even when colon cancer metastasizes to the bone, this is still colon cancer, and the treating physician will recommend treatments that have been shown to address metastatic colon cancer.

[0070] The present invention also relates to the use of a combination of an oncolytic virus and a checkpoint inhibitor for the treatment of cancer. In some embodiments, the cancer is Ewing sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma. In other embodiments, the cancer is B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma or breast cancer (e.g., triple-negative breast cancer). In some embodiments, the cancer is metastatic cancer.

[0071] The present invention also relates to a method of treating a disease or disorder such as cancer by administering (i) a therapeutically effective amount of an oncolytic virus and (ii) a therapeutically effective amount of a GITR agonist. In certain embodiments, the cancer is B-cell lymphoma. In other embodiments, the GITR agonist is AMG 228, TRX518, MEDI1873 or MK-4166.

[0072] The oncolytic virus can be any of those described herein. In some embodiments, the oncolytic virus is herpes simplex virus (e.g., herpes simplex virus type 1). In other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene and does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene, does not contain an intact ICP47 gene, and contains a gene encoding GM-CSF (e.g., human GM-CSF). In a specific embodiment, the oncolytic virus is talimogene laherparepvec.

[0073] A checkpoint inhibitor can be any molecule that blocks or inhibits an inhibitory pathway of the immune system. For example, the following checkpoint molecules can be targeted for blocking or inhibition: CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, expressed on all NK, γδ and memory CD8 + (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR and various B-7 family ligands. Examples of B-7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Examples of checkpoint inhibitors include binding proteins (e.g., antibodies or antibody-binding fragments thereof), biological therapeutics or small molecules that bind to one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049 and block or inhibit the activity of one or more of these.

[0074] In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4, PD-1, PD-L1 or PD-L2. Examples of CTLA-4 inhibitors include tremelimumab, ipilimumab (also known as 10D1, MDX-D010), BMS-986249, AGEN-1884 and anti-CTLA-4 antibodies (described in U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720 and 7,605,238, each incorporated herein by reference).Examples of molecules that inhibit the interaction between PD-1 and a PD-L1 and / or PD-L2 inhibitor include pembrolizumab (anti-PD-1 antibody), nivolumab (BMS 936558; anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), BMS 936558 (anti-PD-1 antibody), BMS-936559 (anti-PD-L1 antibody), CX-072 (anti-PD-L1 antibody), IO-103 (anti-PD-L1), BGB―A333 (anti-PD-L1), WBP-3155 (anti-PD-L1), MDX-1105 (anti-PD-L1), LY-3300054 (anti-PD-L1), KN-035 (anti-PD-L1), FAZ-053 (anti-PD-L1), CK-301 (anti-PD-L1), AK-106 (anti-PD-L1), M-7824 (anti-PD-L1), CA-170 (anti-PD-L1), CS-1001 (anti-PD-L1 antibody), SHR-1316 (anti-PD-L1 antibody), atezolizumab (anti-PD-L1 antibody), AMP 224 (a fusion protein of the extracellular domain of PD-L2 and an IgG1 antibody designed to block the PD-L2 / PD-1 interaction), MEDI4736 (durvalumab; anti-PD-L1 antibody), MSB0010718C (avelumab; anti-PD-L1 antibody), and those described in U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Published Patent Application International Publication Nos. WO 03 / 042402, WO 2008 / 156712, WO 2010 / 089411, WO 2010 / 036959, WO 2011 / 066342, WO 2011 / 159877.Examples of additional anti-PD-1 antibodies include PDR-001, SHR-1210, BGB-A317, BCD-100, JNJ-63723283, PF-06801591, BI-754091, JS-001, AGEN-2034, MGD-013, LZM-009, GLS-010, MGA-012, AK-103, genolimzumab, dostarlimab, semiprimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

[0075] In certain embodiments, the invention relates to a combination of an oncolytic virus and an anti-PD-1 antibody, a combination of an oncolytic virus and an anti-PD-L1 antibody, or a combination of an oncolytic virus and an anti-CTLA-4 antibody. In a specific embodiment, the oncolytic virus is talimogene laherparepvec.

[0076] Often, cancer exists in a patient both as a primary tumor (i.e., a tumor that grows in the anatomical site where tumor progression begins and continues to produce a cancerous mass) and as secondary tumors or metastases (i.e., the spread of the tumor from its primary site to other parts of the body). The oncolytic viruses of the present invention can be effective in the treatment of tumors via a lytic effect and a systemic immune effect. For example, with regard to talimogene laherparepvec, the virus physically lyses the tumor, causing primary tumor cell death. In addition, [1] lysis of tumor cells then releases tumor-derived antigens that are subsequently recognized by the immune system, and [2] production of GM-CSF aids in the induction of an anti-tumor immune response, and both of these mechanisms are thought to result in a systemic immune response, whereby the immune system can recognize and attack both primary and secondary tumors / metastases. In embodiments where the oncolytic virus is combined with a checkpoint inhibitor, the checkpoint inhibitor is thought to further enhance the systemic immune response by enhancing priming and reducing the inhibitory effect of immune checkpoint proteins on immune system cells such as T cells. Further, in embodiments where the oncolytic virus is combined with a GITR agonist, the GITR agonist is thought to further enhance the systemic immune response by expanding the CD8+ T effector memory cell population, driving the elimination or inhibition of Tregs. Accordingly, the present invention contemplates the treatment of primary tumors, metastases (i.e., secondary tumors) or both, using an oncolytic virus (e.g., talimogene laherparepvec) either alone or in combination with a checkpoint inhibitor.

[0077] In some embodiments, the methods of treatment or use described herein do not include treatment by radiation or combination treatment with radiation. In other embodiments, the methods of treatment or use described herein do not include treatment with chemotherapeutic agents (i.e., chemical agents or drugs that are selectively destructive to malignant cells and tissues - typically small molecule compounds), such as cisplatin, or combination treatment with chemotherapeutic agents (e.g., cisplatin). In still other embodiments, the methods of treatment or use described herein do not include treatment by a combination of radiation and chemotherapeutic agents (e.g., cisplatin).

[0078] The methods of the present invention can be used to treat cancer at several different stages. Most disease classification systems include information regarding whether the cancer has spread near lymph nodes, where in the body the tumor is located, the cell type (e.g., squamous cell carcinoma), whether the cancer has spread to different parts of the body, the size of the tumor, and the malignancy of the tumor (i.e., the level of cellular abnormality, the likelihood that the tumor will grow and spread). For example, stage 0 refers to abnormal cells that have not spread near tissue - i.e., the presence of cells that could potentially become cancerous. Stages I, II, and III cancers refer to the presence of cancer. As the stage increases, the cancerous tumor grows larger and spreads more near tissue. Stage IV cancer is cancer that has spread to distant parts of the body. In some embodiments, the methods of the present invention can be used to treat metastatic cancer.

[0079] Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising a oncolytic virus or a combination of an oncolytic virus and a checkpoint inhibitor. The pharmaceutical composition may contain, for example, formulation materials for modifying, maintaining or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. The pharmaceutically active agent can be administered to a patient by various routes, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisternally, rectally, intratumorally, intravascularly, intradermally, or by passive or enhanced absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. In one embodiment, the oncolytic virus (e.g., talimogene laherparepvec) is injected into the tumor (i.e., via intratumoral injection). In another embodiment, the checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA-4 antibody) is administered systemically (e.g., intravenously).

[0080] One of ordinary skill in the art will be able to determine the dosage and treatment duration according to any aspect of the present disclosure. For example, one of ordinary skill in the art can monitor a patient to determine whether to initiate, continue, interrupt, or resume treatment. The effective amount for a particular patient can vary depending on factors such as the condition being treated, the overall health of the patient, and the method, route, and dosage of administration. The clinician makes a determination of an appropriate dosage using parameters known in the art. The effective amount of the pharmaceutically composition used therapeutically will depend, for example, on the context and purpose of the treatment. One of ordinary skill in the art will understand that the appropriate dosage level for treatment will vary in part depending on the molecule so delivered, the indication for which the binding agent molecule is used, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Thus, the clinician can titrate the dosage and change the route of administration to obtain an optimal therapeutic effect.

[0081] Clinical studies have demonstrated that talimogene laherparepvec can be injected directly into visible and palpable skin lesions, subcutaneous lesions, or lymph node lesions, or can be injected using ultrasound guidance. Thus, in one aspect, a pharmaceutical composition comprising talimogene laherparepvec is administered via intralesional injection. Talimogene laherparepvec is currently available in single-use vials of 1 mL at a fixed dosing concentration: 10 6 pfu / mL for the first dose and 10 8 pfu / mL for subsequent doses (Reske, et al. J Immunol, 2008, 180(11): p.7525-36). The volume injected can vary depending on the tumor type. For example, talimogene laherparepvec is administered at a maximum dose of 4.0 mL at 10 6 plaque-forming units / mL (PFU / mL) on day 1 of week 1, followed by a maximum dose of 4.0 mL at 10 8 PFU / mL on day 1 of week 4, and then subsequently by intratumoral injection every two weeks (±3 days) in injectable skin tumors, subcutaneous tumors, and lymph node tumors. In another embodiment, talimogene laherparepvec is administered at a maximum dose of 4.0 mL at 10 6 plaque-forming units / mL (PFU / mL) on day 1 of week 1, followed by a maximum dose of 4.0 mL at 10 7 PFU / mL on day 1 of week 4, and then subsequently by intratumoral injection every two weeks (±3 days) in injectable skin tumors, subcutaneous tumors, and lymph node tumors. The recommended volume of talimogene laherparepvec to be injected into the tumor can be determined according to the size of the tumor, in accordance with the injection volume guidelines shown in Table 1 (and as shown in Patent Application PCT / US Patent Application Publication No. 2013 / 057542, which is incorporated herein by reference).

[0082]

Table 1

[0083] Generally, all appropriately injectable lesions must be injected at the maximum dose effective for an individual administration case. On each treatment day, the injection prioritization is recommended as follows: any new injectable tumor that has appeared since the previous injection; starting with the largest tumor by tumor size; any previously un-injected tumor that is currently injectable.

[0084] The composition of the present invention can include one or more additional components including a physiologically acceptable carrier, excipient or diluent. For example, the composition can include one or more of a buffer solution, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (for example, having less than 10 amino acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose or dextrin, a chelating agent such as EDTA, glutathione, a stabilizer and an excipient. Examples of acceptable carriers include, for example, neutral buffered saline or saline mixed with a specific serum albumin. A preservative such as benzyl alcohol can also be added. The composition can be formulated as a lyophilized product using an appropriate excipient solution (for example, sucrose) as a diluent.

[0085] In certain embodiments, the checkpoint inhibitor is administered at a dose of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg or any combination thereof. In certain embodiments, the checkpoint inhibitor is administered once a week, twice a week, three times a week, once every two weeks or once a month. In certain embodiments, the checkpoint inhibitor is administered as a single dose, two doses, three doses, four doses, five doses or six or more doses.

[0086] In certain embodiments, the anti-PD-1 antibody is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg / kg, such as about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary, for example, from once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 10 to 20 mg / kg every other week.

[0087] In one embodiment, the anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of about 1 mg / kg to 3 mg / kg, such as about 1 mg / kg, 2 mg / kg, or 3 mg / kg every two weeks. In one embodiment, the anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of about 2 mg / kg at 3-week intervals. In one embodiment, nivolumab is administered in an amount of about 1 mg / kg to 5 mg / kg, such as 3 mg / kg, and can be administered over a period of 60 minutes, approximately once a week to once every 2, 3, or 4 weeks.

[0088] In one embodiment, the anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 1 mg / kg to 3 mg / kg, such as about 1 mg / kg, 2 mg / kg, or 3 mg / kg every three weeks. In one embodiment, the anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 2 mg / kg at 3-week intervals. In another embodiment, the anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 100 mg / kg to 300 mg / kg, such as about 100 mg / kg, 200 mg / kg, or 300 mg / kg every three weeks. In one embodiment, the anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 200 mg / kg at 3-week intervals.

[0089] In certain embodiments, an anti-CTLA-4 antibody (e.g., ipilimumab) is administered by injection (e.g., subcutaneous or intravenous) at a dose of about 3 mg / kg IV Q3W for up to 4 doses, at a dose of about 3 mg / kg IV Q6W for up to 4 doses, at a dose of about 3 mg / kg IV Q12W for up to 4 doses, at a dose of about 10 mg / kg IV Q3W for up to 4 doses or at a dose of about 10 mg / kg IV Q12W for up to 4 doses. In certain embodiments, an anti-CTLA-4 antibody (e.g., tremelimumab) is administered by injection (e.g., subcutaneous or intravenous) at a dose of about 10 mg / kg Q4W or at a dose of about 15 mg / kg every 3 months.

[0090] In certain embodiments, an anti-PD-L1 antibody (e.g., atezolizumab) is administered by injection (e.g., subcutaneous or intravenous) at a dose of about 1200 mg IV Q3W until disease progression or unacceptable toxicity.

[0091] Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma, the pharmaceutical composition comprising an oncolytic virus. In another embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma or breast cancer (e.g., triple negative breast cancer), the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor. In a further embodiment, the present invention relates to a pharmaceutical composition for use in a method of treating B-cell lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus and a GITR agonist.

[0092] In other embodiments, the present invention relates to a therapeutically effective amount of an oncolytic virus for use in the treatment of Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumors, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma. In still other embodiments, the present invention relates to a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma or breast cancer (e.g., triple-negative breast cancer). In additional embodiments, the present invention relates to a therapeutically effective amount of an oncolytic virus and a GITR agonist for use in the treatment of B-cell lymphoma.

[0093] In some embodiments, the oncolytic virus is herpes simplex virus (e.g., herpes simplex virus type 1). In other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene and does not contain an intact ICP47 gene. In still other embodiments, herpes simplex virus type 1 is modified such that it does not contain an intact ICP34.5 gene, does not contain an intact ICP47 gene, and contains a gene encoding GM-CSF (e.g., human GM-CSF). In a specific embodiment, the oncolytic virus is talimogene laherparepvec.

[0094] In embodiments where the pharmaceutical composition comprises a checkpoint inhibitor, the checkpoint inhibitor can be any of those discussed herein. For example, the checkpoint inhibitor can be a CTLA-4 blocker, a PD-L1 blocker or a PD-1 blocker. The CTLA-4 blocker can be, for example, an anti-CTLA-4 antibody such as ipilimumab. The PD-L1 blocker can be, for example, an anti-PD-L1 antibody such as atezolizumab. The PD-1 blocker can be, for example, an anti-PD-1 antibody such as nivolumab or pembrolizumab.

[0095] In some embodiments, the GITR agonist is AMG 228, TRX518, MEDI1873 or MK-4166.

[0096] In some embodiments, the pharmaceutical compositions described herein are not used in combination therapy with or in combination with radiation. In other embodiments, the pharmaceutical compositions described herein do not comprise a chemotherapeutic agent (e.g., cisplatin). In still other embodiments, the pharmaceutical compositions described herein are not used in therapy with a combination of radiation and a chemotherapeutic agent (e.g., cisplatin).

[0097] Kit In another aspect, the invention relates to a kit comprising [1] optionally, an oncolytic virus in combination with a checkpoint inhibitor, and [2] instructions for administration to a patient. For example, the kit of the invention can comprise an oncolytic virus (e.g., talimogene laherparepvec) and instructions (e.g., in a package insert or label) for treating a patient with cancer. In some embodiments, the cancer is metastatic cancer. In another embodiment, the kit of the invention can comprise an oncolytic virus (e.g., talimogene laherparepvec), a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA-4 antibody), and instructions (e.g., in a package insert or label) for treating a patient with cancer.

[0098] In another aspect, the invention relates to a kit comprising, optionally, in combination with a GITR agonist, an oncolytic virus, and instructions for administration to a patient. In other embodiments, the kit of the invention may comprise an oncolytic virus (e.g., talimogene laherparepvec), a GITR agonist (e.g., AMG 228 (also referred to as 9H6v3), TRX518, MEDI1873 or MK-4166), and instructions (e.g., in a package insert or label) for treating a cancer patient.

[0099] In some embodiments, a kit comprising talimogene laherparepvec comprises instructions (e.g., in a package insert or label) for administration at a dose of up to 4.0 ml at 10 6 PFU / mL on day 1 of week 1, followed by a dose of up to 4.0 ml at 10 8 PFU / mL on day 1 of week 4, and thereafter, by intratumoral injection every two weeks (e.g., until complete response). In some embodiments, a kit comprising talimogene laherparepvec comprises instructions (e.g., in a package insert or label) for administration at a dose of up to 4.0 ml at 10 6 PFU / mL on day 1 of week 1, followed by a dose of up to 4.0 ml at 10 7 PFU / mL on day 1 of week 4, and thereafter, by intratumoral injection every two weeks (e.g., until complete response).

[0100] In embodiments where the kit comprises an anti-PD-1 antibody, the kit comprises instructions (e.g., in a package insert or label) for intravenous administration at the doses described herein. Examples of anti-PD-1 antibodies include pembrolizumab and nivolumab.

[0101] In embodiments where the kit comprises an anti-PD-L1 antibody, the kit comprises instructions (e.g., in a package insert or label) for intravenous administration at the doses described herein. An example of an anti-PD-L1 antibody is atezolizumab.

[0102] In embodiments where the kit comprises an anti-CTLA-4 antibody, the kit includes instructions (e.g., in the package insert or label) for intravenous administration at the dosages described herein. Examples of anti-CTLA-4 antibodies include ipilimumab.

[0103] In embodiments where the kit comprises a GITR agonist, the kit includes instructions (e.g., in the package insert or label) for intravenous administration at the dosages described herein. Examples of anti-GITR antibodies include AMG 228, TRX518, MEDI1873, or MK-4166.

[0104] In another embodiment, a method of manufacturing the kit of the invention is provided.

[0105] In some embodiments, the kits described herein are not used in combination therapy with or concomitantly with radiation. In other embodiments, the kits described herein do not include a chemotherapeutic agent (e.g., cisplatin). In still other embodiments, the kits described herein are not used in therapy with a combination of radiation and a chemotherapeutic agent (e.g., cisplatin).

Examples

[0106] The following examples are provided for the purpose of exemplifying specific embodiments or features of the invention and are not intended to limit its scope.

[0107] Example 1: Talimogene laherparepvec exhibits anti-tumor activity against a range of tumor types in an in vivo mouse model This example demonstrates that administration of talimogene laherparepvec to tumor-bearing mice results in tumor ablation.

[0108] The antitumor efficacy of talimogene laherparepvec was evaluated in several murine xenograft studies in Balb / c nude mice. Tumor cells (A-673 human pediatric Ewing sarcoma, SJCRH30 human pediatric rhabdomyosarcoma, G-401 human pediatric rhabdomyosarcoma-like tumor, SK-N-AS human pediatric neuroblastoma or SJSA-1 human pediatric osteosarcoma) were transplanted into the right flank of each mouse by subcutaneous injection. In each case, 5×10 6 ~1×10 7 cells in 100 - 200 μL of 50% Matrigel / 50% DMEM were transplanted into the mice.

[0109] Tumor measurements were obtained twice a week. Treatment with talimogene laherparepvec was initiated when the tumors reached an average diameter of 4 - 6 mm. Three talimogene laherparepvec doses (5×10 4 , 5×10 5 or 5×10 6 PFU / dose, 50 μL dose volume) were administered 3 days apart by intratumoral injection. Body weight, general clinical observations and tumor measurements were obtained twice a week. Animals were euthanized when the tumor weight exceeded 10% of the body weight.

[0110] In this experiment, talimogene laherparepvec demonstrated antitumor efficacy against all cell lines tested, with evidence of 65 - 112% tumor growth inhibition and complete regression in 3 - 30% of animals across tumor types.

[0111] Ewing sarcoma A-673 Ewing sarcoma-bearing mice were treated therapeutically with talimogene laherparepvec at 5×10 4 , 5×10 5 or 5×10 6 PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on days 8, 11 and 14 of the study (red arrows in Figure 1). Tumors were measured 2 - 3 times per week. Results are shown as a function of time in days with day 0 being the day of tumor engraftment, in mm 3Represented as the mean tumor volume of the group at the standard error of the mean (n = 10 per group, except as noted for the vehicle control group on days 16, 20, and 23). Asterisks indicate that all Talimogene laherparepvec groups had p < 0.0001 compared to the vehicle control on day 23 of the study.

[0112] The results are shown in Figure 1.

[0113] Neuroblastoma SK-N-AS neuroblastoma-bearing mice were treated therapeutically with Talimogene laherparepvec at 5×10 4 、5×10 5 or 5×10 6 PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on days 6, 9, and 12 of the study (red arrows in Figure 2). Tumors were measured 2-3 times per week. Results are presented as the mean tumor volume of the group at the standard error of the mean, in mm 3 ±, as a function of time in days where day 0 is the day of tumor uptake (n = 10 per group, except as noted for the 5×10 4 PFU / dose group on days 18, 20, and 23). Asterisks indicate that 5×10 6 and 5×10 5 PFU / dose groups had p < 0.0001 compared to the formulation buffer control on day 23 of the study, and the 5×10 4 PFU / dose group had p = 0.0001.

[0114] The results are shown in Figure 2.

[0115] Rhabdomyosarcoma-like tumor G-401 rhabdomyosarcoma-like tumor-bearing mice were treated with Talimogene laherparepvec at 5×10 4 、5×10 5 or 5×10 6Treated therapeutically at the indicated pfu / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on days 14, 17, and 20 of the study (red arrows in Figure 3). Tumors were measured two to three times per week. Results are presented as mean tumor volume of the group in mm 3 ± standard error of the mean as a function of time in days where day 0 is the day of tumor uptake (n = 10 per group unless otherwise noted). Asterisks indicate that all talimogene laherparepvec treatment groups were p < 0.0001 compared to the vehicle control on day 40 of the study.

[0116] Results are shown in Figure 3.

[0117] Osteosarcoma SJSA-1 osteosarcoma-bearing mice were treated therapeutically with talimogene laherparepvec at 5 × 10 4 、5 × 10 5 or 5 × 10 6 pfu / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on days 9, 12, and 15 of the study (red arrows in Figure 4). Tumors were measured two to three times per week. Results are presented as mean tumor volume of the group in mm 3 ± standard error of the mean (n = 10 per group).

[0118] Results are shown in Figure 4.

[0119] Rhabdomyosarcoma SJCRH30 rhabdomyosarcoma-bearing mice were treated therapeutically with talimogene laherparepvec at 5 × 10 4 、5 × 10 5 or 5 × 10 6 pfu / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on days 8, 11, and 14 of the study (red arrows in Figure 5). Tumors were measured two to three times per week. Results are presented as mean tumor volume of the group in mm 3Represented as the mean tumor volume of the group at the standard error of the mean (day 26, day 30, and day 33 for the 5×10 4 PFU / dose group and for the 5×10 6 PFU / dose group, except as noted, n = 10 per group). Asterisks indicate p < 0.0001 for all talimogene laherparepvec - administered groups versus vehicle control on day 26 of the study (the last study day when all control animals were in the study).

[0120] The results are shown in Figure 5.

[0121] Example 2: Talimogene laherparepvec inhibits the growth of a range of human tumor types in cell - based assays Diffuse large B - cell lymphoma (DLBCL or DLBL) Several DLBCL cell lines (SU - DHL - 2, OCI - LY - 3, TMD8, RI - 1 (ABC subtype), and WSU - NHL (GCB subtype)) were seeded at 5,000 cells per well in a 96 - well plate and incubated overnight at 37°C. For each cell line, talimogene laherparepvec was serially diluted in nine wells starting at 100 MOI (1:4 serial dilution). After 72 hours of incubation, the number of cells remaining in each well was quantified using the CellTiter - Glo Luminescent cell viability assay (Promega, Madison, WI).

[0122] Talimogene laherparepvec was effective at MOIs below 100 in 14 out of 21 DLBCL cell lines. Five cell lines (SU - DHL - 2, OCI - LY - 3, TMD8, RI - 1 (ABC subtype), and WSU - NHL (GCB subtype)) had an MOI IC 50showed the highest sensitivity (Table 2). In contrast, OCI-LY-1, KARPAS422, WSU-DLCL2, SU-DHL-4, SU-DHL-10, and OCI-LY-7 (all of the GCB subtype) showed resistance to talimogene laherparepvec at up to 100 MOI (Table 2). Inhibition of cell growth was maximal in most cell lines, showing sensitivity below an MOI of 1 IC 50 and was greatest in most cell lines, showing sensitivity below an MOI of 1 IC. Figure 6 shows the degree of cell growth inhibition achieved by increasing the concentration of talimogene laherparepvec in the WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines. These results demonstrate that treating DLBCL cell lines with talimogene laherparepvec results in a potent inhibition of DLBCL tumor cell growth.

[0123]

Table 2

[0124] Additional solid tumors Various solid tumor cell lines (melanoma, non-small cell lung cancer, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, and triple-negative breast cancer) were seeded in 96-well plates at 2,000 - 10,000 cells per well and incubated overnight at 37°C. For each cell line, talimogene laherparepvec was serially diluted in nine wells starting at 100 MOI (1:4 serial dilution). After 72 hours of incubation, the number of cells remaining in each well was quantified using ATP-Lite (Perkin Elmer, Waltham, MA).

[0125] Talimogene laherparepvec was effective against all 13 melanoma and cancer cell lines tested. All cell lines tested had an MOI IC below 1 50is shown (Table 3). Figure 7 shows the degree of cell growth inhibition achieved by increasing the concentration of talimogene laherparepvec in HCT116 (colorectal cancer) and SK-MEL-5 (melanoma) cell lines. These results demonstrate that treating melanoma, non-small cell lung cancer, colorectal cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, and triple-negative breast cancer cell lines with talimogene laherparepvec results in potent inhibition of tumor cell growth.

[0126]

Table 3

[0127] Cutaneous T-cell lymphoma (CTCL) and multiple myeloma (MM) CTCL and MM cell lines were seeded in 96-well plates at 2,000 - 10,000 cells per well and incubated overnight at 37°C. For each cell line, talimogene laherparepvec was serially diluted in 9 wells starting at 100 MOI (1:4 serial dilution). After 72 hours of incubation, the number of cells remaining in each well was quantified using ATP-Lite (Perkin Elmer, Waltham, MA).

[0128] Talimogene laherparepvec was effective against all 5 cell lines tested (Table 4). Multiple myeloma cell lines showed higher sensitivity than cutaneous T-cell lymphoma lines. Figure 8 shows the degree of cell growth inhibition achieved by increasing the concentration of talimogene laherparepvec in HUT-78 (CTCL) and RPMI 8226 (multiple myeloma) cell lines. These results demonstrate that treating cutaneous T-cell lymphoma (CTCL) and multiple myeloma (MM) cell lines with talimogene laherparepvec results in potent inhibition of tumor cell growth.

[0129]

Table 4

[0130] Example 3: Talimogene laherparepvec inhibits the growth of various murine tumor cell lines in cell-based assays Murine cell lines of melanoma, colorectal cancer, and B-cell lymphoma were seeded at 2,000 - 10,000 cells per well in 96-well plates and incubated overnight at 37°C. For each cell line, talimogene laherparepvec was serially diluted in nine wells starting at 100 MOI (1:4 serial dilution). After 72 hours of incubation, the number of cells remaining in each well was quantified using ATP-Lite (Perkin Elmer, Waltham, MA).

[0131] Talimogene laherparepvec was effective against 4 out of 5 cell lines tested (Table 5). The B16F10 melanoma cell line demonstrated resistance to talimogene laherparepvec. This resistance is mediated by the lack of entry receptor for herpes simplex virus type 1 as previously described by Miller et al., Molecular Therapy, 3(2):160 - 168 (2001). The Cloudman CL M3 (melanoma), CT-26, and MC-38 (colorectal cancer) cell lines showed similar sensitivity at an MOI of approximately 0.2 IC 50 Figure 9 shows the degree of cell growth inhibition achieved by increasing the concentration of talimogene laherparepvec in the CT-26 and MC-38 (colorectal cancer) cell lines. These results demonstrate that treating murine tumor cell lines (melanoma, colorectal cancer, and B-cell lymphoma) with talimogene laherparepvec results in a potent inhibition of tumor cell growth.

[0132]

Table 5

[0133] Example 4: OncoVex mGM-CSF inhibits the growth of B-cell lymphoma and neuroblastoma in a mouse model A20 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6 cells). Tumor volume (mm 3 ) was measured twice weekly (Q2W) using a digital caliper. When the tumors reached an average of approximately 100 mm 3 , the mice were randomized into groups (10 mice per group) such that the average tumor volume (in both flanks) and the variation in tumor volume at the start of treatment were uniform across the treatment groups. Subsequently, the mice received three intratumoral injections of OncoVex mGM-CSF (3 × 10 4 - 3 × 10 6 PFU / dose) or vehicle on days 10, 13, and 16. Clinical signs, body weight changes, and survival time (mice were excluded from the study if the tumors reached 800 mm 3 ) were measured two to three times weekly until the end of the study.

[0134] Treatment of A20 tumor-bearing animals with OncoVex mGM-CSF resulted in complete regression of 100% of all directly injected tumors at all three doses: 3 × 10 4 PFU, 3 × 10 5 PFU, 3 × 10 6 PFU (Figure 10a). Colorectal tumors did not respond at a dose of 3 × 10 4 PFU, showed 50% tumor growth at a dose of 3 × 10 5 PFU, and showed 100% tumor growth inhibition at a dose of 3 × 10 6 PFU (Figure 10b). The median survival time was significantly extended in the 3 × 10 5 PFU (p = 0.0054) and 3 × 10 6 PFU dose groups (38 days vs. 21 days respectively - Figure 10c) compared to vehicle. Decrease in body weight was not observed, indicating that the tested treatment was safe and well-tolerated (Figures 10d and 10e). Table 6 shows the percentage of subjects without tumors in each group.

[0135] In addition, neuro2a neuroblastoma tumor-bearing mice were treated with OncoVex mGM-CSF at 5 × 104 , 5×10 5 or 5×10 6 PFU / dose (n = 10 per group). OncoVex mGM-CSF was administered by intratumoral injection once daily on days 10, 13, and 16 of the study (Figure 10f). The injected tumors were measured twice a week. The results are presented as the individual tumor volumes in mm 3 as a function of time in days where day 0 is the day of tumor uptake. The median survival was significantly prolonged in the 5×10 4 PFU / dose group (p = 0.0056), 5×10 5 PFU / dose group (p < 0.0001), and 5×10 6 PFU / dose group (p < 0.0001) compared to vehicle (Figure 10g).

[0136] The effect of OncoVex on uninjected (“untreated”) tumors in neuro2a neuroblastoma tumor-bearing mice was also evaluated. Neuro2a tumor cells were implanted subcutaneously into the left and right flanks of female A / J mice on day 0. Tumor volume (mm3) was measured twice a week (Q2W) using digital calipers. When the tumors reached an average of approximately 100 mm mGM-CSF , the mice were randomized into groups such that the average tumor volume (in both flanks) and tumor volume variability at the start of treatment were uniform across the treatment groups (10 mice per group). The mice were then administered OncoVex 3 (5×10 mGM-CSF PFU / dose) or vehicle by three intratumoral injections on days 10, 13, and 16 into the right side (“treated” side). Tumor volume and survival (mice were excluded from the study if the tumor reached 800 mm 6 on either side) were measured twice a week until the end of the study (Figure 10h). Treatment with OncoVex in neuro2a tumor-bearing animals resulted in complete regression in 8 out of 10 directly injected tumors (Figure 10h). The contralateral uninjected (“untreated”) tumors showed a significant delay in tumor growth (Figure 10h). The median survival was significantly prolonged in OncoVex 3 compared to vehicle. mGM-CSF compared to vehicle.mGM-CSF Significantly prolonged in the treatment group (32 days vs. 18 days, p < 0.0001, Figure 10i).

[0137] These results demonstrate that treatment with OncoVex in established B-cell lymphoma and neuroblastoma tumor models in vivo results in potent inhibition of tumor growth. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune response) and in non-injected contralateral tumors in the same host (presumably via adaptive immune response). mGM-CSF Significantly prolonged in the treatment group (32 days vs. 18 days, p < 0.0001, Figure 10i).

[0138] [Table 6]

[0139] Example 5: OncoVex mGM-CSF Combination with CTLA-4 or PD-L1 inhibitor inhibits tumor growth of B-cell lymphoma in mouse model A20 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6 cells). Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When tumors reached approximately 100 mm 3 on average, animals were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) and tumor volume variability at the start of treatment were uniform across treatment groups. Then, animals were given three intratumoral injections of OncoVex mGM-CSF (5 × 10 6 PFU / dose) alone or in combination with intraperitoneal injection of anti-PD-L1 mAb or anti-CTLA-4 mAb. Clinical signs, body weight changes, and survival time (mice were excluded from the study when tumors reached 800 mm 3 ) were measured 2 - 3 times a week until the end of the study.

[0140] OncoVex in A20 tumor-bearing animals mGM-CSF , anti-CTLA-4 mAb or OncoVex mGM-CSFTreatment with the combination of OncoVex and anti-CTLA-4 mAb resulted in complete regression of all directly injected tumors (Figure 11a). Tumors treated with intraperitoneal anti-CTL4-4 alone (bilateral flanks) showed approximately 50% tumor inhibition. The contralateral tumors showed some consistent effect with OncoVex alone, while the combination of OncoVex and anti-CTLA-4 mAb regressed all tumors and led to complete cures in 9 out of 10 mice. The median survival time was significantly prolonged in the anti-CTLA-4 mAb group, OncoVex group and the combination group of OncoVex and anti-CTLA-4 mAb compared to the vehicle. In addition, a significant prolongation in median survival time was measured in the combination group compared to either single agent alone (p = 0.012 for OncoVex, p = 0.001 for anti-CTLA-4 mAb). The median survival time was 25.5 days for the vehicle, 36.5 days for the OncoVex group and 32 days for the anti-CTLA-4 mAb group. The median survival time for the combination group remained undetermined beyond day 40, at which point 9 out of 10 mice showed no signs of tumor (Figure 11b and Table 7a). mGM-CSF showed some consistent effect with OncoVex alone, while mGM-CSF the combination of OncoVex and anti-CTLA-4 mAb regressed all tumors and led to complete cures in 9 out of 10 mice. The median survival time was significantly prolonged in the anti-CTLA-4 mAb group, OncoVex mGM-CSF group and OncoVex mGM-CSF and anti-CTLA-4 mAb compared to the vehicle. In addition, a significant prolongation in median survival time was measured in the combination group compared to either single agent alone (OncoVex mGM-CSF p = 0.012, anti-CTLA-4 mAb p = 0.001). The median survival time was 25.5 days for the vehicle, OncoVex mGM-CSF group 36.5 days and the anti-CTLA-4 mAb group 32 days. The median survival time for the combination group remained undetermined beyond day 40, at which point 9 out of 10 mice showed no signs of tumor (Figure 11b and Table 7a).

[0141]

Table 7

[0142] Treatment of A20 tumor-bearing animals with OncoVex mGM-CSF , anti-PD-L1 mAb or the combination of OncoVex mGM-CSF and anti-PD-L1 mAb resulted in complete regression of all directly injected tumors (Figure 11c). Tumors treated with intraperitoneal anti-CTL4-4 alone (bilateral flanks) had no effect on tumor growth. The contralateral tumors showed some consistent effect with OncoVex alone, while OncoVex mGM-CSF showed some consistent effect with OncoVex alone, while OncoVex mGM-CSFThe combination with anti-PD-L1 mAb regressed all tumors and resulted in complete cures in 10 out of 10 mice. The median survival period was significantly extended in the OncoVex mGM-CSF group and the combination group compared to the vehicle. Additionally, a significant extension in the median survival period was measured in the combination group compared to anti-PD-L1 mAb alone. When comparing the combination to OncoVex mGM-CSF , an enhanced trend in the overall survival period was also observed, but statistical superiority was not observed (p = 0.067 for OncoVex mGM-CSF , p = 0.0013 for anti-PD-L1 mAb). The median survival period was 23 days for the vehicle, 48 days for the OncoVex mGM-CSF group, and 26 days for the anti-PD-L1 mAb group. The median survival period for the combination group remained undetermined beyond day 40, and at this point, 10 out of 10 mice showed no signs of tumors (Figure 11d and Table 7b).

[0143]

Table 8

[0144] These results demonstrate that treatment with OncoVex mGM-CSF in combination with either a PD-L1 or CTLA-4 inhibitor for established B-cell lymphoma tumors in an in vivo mouse model results in better inhibition of tumor growth compared to either single agent alone. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune response) and uninjected contralateral tumors in the same host (presumably via adaptive immune response).

[0145] Example 6: OncoVex mGM-CSF inhibits the growth of colorectal tumors in a mouse model either alone or in combination with a CTLA-4 or PD-L1 inhibitor CT-26 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2×10 6Individual cells). Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumor reached approximately 100 mm on average 3 , the animals were randomized into four groups (10 mice per group) such that the average tumor volume (in both flanks) and the variation in tumor volume at the start of treatment administration were uniform across the treatment groups. Subsequently, the animals were administered a) PBS + IgG control, b) OncoVex mGM-CSF + IgG control, c) PBS + anti-CTTLA-4 mAb or PBS + anti-PD-L1 mAb, or d) OncoVex mGM-CSF + anti-CTLA-4 mAb or OncoVex mGM-CSF + anti-PD-L1 mAb. Clinical signs, body weight changes, and survival time (when the tumor reached 800 mm 3 , the mice were excluded from the study) were measured two to three times a week until the end of the study.

[0146] Treatment of CT-26 tumor-bearing animals with OncoVex mGM-CSF , anti-CTLA-4 mAb, or combinations thereof resulted in tumor growth inhibition of approximately 75% of all directly injected tumors by day 20 (data not shown). The contralateral tumors did not show efficacy against OncoVex mGM-CSF alone (approx. 25% TGI), while approximately 75% tumor growth inhibition was observed in the anti-CTLA-4 mAb and combination groups. The median survival time was significantly prolonged in the anti-CTLA-4 mAb and combination groups compared to the vehicle (Figure 12b). The median survival time was 20 days for the vehicle, 22 days for the OncoVex mGM-CSF group, and 41 days for the anti-CTLA-4 group. The median survival time for the combination group was longer than 50 days and remained undetermined at the time the experiment was terminated. The median survival time in the combination group was significantly longer than either the OncoVex mGM-CSF alone group (p = 0.0001) or the anti-CTLA-4 mAb alone group (p = 0.0031) (Figure 12b and Table 8a).

[0147]

Table 9

[0148] OncoVex in CT-26 Tumor-Bearing Animals mGM-CSF or OncoVex mGM-CSF Treatment with a combination of OncoVex and anti-PD-L1 mAb resulted in tumor growth inhibition of approximately 75% of all directly injected tumors by day 18 (data not shown). Intraperitoneal injection of anti-PD-L1 mAb had little effect on tumor growth (in either flank). Contralateral tumors did not respond to OncoVex mGM-CSF or anti-PD-L1 mAb alone, while approximately 75% tumor growth inhibition was observed in the combination group (Figure 12c). The median survival time was significantly extended in the combination group compared to either agent alone (p = 0.012 for OncoVex mGM-CSF and p = 0.007 for anti-PD-L1 mAb). The median survival times were 21 days for vehicle and anti-PD-L1 mAb, 23 days for the OncoVex mGM-CSF group, and 34 days for the combination group (Figure 12d and Table 8b).

[0149]

Table 10

[0150] These results demonstrate that treatment of established colorectal tumors in an in vivo mouse model with 1) OncoVex mGM-CSF , 2) a combination of OncoVex mGM-CSF and an anti-CTLA-4 inhibitor, or 3) a combination of OncoVex mGM-CSF and an anti-PD-L1 inhibitor results in potent inhibition of tumor growth. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune response) with 1) OncoVex mGM-CSF , 2) a combination of OncoVex mGM-CSF and an anti-CTLA-4 inhibitor, and 3) a combination of OncoVex mGM-CSF and an anti-PD-L1 inhibitor. Antitumor activity was observed with 1) OncoVex mGM-CSFin combination with an anti-CTLA-4 inhibitor, and 2) OncoVex mGM-CSF was observed in non-injected contralateral tumors (presumably via an adaptive immune response) in the same host treated with the combination of

[0151] OncoVex mGM-CSF To further understand the anti-tumor activity of the combination of 1) OncoVex mGM-CSF and an anti-CTLA-4 inhibitor, 2) OncoVex mGM-CSF and an anti-CTLA-4 inhibitor, and 3) OncoVex mGM-CSF and an anti-CTLA-4 inhibitor were evaluated for their ability to release tumor antigens and stimulate anti-tumor specific T cell responses. A 96-well ELISpot plate (Millititer HA; Millipore, Temecula, CA) with a nitrocellulose filter base was coated with purified anti-IFN-γ (2 μg / ml) antibody. OncoVex mGM-CSF spleen cells (8 × 10 5 ) from CT-26 tumor-bearing mice treated with OncoVex mGM-CSF , anti-CTLA-4 mAb or the combination on day 10 were incubated with a control peptide (GFP) or an AH1 peptide (SPSYVYHQF) at a final concentration of 1 μM for 20 h at 37°C. The AH1 peptide is an immunodominant Ag derived from the envelope protein (gp70) of endogenous murine leukemia virus presented by MHC class I d molecules (25). Spots were counted using a CTLS6 Fluorospot analyzer (CTL, Shaker Height, OH). Quantification of systemic (spleen) anti-AH1 CD8 + T cells by ELISpot or dextramer staining using FACS demonstrated a significant increase in AH1-reactive T cells in mice treated with OncoVex mGM-CSF , an anti-CTLA-4 inhibitor or the combination of OncoVex mGM-CSF and an anti-CTLA-4 inhibitor (Figure 12e, 12f). Local (tumor) anti-AH1 CD8 +Quantification of T cells showed a significant increase only in the combination group. A significant decrease in Tregs was also observed in the CT-26 model. This effect was greater with the combination with CTLA-4 inhibitor (Figure 12g). This experiment demonstrated that the combination of OncoVex mGM-CSF and anti-CTLA-4 inhibitor resulted in an increase in the presence of effector cells and a decrease in the presence of regulatory T cells, which in turn led to an increase in the efficacy of the combination over either compound alone.

[0152] Example 7: OncoVex mGM-CSF inhibits melanoma tumor growth in a mouse model either alone or in combination with a CTLA-4 inhibitor B16F10 cells (5×10 4 resistant to lysis due to lack of HSV-1 entry receptor) were injected intravenously on day 0. On day 2, B16F10 melanoma cells transfected with mouse nectin 1 (sensitive to lysis by OncoVex mGM-CSF ) were injected subcutaneously into the right flank of female BL6 mice. Tumor volume (mm mGM-CSF ) was measured twice a week (Q2W) using a digital caliper. When the subcutaneous tumors reached an average of approximately 100 mm 3 , the animals were randomized into four groups (10 mice per group) such that the average tumor volume and tumor volume variability at the start of treatment were uniform across the treatment groups. The animals were then treated with three intratumoral injections of OncoVex 3 (5×10 mGM-CSF PFU / dose), four intraperitoneal injections of anti-CTLA-4 mAb, a combination of three intratumoral injections of OncoVex 6 (5×10 mGM-CSF PFU / dose) and four intraperitoneal injections of anti-CTLA-4 mAb, or vehicle alone. Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumor reached 800 mm 6 ) were measured two to three times a week until the end of the study. 3 reached).

[0153] OncoVex in B16F10 nectin 1 tumor-bearing micemGM-CSF or OncoVex mGM-CSF Treatment with the combination of mGM-CSF and anti-CTLA-4 mAb resulted in approximately 85% and 99% tumor (subcutaneous) growth inhibition, respectively. No inhibition was observed with anti-CTLA-4 mAb alone (Figure 13a). Evaluation of lung metastasis burden showed that the combination of OncoVex mGM-CSF and anti-CTLA-4 mAb was significantly more effective than either treatment alone in inhibiting lung metastasis (p = 0.0008 for OncoVex mGM-CSF , p = 0.0007 for anti-CTLA-4 mAb) (Figure 13b). The median survival time was significantly extended in the combination group compared to vehicle (p < 0.0001). The median survival time was 30 days for vehicle and 46 days for the OncoVex mGM-CSF + anti-CTLA-4 mAb group (Figure 13c and Table 9).

[0154]

Table 11

[0155] These results demonstrate that treatment with the combination of OncoVex mGM-CSF or OncoVex mGM-CSF and anti-CTLA-4 mAb results in potent inhibition of tumor growth in an established melanoma tumor in an in vivo mouse model. These results also show that the combination of OncoVex mGM-CSF and anti-CTLA-4 mAb demonstrates a significantly more robust systemic anti-tumor effect compared to either treatment alone, as evident from the ability of the combination to inhibit lung metastasis. Anti-tumor activity was observed in the directly injected tumor (presumably via tumor lysis and immune response) and the non-injected contralateral tumor in the same host (presumably via an adaptive immune response).

[0156] Vehicle, OncoVex mGM-CSFIn the CTLA-4 mAb group, lung tumors were also observed to rarely show scattered T cells and B cells in the tumor periphery and in mild intratumoral macrophages. Interestingly, OncoVex mGM-CSF In the combination group of mGM-CSF and anti-CTLA-4 mAb, tumors showed variable numbers of dominant T cells in the tumor periphery and T cell infiltration into the tumor. Macrophages were dominant in both dense infiltrating cells in the tumor and around the tumor. B cells remained exclusively in the tumor periphery (Figure 13d and data not shown).

[0157] These immune infiltration results of these lung tumors suggest that the combination of OncoVex mGM-CSF and anti-CTLA-4 inhibitor can convert poorly infiltrated B16F10 metastases (i.e., "cold" tumors) into well-infiltrated B16F10 metastases (i.e., "hot" tumors).

[0158] Example 8: OncoVex mGM-CSF inhibits the growth of triple-negative breast cancer tumors in a mouse model 4T1 tumor cells were subcutaneously injected into the right flank of female BALB / c mice on day 0 (2×10 6 cells). Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average of approximately 100 mm 3 , the animals were randomized into four groups (10 mice per group) such that the average tumor volume and tumor volume variation at the start of treatment were uniform across the treatment groups. Then, the animals were given three intratumoral injections of OncoVex mGM-CSF (5×10 4 , 5×10 5 or 5×10 6 PFU / dose) or vehicle. Clinical signs, body weight changes, and survival time (mice were excluded from the study if the tumors reached 800 mm 3 ) were measured two to three times a week until the end of the study.

[0159] Treatment of 4T1 tumor-bearing animals with OncoVex mGM-CSF at 5×106 resulted in approximately 75% tumor growth inhibition at the PFU / dose (p<0.0001). OncoVex mGM-CSF at 5×10 4 and 5×10 5 doses did not result in any measurable tumor growth inhibition (Figure 14).

[0160] These results demonstrate that treatment with OncoVex mGM-CSF results in a strong inhibition of tumor growth in established triple negative breast cancer tumors. Antitumor activity was measured only in the directly injected tumors.

[0161] Example 9: OncoVex mGM-CSF inhibits the growth of B cell lymphoma tumors in a mouse model either alone or in combination with a GITR agonist A20 tumor cells were subcutaneously injected (2×10 6 cells) into the right and left flanks of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using digital calipers. When the tumors reached an average of approximately 100 mm 3 , the animals were randomized into four groups (10 mice per group) such that the average tumor volume (in both flanks) and the variation in tumor volume at the start of treatment implementation were uniform across the treatment groups. The animals were then treated with OncoVex mGM-CSF or a combination of OncoVex mGM-CSF and anti-GITR mAb. Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumors reached 800 mm 3 ) were measured two to three times a week until the end of the study.

[0162] Tumors treated intraperitoneally with anti-GITR mAb were cured in 30% of the tumors (in both the injected and contralateral tumors). OncoVex mGM-CSF cured 6 out of 10 (60%) of the injected tumors while the contralateral tumors showed only a minor response without being cured. OncoVex mGM-CSFThe combination with anti-GITR mAb regressed all tumors (both in the injected tumors and the contralateral tumors), resulting in complete cures in 7 out of 10 mice. See Figure 15a. The median survival time was significantly prolonged in the anti-GITR mAb group, OncoVex mGM-CSF group and the combination group of OncoVex mGM-CSF and anti-GITR mAb compared to the vehicle. In addition, a significant prolongation in the median survival time was measured in the combination group of OncoVex mGM-CSF and anti-GITR mAb compared to either monotherapy alone (p < 0.0001 for OncoVex mGM-CSF , p = 0.039 for anti-GITR mAb). The median survival time was 24 days for the vehicle, 26 days for the OncoVex mGM-CSF group, and 43 days for the anti-GITR mAb group. The median survival time for the combination group remained undetermined beyond day 49, at which point 7 out of 10 mice showed no signs of tumors. See Figure 15b and Table 10.

[0163]

Table 12

[0164] These results demonstrate that the treatment of B-cell lymphoma with the combination of OncoVex mGM-CSF and OncoVex mGM-CSF groups and anti-GITR mAb results in a strong inhibition of tumor growth.

[0165] Example 10: A study to evaluate the combination of OncoVex muGM-CSF group and anti-PD-1 inhibition in a mouse colon (colorectal) adenocarcinoma (MC-38) tumor model This study was designed to evaluate the tolerability and antitumor activity of OncoVex muGM-CSF , anti-PD-1 inhibition or the combination of OncoVex muGM-CSF and anti-PD-1 inhibition in a mouse MC-38 tumor model.

[0166] Syngeneic MC-38 tumor cells were inoculated into both the right and left flanks of C57BL / 6 mice. When the tumors reached an average diameter of 5 mm (60 mm 3 tumor volume), the animals were assigned to six groups (n = 10 per group) on day 10 after inoculation. OncoVEX muGM-CSF (5×10 6 PFU / dose) or formulation buffer control was administered intratumorally to the right tumor once daily, three times in total every three days. The left tumor was not injected.

[0167] Anti-mouse PD-1 (clone RMP1-14, BioXCell) or isotype control antibody (rat IgG2a, clone 2A3, BioXCell) was administered intraperitoneally twice a week at either 1 mg / dose or 300 μg / dose (starting on day 10 of the study and ending on day 30 of the study (7 doses)). Tumor volume, body weight, and overall clinical observations were collected two to three times a week for both the injected (right) tumor and the non-injected (left) tumor. Animals were euthanized if the total tumor volume (right + left) reached >10% of body weight for IACUC-mandated end-of-study or if the animals showed signs of distress. Peripheral blood was collected for immunophenotyping analysis on days 14 and 20 of the study (4 days and 10 days after the start of dosing, respectively). After erythrocyte lysis, white blood cells were stained for the following markers: CD3, CD4, CD8, CD25, CD49b (NK marker), FoxP3, GITR, PD-1, and PD-L1 and analyzed by flow cytometry.

[0168] All animals survived during the experimental period and showed no evidence of harmful health effects related to treatment, as evident from body weight (Figure 16a) or survival duration, and had no noted harmful clinical signs identified by daily health monitoring examinations. The slightly lower body weight in the treatment groups compared to the control group was due to more aggressive tumor growth in the control animals compared to the treated animals, as discussed below.

[0169] Tumor growth inhibition was observed with the anti-mPD-1 antibody (at both tested doses of 300 μg and 1 mg per dose) or OncoVexmuGM-CSF were seen in response to monotherapy treatment with any of (Figure 16b). Table 11 summarizes the number of animals without tumors (regressed) at the end of the experiment on either the right (injected side) or left (non-injected side) flank. Monotherapeutic activity with any of the agents was limited to 10 - 20% complete regression in the injected tumors (and no complete regression of non-injected tumors), whereas the combination resulted in 80 - 90% regression in the injected tumors (and 10 - 20% complete regression of non-injected tumors). These data show that OncoVex muGM-CSF in combination with anti-PD-1 resulted in substantially improved tumor clearance in the mouse MC-38 tumor model.

[0170]

Table 13

[0171] These results demonstrate that OncoVex muGM-CSF inhibits the growth of colorectal MC-38 tumors either alone or in combination with a PD-1 inhibitor.

[0172] Example 11: Study to Evaluate the Combination of OncoVex muGM-CSF Group with Anti-PD-L1 Inhibition This study was designed to evaluate the tolerability and antitumor activity of OncoVex muGM-CSF , anti-PD-L1 inhibition or the combination of OncoVex muGM-CSF with anti-PD-L1 inhibition in the mouse MC-38 tumor model.

[0173] MC-38 tumor cells were subcutaneously injected into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice weekly (Q2W) using a digital caliper. The tumors averaged approximately 100 mm 3Once reached, the animals were randomized into four groups (10 mice per group) such that the mean tumor volume and the variation in tumor volume at the start of treatment implementation were uniform across all treatment groups. OncoVex mGM-CSF (5×10 6 PFU / dose) or formulation buffer control alone or in combination with anti-PD-L1 mAb (clone MIH5, mouse IgG1) or control IgG1 (the mAb was administered a total of 4 times), and a total of 3 injections were administered intratumorally every 3 days. The non-injected tumors (contralateral; on the left side of the animal) did not receive the injection. Clinical signs, body weight changes, and survival time (when the tumor reached 800 mm 3 , the mice were excluded from the study) were measured 2 - 3 times per week until the end of the study.

[0174] All animals survived during the experimental period and showed no evidence of harmful health effects related to treatment, as evident from body weight, and had no noted harmful clinical signs identified by daily health monitoring examinations.

[0175] Tumor growth inhibition was seen in response to monotherapy treatment with either OncoVex mGM-CSF or anti-PD-L1 mAb in both the injected tumors and the contralateral tumors (Figure 17), while the combination of OncoVex mGM-CSF and anti-PD-L1 mAb regressed all of the injected tumors and 7 out of 10 injected tumors.

[0176] Example 12: Study to Evaluate the Combination of OncoVex muGM-CSF Group with Anti-PD-1 Inhibition in a Mouse Melanoma (B16F10) Tumor Model This study was designed to evaluate the tolerance and antitumor activity of OncoVex muGM-CSF , anti-PD-1 inhibition, or the combination of OncoVex muGM-CSF and anti-PD-1 inhibition in a mouse melanoma (B16F10) tumor model.

[0177] B16F10 tumor cells engineered to express mNectin were subcutaneously injected into the right flank of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice weekly (Q2W) using a digital caliper. When the tumors reached an average of approximately 100 mm 3 , the animals were randomized into four groups (10 mice per group) such that the average tumor volume and tumor volume variability at the start of treatment were uniform across the treatment groups. OncoVex mGM-CSF (5×10 6 PFU / dose) or formulation buffer control was administered intratumorally three times every three days, either alone or in combination with anti-PD-1 mAb (clone 29F1A12, mouse IgG1) or control IgG1 (the mAb was administered a total of four times). Clinical signs, body weight changes, and survival time (mice were excluded from the study if the tumors reached 800 mm 3 ) were measured two to three times weekly until the end of the study.

[0178] All animals survived during the experimental period and showed no evidence of treatment-related adverse health effects as evident from body weight and had no noted adverse clinical signs identified by daily health monitoring examinations.

[0179] Tumor growth inhibition was seen in response to monotherapy treatment with OncoVex mGM-CSF monotherapy alone (3 out of 10 mice showed tumor regression), while anti-PD-1 mAb monotherapy had no inhibitory effect on tumor growth (Figure 18). The combination of OncoVex mGM-CSF and anti-PD-1 mAb regressed 5 out of 10 injected tumors (Figure 18), demonstrating that the combination of OncoVex mGM-CSF and anti-PD-1 mAb had superior antitumor activity compared to monotherapy.

[0180] Example 13: A Phase 1, Multicenter, Open-Label, Dose De-escalation Trial to Evaluate the Safety and Efficacy of Talimogene Laherparepvec in Pediatric Subjects with Progressive Non-CNS Tumors that are Easy to Inject This example describes a Phase 1, multi-center, open-label, dose-escalation study to evaluate the safety and efficacy of talimogene laherparepvec in pediatric subjects with progressive non-central nervous system tumors that are amenable to direct injection. See the U.S. National Institutes of Health website (clinicaltrials.gov), trial identifier: NCT 02756845 (which is incorporated herein by reference).

[0181] The primary objective of this study is to determine the safety and efficacy of talimogene laherparepvec, as evaluated by the occurrence of dose-limiting toxicity (DLT), in pediatric subjects with progressive non-CNS tumors that are amenable to direct injection.

[0182] Talimogene laherparepvec will be administered to approximately 18 to 36 pediatric subjects with progressive non-CNS tumors that are amenable to direct injection. Pediatric subjects will be enrolled overall into cohorts stratified by age and baseline herpes simplex virus type 1 (HSV-1) serostatus (3 to 6 subjects / cohort). DLT will be evaluated based on 3 to 6 DLT-evaluable subjects in that cohort.

[0183] The tumor evaluation item is to determine the safety and efficacy of talimogene laherparepvec, as evaluated by the occurrence of dose-limiting toxicity (DLT), in pediatric subjects with progressive non-central nervous system (CNS) tumors that are amenable to direct injection.

[0184] The secondary evaluation items are: (1) evaluating talimogene laherparepvec by overall response rate (ORR), duration of response (DOR), time to first response (TTR), time to progression (TTP), progression-free survival (PFS) and overall survival (OS) using immune-related response criteria simulating response evaluation criteria in solid tumors (irRC-RECIST) in modified solid tumors, and (2) evaluating the relationship between granulocyte macrophage colony-stimulating factor (GM-CSF) receptor / subunit in archival tumor tissue and clinical outcomes (safety endpoints and efficacy endpoints such as ORR, DOR, TTR, TTP, PFS and OS).

[0185] Selection Criteria · If the subject is legally too young to provide informed consent / assent, the subject's legally recognized proxy provides informed consent / assent, and the subject has been provided with assent written in accordance with regulations and / or guidelines before any study-specific activities / treatments are initiated. · The subject is male or female and is between 0 and less than 18 years old at the time of informed consent / assent. · Should be willing to submit local HSV-1 serostatus within 28 days prior to enrollment. · Is a histologically or cytologically confirmed non-CNS solid tumor that has recurred after standard treatment or for which no standard treatment is available. · There are measurable (as defined by irRC-RECIST) or non-measurable lesions as defined by modified irRC-RECIST. · Subjects who are candidates for intralesional injection defined as one or more of the following: - At least one injectable lesion with a longest diameter of ≥10 mm - Multiple injectable lesions with an overall longest diameter of ≥10 mm Note: The internal lesions are not the injection targets. Further, bone lesions are not the injection targets unless there is a soft tissue component that is easy to inject. · Performance status: - For those aged 12 to less than 18 years, Karnofsky performance status is ≥ 70% - For children aged 0 to less than 12 years, Lansky play scale is ≥ 70% · The average remaining life is > 4 months from the registration date and time · There is sufficient organ function defined as follows: - Blood function (without the need for hematopoietic growth factors) Absolute neutrophil count (ANC) is ≥ 1.0×10 9 / L Platelet count is ≥ 75×10 9 / L Hemoglobin is ≥ 8 g / dL of hemoglobin (without blood transfusion support) - Renal function Serum creatinine is ≤ 1.5× the upper limit of normal value (ULN) of age, or for subjects with a creatinine level > 1.5× the ULN of age, creatinine clearance is ≥ 60 mL / min / 1.73 m 2 (Note that creatinine clearance does not need to be determined when the baseline serum creatinine is ≤ 1.5× the ULN of age. Creatinine clearance should be determined according to the in-facility standards). - Liver function Serum bilirubin is ≤ 1.5× the ULN of age, or for subjects with a total bilirubin level > 1.5× the ULN of age, direct bilirubin is ≤ the ULN of age Aspartate aminotransferase (AST) is ≤ 2.5× the ULN of age, or for subjects with liver metastasis, ≤ 5× the ULN of age Alanine aminotransferase (ALT) is ≤ 2.5× the ULN of age, or for subjects with liver metastasis, ≤ 5× the ULN of age - Coagulation function The international normalized ratio (INR) or prothrombin time (PT) is ≤ 1.5 × the upper limit of normal (ULN) for age The partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT) is ≤ 1.5 × the ULN for age · Women of childbearing potential must have a negative urine or serum pregnancy test within 72 hours before dosing. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required

[0186] Exclusion criteria · Diagnosis of leukemia, non-Hodgkin lymphoma, Hodgkin disease or other hematologic malignancies · Radiation therapy to the bone marrow within 6 weeks before enrollment or within 3 months before enrollment if prior radiation to at least 60% of the craniospinal axis or pelvis was received within 6 weeks before enrollment, or within 2 weeks before enrollment if palliative local radiation therapy was received · Presence of a CNS tumor or clinically active brain metastases · Presence of primary intraocular melanoma or mucosal melanoma · History or evidence of giant congenital melanocytic nevi, dysplastic nevus syndrome or xeroderma pigmentosum · History of other malignancies within the past 5 years, except for the following exceptions: - Malignancies treated for curative purposes, without known active disease, not receiving chemotherapy for > 5 years before enrollment, and for which the treating physician feels the risk of recurrence is low · History or evidence of active autoimmune disease requiring systemic treatment (i.e., using disease-modifying agents, corticosteroids or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment · Clinically significant immunosuppression as follows: - Primary immunodeficiencies such as severe combined immunodeficiency - Concurrent opportunistic infections - Receiving systemic immunosuppressive therapy including oral steroid administration (more than 2 weeks before enrollment) (excluding maintenance physiological replacement therapy). Subjects who require discontinuous use of steroids for inhalation or local steroid injection are not excluded from this study. · Having active herpes skin lesions or a history of previous herpes infection complications (e.g., herpes keratitis or herpes encephalitis) · Having previously received treatment with talimogene laherparepvec or any other oncolytic virus · Having previously received treatment with a tumor vaccine · Requiring discontinuous or chronic treatment with anti-herpes drugs (e.g., acyclovir) other than discontinuous topical use · Having received chemotherapy, radiotherapy, or biological cancer therapy within 28 days before enrollment, or not having recovered to grade 1 or higher of the Common Terminology Criteria for Adverse Events (CTCAE) from adverse events caused by cancer therapy administered more than 28 days before enrollment · Currently receiving treatment with another investigational medical device or drug, or less than 28 days since the end of treatment with another investigational medical device or drug. Participating in this study while receiving other investigational treatments is excluded. · Having undergone major surgery within ≤28 days before enrollment · Anticipating the need for other cancer therapies during this study, except for local palliative radiotherapy · Having acute or chronic active hepatitis B virus or hepatitis C virus infection, or having received treatment with nucleotide analogs such as those used in the treatment of hepatitis B virus (e.g., lamivudine, adefovir, tenofovir, telbivudine, and entecavir), ribavirin, or interferon alpha within 12 weeks from the start of the test treatment · Known or suspected human immunodeficiency virus (HIV) infection · Having received a live vaccine within 28 days before enrollment ·No antiplatelet or anticoagulant drugs were permitted within 7 days prior to the injection of talimogene laherparepvec, except for the low-dose heparin necessary to maintain the patency of the venous catheter. ·Female subjects are pregnant, breastfeeding, or planning to become pregnant during the test procedure and for 3 months after the last administration of talimogene laherparepvec. ·Female subjects of childbearing potential who do not wish to use an effective and acceptable method of contraception during the test procedure and for 3 months after the last administration of talimogene laherparepvec. Note: Effective and acceptable methods of contraception are defined in the Informed Consent / Assent Form. Where required by regulations and rules, additional country-specific contraceptive requirements are outlined in the protocol addendum for the country at the end of the appendix section of the protocol. ·Sexually active subjects and their partners who do not wish to use male or female latex condoms to avoid viral transmission during sexual contact during the treatment and within 30 days after treatment with talimogene laherparepvec. ·Subjects known to be sensitive to any of the administered formulations or components during dosing. ·Subjects who are unable to complete the required hospital visits or treatments during the test as required by all protocols and / or are likely unable to comply with all required test procedures to the extent known to the subject and the investigator. ·In the opinion of the investigator or an Amgen physician, if the subject is participating in the consultation, there is a history or evidence of any mental disorder, substance abuse, or any other clinically significant disorder, condition, or disease (excluding those outlined above) that is thought to pose a risk to the subject's safety or interfere with the test evaluation, treatment, or completion. ·Subjects who do not wish to minimize the exposure of their blood or body fluids to individuals at higher risk of HIV-1-induced complications (immunocompromised individuals, HIV-positive individuals, pregnant women, or infants under 1 year of age) during the talimogene laherparepvec treatment and for 28 days after the last administration of talimogene laherparepvec.

[0187] Based on the definitions of the selection criteria and exclusion criteria, talimogene laherparepvec will be administered by intralesional injection only into injectable skin, subcutaneous, lymph node, and other non-implanted tumors. Therefore, the eligible tumor types expected for this trial are as follows. · Bone sarcomas: Ewing sarcoma and osteosarcoma · Soft tissue sarcomas: Rhabdomyosarcoma and non-rhabdomyosarcoma soft tissue sarcomas · Neuroblastoma · Melanoma

[0188] The first dose of talimogene laherparepvec is 10 6 PFU / mL up to a maximum of 4.0 mL, administered on Day 1. 10 8 PFU / mL up to a maximum of 4.0 mL (or 10 6 PFU / mL up to a maximum of 4.0 mL for the dose de-escalation cohort) of the second injection is administered on Day 21 (+3) after the first injection (i.e., on or after Day 22, but not delayed more than 3 days from Day 21). 10 8 PFU / mL up to a maximum of 4.0 mL (or 10 6 PFU / mL up to a maximum of 4.0 mL for the dose de-escalation cohort) of all subsequent injections will be administered every 14 (±3) days. The treatment cycle interval can be extended due to toxicity. At any dose, the maximum amount of talimogene laherparepvec administered is 4.0 mL for any individual lesion and in any treatment. The recommended amount of talimogene laherparepvec injected into the tumor will be determined according to the size of the tumor, following the injection volume guidelines in Table 12. Using the following priority model and injection volume guidelines based on tumor size, each lesion should receive the maximum amount that can be injected due to the tumor characteristics at each visit before moving on to the next lesion.

[0189]

Table 14

[0190] On each treatment date, the injection priority is recommended as follows. 1. Any new injectable tumor that has appeared since the previous injection 2. Start with the largest tumor by tumor size 3. Any previously seen tumor that was too small to inject at the time of tumor evaluation but is now large enough to inject

[0191] The subject is administered talimogene laherparepvec until the subject achieves a complete response (CR), there are no injectable tumors remaining, disease progression (PD) is confirmed according to modified irRC-RECIST, the tolerance of the study treatment deteriorates, 24 months have elapsed since the date of the first administration of talimogene laherparepvec, and alternative anticancer therapy or termination of the study is required at the earlier of either. Due to the mechanism of action, the subject may experience growth of existing tumors or the appearance of new tumors before the maximum clinical utility of talimogene laherparepvec. Therefore, modified irRC-RECIST will be used for response assessment.

Claims

1. A method of treating Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B cell lymphoma (e.g., diffuse large B cell lymphoma), non-small cell lung cancer, colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple negative breast cancer), cutaneous T cell lymphoma, or multiple myeloma by administering a therapeutically effective amount of an oncolytic virus.

2. The method of claim 1 , wherein the oncolytic virus is a herpes simplex virus.

3. The method of claim 1 or 2, wherein the oncolytic virus is talimogene laherparepvec.

4. (i) a therapeutically effective amount of an oncolytic virus; (ii) a therapeutically effective amount of a checkpoint inhibitor; A method of treating B cell lymphoma, colorectal cancer, head and neck squamous cell carcinoma, or breast cancer (e.g., triple negative breast cancer) by administering

5. 5. The method of claim 4, wherein the checkpoint inhibitor is CTLA-4, PD-L1 or a PD-L1 blocker.

6. The method of claim 4 or 5, wherein the oncolytic virus is a herpes simplex virus.

7. 7. The method of claim 6, wherein the herpes simplex virus is talimogene laherparepvec.

8. A therapeutically effective amount of an oncolytic virus for use in the treatment of Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B cell lymphoma (e.g., diffuse large B cell lymphoma), non-small cell lung cancer, colorectal cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple negative breast cancer), cutaneous T cell lymphoma or multiple myeloma.

9. 1. A pharmaceutical composition for use in a method for treating Ewing's sarcoma, neuroblastoma, rhabdomyosarcoma-like tumor, osteosarcoma, rhabdomyosarcoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, colorectal cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, gastric cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma or multiple myeloma, comprising an oncolytic virus.

10. The oncolytic virus according to claim 8 or 9, which is a herpes simplex virus.

11. The oncolytic virus of claim 10, wherein the herpes simplex virus is talimogene laherparepvec.

12. A therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor for use in the treatment of B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, head and neck squamous cell carcinoma or breast cancer (e.g., triple negative breast cancer).

13. A pharmaceutical composition for use in a method for treating B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, head and neck squamous cell carcinoma or breast cancer (e.g., triple-negative breast cancer), comprising a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor.

14. The checkpoint inhibitor of claim 12 or 13, which is a CTLA-4, PD-1 or PD-L1 blocker.

15. 15. The oncolytic virus of claim 13 or 14, which is a herpes simplex virus.

16. 16. The oncolytic virus of claim 15, wherein the herpes simplex virus is talimogene laherparepvec.

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