Use of oncolytic viruses alone or in combination with checkpoint inhibitors to treat cancer

Abstract

To provide effective cancer therapies with diminished side effects and cancer therapies effective against metastatic cancers.SOLUTION: The invention relates to the use of an oncolytic virus (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 an antibodies) for the treatment of various types of cancer. In addition, the present invention relates to compositions and kits relating to such a use of an oncolytic virus, 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 under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 471,875, filed March 15, 2017, which is incorporated herein by reference. [Background technology]

[0002] In light of advances in cancer treatment over the past several decades, cancer mortality rates for the most common types of cancer, such as lung, colon, breast, and prostate, continue to decline for both men and women. This improvement in survival is likely the result of advances in diagnosing certain cancers at earlier stages, improved treatments, and public health efforts that encourage preventive measures and screening.

[0003] Nonetheless, cancer remains a major public health problem with over 16 million people diagnosed each year. In addition, a cancer diagnosis has serious consequences for patients and their families and friends. In fact, in the United States, cancer remains the second leading cause of death (surpassed only by heart disease) and accounts for nearly 1 in 4 deaths. See progressreport.cancer.gov / introduction, accessed March 8, 2017.

[0004] The desired goal of cancer therapy is the preferential killing of cancer cells without adversely affecting normal cells. Several methods have been used in an attempt to achieve this goal, including surgery, radiation therapy, chemotherapy, and oncolytic virus therapy.

[0005] Localized treatments such as radiation therapy and surgery provide a way to reduce tumor burden in areas of the body that are accessible through surgical techniques or high-dose radiation therapy. However, the primary approach to cancer treatment is chemotherapy. However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. This failure is due in part to drug resistance (whether acquired or intrinsic) in many tumor cells. A significant obstacle to the use of chemotherapeutic agents are their severe side effects. These include bone marrow suppression, 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 anticancer activity. 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 require a helper virus to allow integration and / or replication in host cells. These viruses are limited in their effectiveness because each replication-deficient retroviral particle can only enter a single cell and cannot subsequently productively infect other cells. Thus, they cannot spread away from producer cells and cannot fully invade many tumor cells in vivo. More recently, engineering of oncolytic viruses has focused on the generation of "replication-restricted" viruses in an attempt to avoid systemic infection while allowing the virus to spread to other tumor cells.

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

[0008] Therapeutic combinations of oncolytic viruses with checkpoint inhibitors are being explored. For example, the combination of talimogene laherparepvec with immunotherapy (e.g., ipilimumab and pembrolizumab) is currently being explored in clinical trials for melanoma (NCT01740297 and NCT02263508) and head and neck squamous cell carcinoma (NCT02626000). Checkpoint inhibitors such as ipilimumab (CTLA-4 antibody), pembrolizumab, and nivolumab (anti-PD-1 antibody) and atezolizumab (anti-PD-L1 antibody) have demonstrated efficacy in a variety of 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 literature]

[0009] [Non-Patent Document 1] Lie et al.,Gene Ther.,10:292-303,2003 [Non-Patent Document 2] Grosso et al., Cancer Immun.,13:5(2013);Pardoll, Nat Rev Cancer,12:252-264(2012) [Non-Patent Document 3] Chen et al.,Immunity,39:1-10(2013) Summary of the Invention [Problem to be solved by the invention]

[0010] However, there remains a need to further develop effective cancer therapies that have 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 problem]

[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 a metastatic cancer. In some embodiments, the oncolytic virus is a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the 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 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.

[0012] The present invention also relates to a method of 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 a herpes simplex virus. The herpes simplex virus may be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. The herpes simplex virus type 1 may 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 yet other embodiments, the 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 invention also relates to a method of treating B-cell lymphoma, colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of an 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 invention relates to a method of treating B-cell lymphoma, colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of an 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 another embodiment, the invention relates to a method of treating B-cell lymphoma, colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective amount of an 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 WO2015031667 and U.S. Patent No. 9,464,139, both of which are incorporated by reference in their entirety. In some embodiments, the oncolytic virus is a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified (i) such that 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 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.

[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 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, 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, 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, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, the herpes simplex virus type 1 is also modified such that (i) it does not contain an intact ICP34.5 gene, and (ii) it does not contain an intact ICP47 gene. In yet other embodiments, the 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 invention relates to a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor for use in treating B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple-negative breast cancer). In some embodiments, the cancer is metastatic cancer. In another embodiment, the 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, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple-negative breast cancer), comprising a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor. In such an embodiment, the checkpoint inhibitor is a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody). In a particular embodiment, 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 a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, the herpes simplex virus type 1 is also modified such that (i) it does not contain an intact ICP34.5 gene, and (ii) it does not contain an intact ICP47 gene. In yet other embodiments, the 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] Additionally, 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) for use in the treatment of B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, 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) for use in the treatment of B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, 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. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, the herpes simplex virus type 1 is also modified such that (i) it does not contain an intact ICP34.5 gene, and (ii) it does not contain an intact ICP47 gene. In yet other embodiments, the 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 invention relates to a pharmaceutical composition for use in a method for 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), 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 for treating B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple negative breast cancer), 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 for treating B cell lymphoma (e.g., diffuse large B cell lymphoma), colorectal cancer, melanoma, squamous cell carcinoma of the head and neck, or breast cancer (e.g., triple negative breast cancer), 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 a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, the herpes simplex virus type 1 is also modified such that (i) it does not contain an intact ICP34.5 gene, and (ii) it does not contain an intact ICP47 gene. In yet other embodiments, the 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 a particular embodiment, the oncolytic virus is talimogene laherparepvec. [Brief description of the drawings]

[0019] [Figure 1] Figure 2 shows the effect of intratumoral administration of talimogene laherparepvec on tumor volume of A-673 Ewing sarcoma in Balb / c nude mice. [Diagram 2] 1 shows the effect of intratumoral administration of talimogene laherparepvec on tumor volume of SK-N-AS neuroblastoma in Balb / c nude mice. [Diagram 3] 1 shows the effect of intratumoral administration of talimogene laherparepvec on the tumor volume of G-401 rhabdomyosarcoma-like tumors in Balb / c nude mice. [Figure 4] Figure 2 shows the effect of intratumoral administration of talimogene laherparepvec on tumor volume of SJSA-1 osteosarcoma in Balb / c nude mice. [Diagram 5] 1 shows the effect of intratumoral administration of talimogene laherparepvec on tumor volume of SJCRH30 rhabdomyosarcoma in Balb / c nude mice. [Figure 6] 1 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines. [Figure 7] 1 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in HCT-116 (colorectal) and SK-MEL-5 (melanoma) cell lines. [Figure 8] 1 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in HUT-78 (CTCL) and RPMI 8226 (multiple myeloma) cell lines. [Figure 9] 1 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in CT-26 and MC-38 (colorectal) cell lines. [Figure 10a] 1 shows the effect of OncoVex mGM-CSF on injected tumor volume in A20 tumor-bearing animals treated with three doses: 3×10 4 PFU, 3×10 5 PFU, and 3×10 6 PFU. [Figure 10b] FIG. 1 shows the effect of OncoVex mGM-CSF on the volume of non-injected (contralateral) tumors in A20 tumor-bearing animals treated with three doses: 3×10 4 PFU, 3×10 5 PFU, and 3×10 6 PFU. [Figure 10c] 1 shows the effect of OncoVex mGM-CSF on the median survival of A20 tumor-bearing animals treated with three doses: 3×10 4 PFU, 3×10 5 PFU and 3×10 6 PFU. [Figure 10d] 1 shows the effect of administration of OncoVex mGM-CSF, anti-CTLA-4 mAb, and the combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on mouse body weight. [Figure 10e] 1 shows the effect of administration of OncoVex mGM-CSF, anti-CTLA-4 mAb, and the combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on mouse body weight. [Figure 10f] 1 shows the effect of OncoVex mGM-CSF on injected tumor volume in neuro2a neuroblastoma tumor-bearing mice treated with three doses: 5×10 4 PFU, 5×10 5 PFU, and 5×10 6 PFU. [Figure 10g] FIG. 1 shows the effect of OncoVex mGM-CSF on median survival of neuro2a neuroblastoma tumor-bearing mice treated with three doses: 5×10 4 PFU, 5×10 5 PFU, and 5×10 6 PFU. [Figure 10h] FIG. 1 shows the effect of OncoVexmGM-CSF on injected (treated) and non-injected (contralateral / untreated) tumor volumes in neuro2a neuroblastoma tumor-bearing mice treated with 5×10 6 PFU. [Figure 10i] 1 shows the effect of OncoVexmGM-CSF on median survival of neuro2a neuroblastoma tumor-bearing mice treated with 5×10 6 PFU. [Figure 11a] FIG. 1 shows the effect of treatment of A20 tumor-bearing animals with OncoVex mGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on the volumes of directly injected and non-injected (contralateral) tumors. [Figure 11b] FIG. 1 shows the effect of treatment of A20 tumor-bearing animals with OncoVex mGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on median mouse survival. [Figure 11c] Figure 2 shows the effect of 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, on the volume of directly injected and non-injected (contralateral) tumors. [Figure 11d] Figure 2 shows the effect of 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 on median survival of mice. [Figure 12a] FIG. 1 shows the effect of treatment of CT-26 tumor-bearing animals with OncoVex mGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on the volume of directly injected and non-injected (contralateral) tumors. [Figure 12b] FIG. 1 shows the effect of treatment of CT-26 tumor-bearing animals with OncoVex mGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVex mGM-CSF and anti-CTLA-4 mAb on median mouse survival. [Figure 12c] Figure 2 shows the effect of treatment of CT-26 tumor-bearing animals with OncoVex mGM-CSF, anti-PD-L1 mAb, or the combination of OncoVex mGM-CSF and anti-PD-L1 mAb on the volumes of directly injected and non-injected (contralateral) tumors. [Figure 12d]Figure 2 shows the effect of treatment of CT-26 tumor-bearing animals with OncoVex mGM-CSF, anti-PD-L1 mAb, or the combination of OncoVex mGM-CSF and anti-PD-L1 mAb on median survival of mice. [Figure 12e] Quantification of systemic (splenic) anti-AH1 CD8+ T cells by ELISpot or by dextramer staining using FACS in CT-26 tumor-bearing mice treated with OncoVex mGM-CSF, CTLA-4 inhibitor, or a combination of OncoVex mGM-CSF and CTLA-4 inhibitor. [Figure 12f] Quantification of systemic (splenic) anti-AH1 CD8+ T cells by ELISpot or by dextramer staining using FACS in CT-26 tumor-bearing mice treated with OncoVex mGM-CSF, CTLA-4 inhibitor, or a combination of OncoVex mGM-CSF and CTLA-4 inhibitor. [Figure 12g] Quantification of local (tumor) anti-AH1 CD8+ T cells in CT-26 tumor-bearing mice treated with OncoVex mGM-CSF, CTLA-4 inhibitor, or a combination of OncoVex mGM-CSF and CTLA-4 inhibitor. [Figure 13a] FIG. 1 shows the effect of treatment of B16F10 Nectin1 tumor-bearing mice treated with control, OncoVex mGM-CSF, CTLA-4 inhibitor or a combination of OncoVex mGM-CSF and CTLA-4 inhibitor on the volume of injected tumors. [Figure 13b] 1 shows an assessment of pulmonary metastatic burden (as evidenced by the number of pulmonary metastases) in tumor-bearing mice following treatment with control, OncoVex mGM-CSF, CTLA-4 inhibitor or a combination of OncoVex mGM-CSF and CTLA-4 inhibitor. [Figure 13c] The effect of treatment with control or the combination of OncoVexmGM-CSF and a CTLA-4 inhibitor on the median survival of tumor-bearing mice is shown. [Figure 13d]We show that after treatment with control, OncoVex mGM-CSF, CTLA-4 inhibitor, or the combination of OncoVex mGM-CSF and CTLA-4 inhibitor, macrophages were prominent both within the tumor and within the dense cellular infiltrate at the tumor periphery, while B cells remained exclusively at the tumor periphery. [Figure 14] FIG. 1 shows the effect of treatment of 4T1 tumor-bearing mice with control or OncoVexmGM-CSF on the volume of injected tumors. [Figure 15a] FIG. 1 shows the effect of treatment of A20 tumor-bearing animals with OncoVex mGM-CSF, anti-GITR mAb, or a combination of OncoVex mGM-CSF and anti-GITR mAb on the volume of directly injected and non-injected (contralateral) tumors. [Figure 15b] FIG. 1 shows the effect of treatment of A20 tumor-bearing animals with OncoVex mGM-CSF, anti-GITR mAb or a combination of OncoVex mGM-CSF and anti-GITR mAb on median survival of mice. [Figure 16a] Shows the effect of administration of OncoVex mGM-CSF, anti-PD-1 mAb, and the combination of OncoVex mGM-CSF and anti-PD-1 mAb on mouse body weight. [Figure 16b] Figure 2 shows the effect of treatment of MC-38 tumor-bearing animals with OncoVex mGM-CSF, anti-PD-1 mAb, or the combination of OncoVex mGM-CSF and anti-PD-1 mAb on the volume of directly injected and non-injected (contralateral) tumors. [Figure 17] Figure 2 shows the effect of treatment of MC-38 tumor-bearing animals with OncoVex mGM-CSF, anti-PD-L1 mAb, or the combination of OncoVex mGM-CSF and anti-PD-L1 mAb on the volume of directly injected and non-injected (contralateral) tumors. [Figure 18] 1 shows the effect of treatment of B16F10 tumor-bearing animals with OncoVex mGM-CSF, anti-PD-1 mAb, or the combination of OncoVex mGM-CSF and anti-PD-1 mAb on the volume of directly injected tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0021] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques can be generally performed 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. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3rd ed., Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature used in connection with and laboratory procedures and techniques of analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery and treatment of patients.

[0022] Oncolytic viruses As discussed herein, the present invention demonstrates that oncolytic viruses can provide antitumor effects in various tumor types, either alone or in combination with checkpoint inhibitors. A notable benefit of the oncolytic viruses of the present invention is that the antitumor effects are accompanied by less severe / negative side effects, for example, compared to 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 to treat 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 another embodiment, the invention relates to the use of a combination of an oncolytic virus and a checkpoint inhibitor to treat 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 a herpes simplex virus. The herpes simplex virus can be herpes simplex virus type 1. In some embodiments, the herpes simplex virus type 1 is modified such that (i) it does not contain an intact ICP34.5 gene. In other embodiments, the 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 yet other embodiments, the 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-CSFHSV-1 was derived from the JS1 strain deposited with the European Cell Culture Authority (ECAAC) under registration number 01010209. In talimogene laherparepvec, the HSV-1 viral gene encoding ICP34.5 is functionally deleted. Functional deletion of ICP34.5, which acts as a virulence factor during HSV infection, restricts replication in non-dividing cells and renders the virus non-pathogenic. The safety of HSV with a functional deletion of 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, has been functionally deleted from talimogene laherparepvec. Functional deletion of ICP47 also results in early expression of US11, a gene that promotes viral growth in tumor cells, without reducing tumor selectivity. As used herein, "lacking a functional viral gene" means that a gene has been partially or completely deleted, replaced, rearranged, or otherwise altered in the herpes simplex virus genome such that a functional viral protein can no longer be expressed from that gene by the herpes simplex virus. The coding sequence for human GM-CSF, a cytokine involved in stimulating the immune response, has been inserted into the talimogene laherparepvec viral genome (at two previous sites in the ICP34.5 gene).The insertion of the gene encoding human GM-CSF was made so that it replaces almost all of the ICP34.5 gene, ensuring that any potential recombination events between talimogene laherparepvec and wild-type virus can only result in a disabled, non-pathogenic virus, and cannot result in the generation of wild-type virus carrying the gene for human GM-CSF. The HSV thymidine kinase (TK) gene remains intact in talimogene laherparepvec, which makes the virus sensitive to antiviral agents such as acyclovir. Acyclovir can therefore be used to block talimogene laherparepvec replication, if necessary.

[0025] Examples of additional HSV genes that may 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. The immediate early genes encoding thymidine kinase, which is responsible for the phosphorylation of acyclovir to acyclovir monophosphate, the virion transactivator protein vmw65, glycoprotein H, vhs, ICP43 and ICP4, ICP27, ICP22, and / or ICP0 may also be modified.

[0026] Modifications can also be made to change 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 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 will be appreciated 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 induction of an antitumor immune response that is enhanced by a direct oncolytic effect due to viral replication in the tumor and by the release of tumor-derived antigens via local expression and lysis of GM-CSF. Since many cancers present in patients as primary and secondary (i.e. metastatic) tumors, this dual activity is beneficial as a therapeutic treatment. The intended clinical effects include destruction of the injected tumor, destruction of localized locoregional and distant non-injected tumors, reduction in the formation of new metastases, reduction in the overall rate of progression and recurrence rate after treatment of the initially existing disease, and increased overall survival.

[0029] As used herein, the terms "patient" or "subject" are used interchangeably and refer to a mammal, including, but not limited to, a human, a non-human mammal, such as a cow, a horse, a dog, a sheep, or a cat. Preferably, the patient is a human.

[0030] Talimogene-Laherparepvec and OncoVex mGM-CSF (HSV-1 virus with the same genetic modifications 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 generated immune response, enhancing the response of both injected and non-injected tumors, and that increased surface levels of MHC class I molecules result from the deletion of ICP47. Talimogene laherparepvec has been injected into normal and tumor-bearing mice to evaluate its safety. Overall, the virus has been well tolerated, with up to 1 × 10 8 The dose of PFU / dose did not pose any indication of safety concerns (see, e.g., Liu et al., Gene Ther 10:292-303, 2003).

[0031] Clinical trials have been or are currently being conducted in several advanced tumor types, with over 400 subjects being 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 benefit for patients with advanced melanoma. In particular, a high rate of complete responses was achieved in stage IIIc-IV melanoma (Scenzer et al., J. Clin. Oncol. 271(12):907-913, 2009). In addition, responses were observed at both injected and non-injected sites, including visceral sites.

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

[0033] The herpes simplex virus strain may be derived from a clinical isolate. Such a strain is isolated from an infected individual, such as one with 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), the clinical isolate may be screened for desired capabilities or characteristics, such as enhanced replication in tumors and / or other cells in vitro and / or in vivo, compared to standard laboratory strains. In one embodiment, the herpes simplex virus is a clinical isolate from recurrent herpes simplex.

[0034] Herpes simplex virus type 1 strains include, but are not limited to, JS1 strain, 17+ strain, F strain, KOS strain, and 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 a BamHI fragment (0 to 0-02 and 0-81 to 0.83 map units) of the long repeat region of the HSV genome, removing one complete copy from the 18 bp DR through the "a" sequence element, terminating 1105 bp upstream of the 5' end of 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 F strain of wild-type HSV-1 with a deletion of both copies of the ICP34.5 gene, a major determinant of HSV neurovirulence, and an inactivating insertion of the 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 with a deletion 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 has been deleted, including one copy of ICP34.5, UL24, and UL56.34, 35. The deleted region was 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 a deletion 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) with functional deletions of the genes encoding vhs, ICP47, ICP34.5, UL43 and US5.

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

[0042] Additional examples of modified herpes simplex viruses include G47delta, G47delta 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 for producing such strains are described in U.S. Pat. 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, WIP and in WO 199600007, WO 199639841, WO 199907394, WO 200054795, WO 2006002394, WO 201306795, Chinese Patents Nos. 128303, 10230334 and 10230335, 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 control certain types of immune system cells, such as T cells (which play a central role in cell-mediated immunity). Although immune checkpoints help to suppress immune responses, they can also stop T cells from killing cancer cells. Immune checkpoint inhibitors (or simply "checkpoint inhibitors") can block immune checkpoint protein activity, releasing the "brakes" on the immune system and allowing T cells to better kill cancer cells.

[0045] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely 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 along with 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 T cell responses. Immune checkpoint proteins control and maintain the duration and amplitude of self-tolerance and physiological immune responses. Immune checkpoint inhibitors can include or be derived from antibodies.

[0046] Checkpoint inhibitors may include small molecule inhibitors, or may include 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 may 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, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also called BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 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, other binding proteins, biotherapeutics or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049.

[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 to enhance anti-tumor immune response to tumor antigens released after cytolytic replication of the virus in tumors. Thus, the combination of herpes simplex virus and anti-CTLA-4 antibody can increase the destruction of injected tumors and non-injected / distant tumors, improve overall tumor response, and increase overall survival, especially when the increase in overall survival is 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 expressed on T cells and pro-B cells and plays a role in their fate / differentiation. PD-1's two ligands, PD-L1 and PD-L2, are members of the B7 family. PD-1 limits the activity of T cells in peripheral tissues during inflammatory responses to infection, and limiting autoimmunity PD-1 blockade in vitro enhances T cell proliferation and cytokine production in response to challenge with specific antigen targets or with allogeneic cells in a mixed lymphocyte reaction. A strong correlation between PD-1 expression and response has been shown with PD-1 blockade (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). PD-1 blockade can be achieved by a variety of mechanisms, including antibodies that bind to PD-1 or PD-L1.

[0049] Programmed cell death-ligand 1 (PD-L1), also called cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene. See Entrez Gene:CD274 CD274 molecule. PD-L1 is a 40 kDa type 1 transmembrane protein that plays a role in suppressing the immune system and binds to its receptor (PD-1) 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 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 invention relates to a pharmaceutical composition comprising a combination of an oncolytic virus and a checkpoint inhibitor.

[0052] Thus, in one aspect of the 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. Pat. 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 of which is incorporated herein by reference).In some embodiments, the checkpoint inhibitor is a PD-L1 or PD-1 blocker or inhibitor (e.g., a molecule that inhibits the interaction of PD-1 with PD-L1 and / or a PD-L2 inhibitor), such as pembrolizumab (anti-PD-1 antibody), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 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-30 1 (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), BMS 936558 (anti-PD-1 antibody), BMS-936559 (anti-PD-1 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; an anti-PD-L1 antibody), MSB0010718C (an anti-PD-L1 antibody), and U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT published patents. Examples include those described in International Publication Nos. WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400 and WO 2011161699 (each of which is incorporated by reference herein).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, cemiplimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

[0053] BMS 936558 is a fully human IgG4 monoclonal antibody targeting PD-1. In a Phase 1 clinical trial, biweekly administration of BMS-936558 in subjects with advanced refractory malignancies demonstrated sustained partial or complete regressions. The most significant response rates were observed in subjects with melanoma (28%) and renal cell carcinoma (27%), but substantial clinical activity was also observed in subjects with non-small cell lung cancer (NSCLC), with some responses lasting for more than one year.

[0054] BMS 936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1. Phase 1 study results showed that biweekly dosing of the agent produced 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, with some subjects experiencing responses lasting for more than a year.

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

[0056] MEDI4736 is an anti-PD-L1 antibody that has demonstrated a tolerable safety profile and durable clinical activity in this dose-escalation study. Expansion of MEDI4736 in multiple cancers and development of MEDI4736 as monotherapy and in combinations is ongoing.

[0057] GITR agonists Glucocorticoid-Inducible TNFR-Related Gene (GITR:TNFRSF 18), 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 that contains 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 tested as a means to expand CD8+ T effector memory cell populations while simultaneously promoting the loss or inhibition of Tregs.

[0058] The suppression of the costimulatory activity of responder T cells and the suppressive activity of regulatory T cells means that GITR activation leads to an enhanced immune response. Such activation has the potential to restore immune responses to infections and tumors. Thus, molecules capable of activating GITR would be of value as immunostimulants in situations where it is desirable to induce an enhanced immune response.

[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 known as 9H6v3), TRX518, MEDI1873, MK-4166, BMS-986156, MK-1248, INCAGN01876, or GWN323.

[0061] TRX518 is a humanized Fc-disabled anti-GITR monoclonal antibody that has been shown to block GITR interactions and act synergistically with chemotherapeutic agents in cancer models. TRX518 is currently being investigated in clinical trials, including NCT01239134 (stage 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 antineoplastic activity. MEDI1873 is currently being investigated in clinical trials, including NCT02583165 (advanced solid tumors).

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

[0064] BMS-986156 is an anti-GITR agonist monoclonal antibody. BMS-986156 is currently being investigated 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 agonist monoclonal antibody. MK-1248 is currently being investigated in clinical trials, including NCT02553499 (as monotherapy and in combination with pembrolizumab in subjects with advanced solid tumors).

[0066] INCAGN01876 is an anti-GITR agonist monoclonal antibody. INCAGN01876 is currently being investigated in clinical trials, including NCT02697591 (in subjects with advanced or metastatic solid tumors).

[0067] GWN323 is an anti-GITR agonist monoclonal antibody. GWN323 is currently being investigated in clinical trials, including NCT02697591 (as monotherapy and in combination with PDR001 in subjects with advanced cancer or lymphoma).

[0068] Methods for Treating Disease or Disorders The present invention also relates to methods of treating diseases or disorders 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 a 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 spread to the pancreas is referred to as "metastatic colon cancer to pancreas" rather than pancreatic cancer. Treatment is also based on where the cancer originated. If colon cancer metastasizes to the bone, it is still colon cancer, and the relevant doctor will recommend treatments shown to address metastatic colon cancer.

[0070] The present invention also relates to the use of a combination of oncolytic viruses and checkpoint inhibitors for the treatment of 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, 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 a herpes simplex virus (e.g., herpes simplex virus type 1). In other embodiments, the herpes simplex virus type 1 is modified so that it does not contain an intact ICP34.5 gene. In other embodiments, the herpes simplex virus type 1 is modified so that it does not contain an intact ICP34.5 gene and it does not contain an intact ICP47 gene. In yet other embodiments, the herpes simplex virus type 1 is modified so that it does not contain an intact ICP34.5 gene, it does not contain an intact ICP47 gene, and it 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 (belongs to the CD2 family of molecules and inhibits all NK, gamma delta and memory CD8 + (expressed on αβ)T cells), CD160 (also called BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 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), biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049.

[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 of which is incorporated herein by reference).Examples of molecules that inhibit the interaction of PD-1 with PD-L1 and / or PD-L2 inhibitors 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-03 5 (anti-PD-L1), FAZ-053 (anti-PD-L1), CK-301 (anti-PD-L1), AK-106 (anti-PD-L1), M-7824 (anti-PD-L1), C A-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; an anti-PD-L1 antibody), MSB0010718C (avelumab; an anti-PD-L1 antibody), and the antibodies described in U.S. Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8, Examples of such compounds include those described in PCT published patent applications WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, and WO 2011159877.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, cemiplimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

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

[0076] In many cases, cancers are present in patients both as primary tumors (i.e., tumors growing at the anatomical site where tumor progression begins and continues to result in a cancerous mass) and as secondary tumors or metastases (i.e., tumors spreading from their primary site to other parts of the body). The oncolytic viruses of the present invention can be effective in treating tumors through lytic effects and systemic immune effects. For example, with respect to talimogene laherparepvec, the virus physically lyses the tumor, causing primary tumor cell death. In addition, it is believed that [1] lysis of tumor cells releases tumor-derived antigens that are then recognized by the immune system, and [2] production of GM-CSF helps induce an anti-tumor immune response, and both of these mechanisms result in a systemic immune response that allows the immune system to recognize and attack both primary and secondary tumors / metastases. In embodiments in which the oncolytic virus is combined with a checkpoint inhibitor, the checkpoint inhibitor is believed 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. Additionally, in embodiments in which the oncolytic virus is combined with a GITR agonist, the GITR agonist is believed to further enhance the systemic immune response by expanding the CD8+ T effector memory cell population, driving the loss or inhibition of Tregs.Thus, the present invention contemplates the treatment of primary tumors, metastases (i.e., secondary tumors), or both, with oncolytic viruses (e.g., talimogene laherparepvec) either alone or in combination with checkpoint inhibitors.

[0077] In some embodiments, the methods of treatment or use described herein do not include treatment with or in combination with radiation. In other embodiments, the methods of treatment or use described herein do not include treatment with or in combination with a chemotherapeutic agent (i.e., a chemical agent or drug that is selectively destructive to malignant cells and tissues - typically a small molecule compound), such as cisplatin. In yet other embodiments, the methods of treatment or use described herein do not include treatment with a combination of radiation and a chemotherapeutic agent (e.g., cisplatin).

[0078] The method of the present invention can be used to treat several different stages of cancer. Most disease classification systems include information on whether the cancer has spread to nearby lymph nodes, where the tumor is located in the body, 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 grade of the tumor (i.e., the level of cell abnormality, the likelihood of the tumor growing and spreading). For example, stage 0 refers to the presence of abnormal cells that have not spread to nearby tissues - i.e., cells that can become cancerous. Stages I, II, and III cancer refer to the presence of cancer. The higher the stage, the larger the cancerous tumor becomes and the more it spreads to nearby tissues. Stage IV cancer is cancer that has spread to distant parts of the body. In some embodiments, the method of the present invention can be used to treat metastatic cancer.

[0079] Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising an oncolytic virus or a combination of an oncolytic virus and a checkpoint inhibitor. The pharmaceutical composition may contain formulation materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. The pharma- cetically active agent may be administered to the patient by various routes including, for example, oral or parenteral, e.g., intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intracisternal, intrarectal, intratumoral, intravascular, intradermal, or by passive or enhanced absorption through the skin, e.g., using 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] A person skilled in the art would be able to determine the dosage and duration of treatment according to any aspect of the disclosure. For example, a person skilled in the art would be able to monitor a patient to determine whether treatment should be initiated, continued, discontinued, or resumed. The effective amount for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, and the method, route, and dose of administration. A clinician will use parameters known in the art to make appropriate dosage decisions. The effective amount of a pharmaceutical composition to be used therapeutically will depend, for example, on the context and purpose of the treatment. A person skilled in the art will understand that appropriate dosage levels for treatment will vary in part depending on the molecule thus delivered, the indication for which the conjugated drug molecule is used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and general health) of the patient. Thus, a clinician may titrate the dosage and vary the route of administration to obtain optimal therapeutic effect.

[0081] Clinical studies have demonstrated that talimogene laherparepvec can be injected directly into skin lesions, subcutaneous lesions, or lymph node lesions that are visible and palpable, or can be injected using ultrasound guidance. Thus, in one embodiment, a pharmaceutical composition containing talimogene laherparepvec is administered via intralesional injection. Talimogene laherparepvec is currently available in fixed dosage concentrations: 10 mg for initial administration; 10 mg for subcutaneous administration; 10 mg for intralesional administration; 10 mg for subcutaneous administration. 6 pfu / mL and 10 for subsequent administrations 8 It is provided in 1 mL disposable vials at pfu / mL (Reske, et al. J Immunol, 2008, 180(11):p.7525-36). The volume injected may vary depending on the tumor type. For example, talimogene laherparepvec is administered at 10 mL on day 1 of week 1 in injectable skin, subcutaneous and lymph node tumors. 6 at a dose of up to 4.0 mL of plaque-forming units / mL (PFU / mL), followed by 10 8 In another embodiment, talimogene laherparepvec is administered by intratumoral injection at a dose of 10 PFU / mL on day 1 of week 1 in injectable skin tumors, subcutaneous tumors, and lymph node tumors. 6 at a dose of up to 4.0 mL of plaque-forming units / mL (PFU / mL), followed by 10 7 A maximum dose of 4.0 mL of PFU / mL is administered by intratumoral injection every 2 weeks (± 3 days) thereafter. The recommended volume of talimogene laherparepvec injected into the tumor depends on the size of the tumor and can be determined according to the injection volume guidelines in Table 1 (and as set forth in patent application PCT / US2013 / 057542, which is incorporated herein by reference).

[0082] [Table 1]

[0083] In general, all suitably injectable lesions should be injected at the maximum dose available for the individual dosing instance. On each treatment day, prioritization of injections is recommended as follows: any new injectable tumors that have appeared since the previous injection; starting with the largest tumor by tumor size; any previously uninjected tumors that are now injectable.

[0084] The compositions of the present invention may include one or more additional components, including physiologically acceptable carriers, excipients or diluents. For example, the compositions may include one or more of buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (e.g., those with less than 10 amino acids), proteins, amino acids, carbohydrates such as glucose, sucrose or dextrin, chelating agents such as EDTA, glutathione, stabilizers and excipients. Acceptable carriers include, for example, neutral buffered saline or saline mixed with specific serum albumin. Preservatives such as benzyl alcohol may also be added. The compositions may be formulated as lyophilizates using appropriate excipient solutions (e.g., sucrose) as diluents.

[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 in a single dose, two doses, three doses, four doses, five doses, or more than six doses.

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

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

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

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

[0090] In a particular embodiment, the anti-PD-L1 antibody (e.g., atezolizumab) is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1200 mg IV Q3W until disease progression or unacceptable toxicity.

[0091] Thus, in one embodiment, the present invention relates to 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, 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 for 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 an additional embodiment, the invention relates to a pharmaceutical composition for use in a method for treating B cell lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of an oncolytic virus and a GITR agonist.

[0092] In another embodiment, the present invention relates to a therapeutically effective amount of an oncolytic virus for use in 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. In yet another embodiment, the present invention relates to a therapeutically effective amount of an oncolytic virus and a checkpoint inhibitor for use in 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). In an additional embodiment, the present invention relates to a therapeutically effective amount of an oncolytic virus and a GITR agonist for use in treating B-cell lymphoma.

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

[0094] In embodiments in which 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 conjunction with or in combination therapy with radiation. In other embodiments, the pharmaceutical compositions described herein do not include a chemotherapeutic agent (e.g., cisplatin). In yet other embodiments, the pharmaceutical compositions described herein are not used in combination therapy with radiation and a chemotherapeutic agent (e.g., cisplatin).

[0097] kit In another aspect, the present invention relates to a kit comprising: [1] an oncolytic virus, optionally in combination with a checkpoint inhibitor; and [2] instructions for administration to a patient. For example, the kit of the present invention may 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 present invention may 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: [1] an oncolytic virus, optionally in combination with a GITR agonist; and [2] 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., AMG228 (also known as 9H6v3), TRX518, MEDI1873, or MK-4166), and instructions (e.g., in a package insert or label) for treating a patient with cancer.

[0099] In some embodiments, the kit containing talimogene laherparepvec is administered at 10 6 at a maximum dose of 4.0 ml PFU / mL, followed by 10 on day 1 of week 4 8 In some embodiments, the kits containing talimogene laherparepvec include 100 mg of 1000 PFU / mL of 10 ... 6 at a maximum dose of 4.0 ml PFU / mL, followed by 10 on day 1 of week 4 7 Include instructions (e.g., in the package insert or label) for administration by intratumoral injection at a dose of up to 4.0 ml of PFU / mL, and then every 2 weeks (e.g., until complete response).

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

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

[0102] In embodiments in which the kit includes an anti-CTLA-4 antibody, the kit includes instructions (e.g., on a package insert or label) for intravenous administration in the dosages described herein. An example of an anti-CTLA-4 antibody includes ipilimumab.

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

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

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

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

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

[0108] The antitumor efficacy of talimogene laherparepvec was evaluated in several mouse 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 implanted by subcutaneous injection into the right flank of each mouse. In each case, 5 × 10 6 ~1×10 7 The cells were transplanted into mice.

[0109] Tumor measurements were obtained twice weekly. Treatment with talimogene laherparepvec was initiated when tumors reached a mean diameter of 4–6 mm. Three doses of talimogene laherparepvec (5 × 10 4 , 5×10 5 Or 5 x 10 6 PFU / dose, 50 uL dose volume) were administered 3 days apart by intratumoral injection. Body weights, gross clinical findings, and tumor measurements were obtained twice weekly. Animals were euthanized when tumor weight exceeded 10% of body weight.

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

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

[0112] The results are shown in Figure 1.

[0113] Neuroblastoma SK-N-AS neuroblastoma-bearing mice were inoculated with talimogene laherparepvec at 5 × 10 4 , 5×10 5 Or 5 x 10 6 Therapeutic treatment was in PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on study days 6, 9, and 12 (red arrows in Figure 2). Tumors were measured 2-3 times per week. Results are reported as a function of time in days, with day 0 being the day of tumor inoculation, mm 3 The tumor volumes are expressed as group mean tumor volumes ± standard error of the mean (5 × 10 4 (n=10 per group unless otherwise noted for PFU / dose group. Asterisks indicate a 5×10 vs. formulation buffer control on study day 23. 6 and 5 x 10 5 p<0.0001 for PFU / dose group, 5 × 10 4 Shown is p=0.0001 for PFU / dose group.

[0114] The results are shown in Figure 2.

[0115] Rhabdomyosarcoma-like tumors G-401 rhabdomyosarcoma-bearing mice were treated with talimogene laherparepvec at 5 × 10 4 , 5×10 5 Or 5 x 10 6Therapeutic treatment was in PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on study days 14, 17, and 20 (red arrows in Figure 3). Tumors were measured 2-3 times per week. Results are presented as a function of time in days, with day 0 being the day of tumor inoculation, mm 3 Expressed as group mean tumor volume ± standard error of the mean (n=10 per group unless noted). Asterisks indicate p<0.0001 for all talimogene laherparepvec-treated groups versus vehicle control on study day 40.

[0116] The results are shown in Figure 3.

[0117] Osteosarcoma SJSA-1 osteosarcoma-bearing mice were treated with talimogene laherparepvec at 5 × 10 4 , 5×10 5 Or 5 x 10 6 Therapeutic treatment was in PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on study days 9, 12, and 15 (red arrows in Figure 4). Tumors were measured 2-3 times per week. Results are presented as a function of time in days, with day 0 being the day of tumor inoculation, in mm 3 Expressed as group mean tumor volume ± standard error of the mean (n=10 per group).

[0118] The results are shown in Figure 4.

[0119] Rhabdomyosarcoma SJCRH30 rhabdomyosarcoma-bearing mice were inoculated with talimogene laherparepvec at 5 × 10 4 , 5×10 5 Or 5 x 10 6 Therapeutic treatment was in PFU / dose. Talimogene laherparepvec was administered by intratumoral injection once daily on study days 8, 11, and 14 (red arrows in Figure 5). 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 inoculation, mm 3The tumor volumes are expressed as group mean tumor volumes ± standard error of the mean (5 × 10 4 PFU / dose group and 5 × 10 6 (N=10 per group unless otherwise noted for PFU / dose group. Asterisks indicate p<0.0001 for all talimogene laherparepvec-treated groups versus vehicle control on study day 26 (last study day that all control animals were on 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 96-well plates and incubated overnight at 37°C. For each cell line, talimogene laherparepvec was serially diluted in nine wells starting at an MOI of 100 (1:4 serial dilutions). After 72 h of incubation, the number of cells remaining in each well was quantified using a CellTiter-Glo Luminescent cell viability assay (Promega, Madison, WI).

[0122] Talimogene laherparepvec was effective in 14 of 21 DLBCL cell lines at an MOI below 100. Five cell lines (SU-DHL-2, OCI-LY-3, TMD8, RI-1 (ABC subtype) and WSU-NHL (GCB subtype)) had an MOI IC of 1 or less. 50In contrast, OCI-LY-1, KARPAS422, WSU-DLCL2, SU-DHL-4, SU-DHL-10 and OCI-LY-7 (all of the GCB subtype) were resistant to talimogene laherparepvec at MOIs up to 100 (Table 2). Inhibition of cell proliferation was observed at an MOI IC of 1. 50 The concentration of talimogene laherparepvec in WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines was maximal, with most cell lines showing sensitivity below 100%. Figure 6 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines. These results demonstrate that treatment of DLBCL cell lines with talimogene laherparepvec results in 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 into 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 an MOI of 100 (1:4 serial dilutions). After 72 h 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. 50(Table 3). Figure 7 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in HCT116 (colorectal cancer) and SK-MEL-5 (melanoma) cell lines. These results demonstrate that treatment of 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 into 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 an MOI of 100 (1:4 serial dilutions). After 72 h 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 five 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 concentrations of talimogene laherparepvec in HUT-78 (CTCL) and RPMI 8226 (multiple myeloma) cell lines. These results demonstrate that treatment of 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 proliferation of various murine tumor cell lines in cell-based assays Melanoma, colorectal carcinoma, and B-cell lymphoma murine cell lines were seeded into 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 an MOI of 100 (1:4 serial dilutions). After 72 h 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 four of the five cell lines tested (Table 5). The B16F10 melanoma cell line demonstrated resistance to talimogene laherparepvec. This resistance is mediated by the lack of an entry receptor for herpes simplex virus type 1, as previously described by Miller et al., Molecular Therapy, 3(2):160-168 (2001). Cloudman CL M3 (melanoma), CT-26 and MC-38 (colorectal carcinoma) cell lines demonstrated an MOI IC of approximately 0.2. 50 Figure 9 shows the degree of cell growth inhibition achieved by increasing concentrations of talimogene laherparepvec in CT-26 and MC-38 (colorectal carcinoma) cell lines. These results demonstrate that treatment of murine tumor cell lines (melanoma, colorectal carcinoma, and B-cell lymphoma) with talimogene laherparepvec results in 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 mouse models A20 tumor cells were injected subcutaneously into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6 Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100 mg / kg, mice were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume were uniform across treatment groups. Mice were then administered OncoVex mGM-CSF (3×10 4 ~3×10 6 Three intratumoral injections of either PFU / dose or vehicle were administered on days 10, 13, and 16. Clinical signs, body weight changes, and survival time (tumors >800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0134] OncoVex in A20 tumor-bearing animals mGM-CSF Treatment with 3x10 4 PFU, 3 × 10 5 PFU, 3 × 10 6 PFU resulted in 100% complete regression of all directly injected tumors (Figure 10a). Colorectal tumors were 4 No response was observed at a dose of 3 × 10 5 PFU dose showed 50% tumor growth, and 3×10 6 A dose of 100 PFU demonstrated 100% tumor growth inhibition (Figure 10b). Median survival was 3×10 compared to vehicle. 5 PFU (p=0.0054) and 3×10 6 PFU dose (p=0.0004) group (38 days vs. 21 days, respectively - FIG. 10c). No weight loss was observed, indicating that the tested treatments were safe and tolerable (FIGS. 10d and 10e). Table 6 shows the percentage of tumor-free subjects in each group.

[0135] In addition, mice bearing neuro2a neuroblastoma tumors were cultured using OncoVex mGM-CSF 5×104 , 5×10 5 Or 5 x 10 6 Treatment was at 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 (FIG. 10f). Injected tumors were measured twice weekly. Results are shown in mm as a function of time in days, with day 0 being the day of tumor inoculation. 3 The median survival time was 5×10 compared to vehicle. 4 PFU / dose group (p = 0.0056), 5 × 10 5 PFU / dose group (p<0.0001) and 5×10 6 The increase was significant in the PFU / dose group (p<0.0001) (FIG. 10g).

[0136] OncoVex on non-injected ("naive") tumors in neuro2a neuroblastoma tumor-bearing mice mGM-CSF The efficacy of treatment was also evaluated. Neuro2a tumor cells were implanted subcutaneously into the left and right flanks of female A / J mice on day 0. Tumor volumes (mm3) were measured twice weekly (Q2W) using electronic calipers. Tumors averaged approximately 100 mm 3 Once tumor volume reached 100 mg / kg, mice were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume were uniform across treatment groups. Mice were then administered OncoVex mGM-CSF (5×10 6 Three intratumoral injections of either PFU / dose or vehicle were administered on the right side (the "treated" side) on days 10, 13, and 16. Tumor volume and survival time (when tumors were >800 mm on either side) 3 Mice were removed from the study if the tumor size reached 0.05 mg / kg / day (Figure 10h) twice weekly until the end of the study. mGM-CSF Treatment with OncoVex resulted in complete regression in 8 of 10 directly injected tumors (Fig. 10h). Contralateral uninjected ("untreated") tumors showed a significant delay in tumor growth (Fig. 10h). Median survival was significantly longer with OncoVex compared to vehicle.mGM-CSF The increase was significant in the treated group (32 days vs. 18 days, p<0.0001, FIG. 10i).

[0137] These results demonstrate that OncoVex expression of established B cell lymphoma and neuroblastoma tumors in in vivo mouse models mGM-CSF have demonstrated that treatment with EGFR results in potent inhibition of tumor growth. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune responses) and in non-injected contralateral tumors in the same host (presumably via adaptive immune responses).

[0138] [Table 6]

[0139] Example 5: OncoVex mGM-CSF Combined with CTLA-4 or PD-L1 inhibitors inhibits tumor growth in B-cell lymphoma in mouse models A20 tumor cells were injected subcutaneously into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6 Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100 mg / kg, animals were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume were uniform across treatment groups. Animals were then administered OncoVex mGM-CSF (5×10 6 The mice were given three intratumoral injections of 100 PFU / dose (1000 mg / kg) alone or in combination with intraperitoneal injections of anti-PD-L1 mAb or anti-CTLA-4 mAb. Clinical signs, body weight changes, and survival time (tumors >800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was 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 a combination of anti-CTLA-4 mAb and anti-CTLA-4 mAb resulted in complete regression of all directly injected tumors (Fig. 11a). Tumors treated with intraperitoneal anti-CTLA-4 alone (both flanks) showed approximately 50% tumor inhibition. Contralateral tumors were treated with OncoVex mGM-CSF While it showed some consistent efficacy alone, OncoVex mGM-CSF The combination of OncoVex and anti-CTLA-4 mAb resulted in regression of all tumors and complete cure in 9 of 10 mice. Median survival time was significantly longer in the anti-CTLA-4 mAb group, OncoVex, and OncoVex+ groups compared to vehicle. mGM-CSF Group and OncoVex mGM-CSF In addition, a significant extension in median survival was measured in the combination arm compared with either agent alone (OncoVex mGM-CSF p=0.012 vs. anti-CTLA-4 mAb and p=0.001 vs. anti-CTLA-4 mAb). Median survival was 25.5 days in the vehicle group and 26.7 days in the OncoVex group. mGM-CSF The median survival time for the combination group was 36.5 days in the IL-16 group and 32 days in the anti-CTLA-4 mAb group. The median survival time for the combination group remained undetermined beyond day 40, at which point 9 of 10 mice showed no signs of tumor (Figure 11b and Table 7a).

[0141] [Table 7]

[0142] OncoVex in A20 tumor-bearing animals mGM-CSF , anti-PD-L1 mAb or OncoVex mGM-CSF Treatment with a combination of anti-PD-L1 mAb and intraperitoneal anti-CTL4-4 alone (both flanks) resulted in complete regression of all directly injected tumors (Fig. 11c). Tumors treated with intraperitoneal anti-CTL4-4 alone (both flanks) showed no effect on tumor growth. Contralateral tumors were treated with OncoVex mGM-CSF While it showed some consistent efficacy alone, OncoVex mGM-CSFThe combination of OncoVex and anti-PD-L1 mAb caused regression of all tumors and complete cure in 10 of 10 mice. Median survival was 1.1-fold longer than that of the vehicle group. mGM-CSF In addition, a significant extension in median survival was measured in the combination group compared to anti-PD-L1 mAb alone. mGM-CSF A trend towards improved overall survival was also observed when compared with , but no statistical significance was observed (OncoVex mGM-CSF p=0.067 for OncoVex and p=0.0013 for anti-PD-L1 mAb. Median survival was 23 days in the vehicle group and 10 days in the OncoVex group. mGM-CSF The median survival time for the combination group was 48 days and 26 days for the anti-PD-L1 mAb group. The median survival time for the combination group remained undetermined beyond day 40, at which point 10 of 10 mice showed no signs of tumor (Figure 11d and Table 7b).

[0143] [Table 8]

[0144] These results suggest that OncoVex® in combination with either PD-L1 or CTLA-4 inhibitors in established B-cell lymphoma tumors in an in vivo mouse model mGM-CSF have demonstrated that treatment with EGFR-1 resulted in better inhibition of tumor growth than either single agent alone. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune responses) and in non-injected contralateral tumors in the same host (presumably via adaptive immune responses).

[0145] Example 6: OncoVex mGM-CSF Inhibits colorectal tumor growth in mouse models, either alone or in combination with CTLA-4 or PD-L1 inhibitors CT-26 tumor cells were injected subcutaneously into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100 mg / kg / day, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume was uniform across treatment groups. Animals were then randomized into 4 groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume were uniform across treatment groups. 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 (tumors >800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0146] OncoVex in CT-26 tumor-bearing animals mGM-CSF Treatment with anti-CTLA-4 mAb or the combination resulted in approximately 75% tumor growth inhibition of all directly injected tumors by day 20 (data not shown). Contralateral tumors were injected with OncoVex. mGM-CSF While no response was observed against OncoVex alone (TGI of about 25%), about 75% tumor growth inhibition was observed in the anti-CTLA-4 mAb and combination group. Median survival was significantly increased in the anti-CTLA-4 mAb and combination group compared to vehicle (Figure 12b). Median survival was 20 days in the vehicle group and 10 days in the OncoVex group. mGM-CSF The median survival time for the combination arm was 22 days in the OncoVex group and 41 days in the anti-CTLA-4 group. The median survival time for the combination arm was greater than 50 days and remained undetermined when the study was stopped. mGM-CSF This was significantly longer than either the anti-CTLA-4 mAb 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 anti-PD-L1 mAb and OncoVex resulted in approximately 75% tumor growth inhibition of all directly injected tumors by day 18 (data not shown). Intraperitoneal injection of anti-PD-L1 mAb had little effect on tumor growth (either flank). Contralateral tumors were treated with OncoVex mGM-CSF While no response was observed to either anti-PD-L1 or anti-PD-L1 mAb alone, approximately 75% tumor growth inhibition was observed in the combination group (Figure 12c). Median survival was significantly longer in the combination group compared to either agent alone (OncoVex mGM-CSF p=0.012 for OncoVex and p=0.007 for anti-PD-L1 mAb. Median survival was 21 days for vehicle and anti-PD-L1 mAb, and 10 days for OncoVex. mGM-CSF The mean time to treatment was 23 days in the control group and 34 days in the combination group (Figure 12d and Table 8b).

[0149] [Table 10]

[0150] These results demonstrate that 1) OncoVex is a novel therapeutic option for established colorectal tumors in an in vivo mouse model. mGM-CSF 2) OncoVex mGM-CSF and anti-CTLA-4 inhibitors, or 3) OncoVex mGM-CSF We have demonstrated that combined treatment with OncoVex and anti-PD-L1 inhibitors results in potent inhibition of tumor growth. mGM-CSF 2) OncoVex mGM-CSF and anti-CTLA-4 inhibitors, and 3) OncoVex mGM-CSF Antitumor activity was observed in 1) OncoVex in combination with an anti-PD-L1 inhibitor injected directly into tumors (possibly via tumor lysis and immune response). mGM-CSFand anti-CTLA-4 inhibitors, and 2) OncoVex mGM-CSF This was observed in non-injected contralateral tumors (likely via an adaptive immune response) in the same hosts treated with a combination of PD-L1 and anti-PD-L1 inhibitors.

[0151] OncoVex mGM-CSF To further understand the antitumor activity of the combination of OncoVex and anti-CTLA-4 inhibitors, mGM-CSF , 2) anti-CTLA-4 inhibitors, and 3) OncoVex mGM-CSF The ability of OncoVex in combination with an anti-CTLA-4 inhibitor to release tumor antigens and stimulate anti-tumor specific T cell responses was evaluated. 96-well ELISpot plates (Millipore, Temecula, CA) with a nitrocellulose filter base were coated with purified anti-IFN-γ (2 μg / ml) antibody. mGM-CSF Spleen cells (8 × 10) from CT-26 tumor-bearing mice treated with CTLA-4 mAb or the combination on day 10 5 ) were incubated with a control peptide (GFP) or the 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 an endogenous murine leukemia virus presented by MHC class ILD molecules (25). Spots were counted using a CTLS6 Fluorospot analyzer (CTL, Shaker Heights, OH). Systemic (splenic) anti-AH1 CD8 + Quantification of T cells by ELISpot or by dextramer staining using FACS was performed using OncoVex mGM-CSF , anti-CTLA-4 inhibitors or OncoVex mGM-CSF We demonstrated a significant increase in AH1-reactive T cells in mice treated with the combination of anti-CTLA-4 inhibitors and 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 in combination with a CTLA-4 inhibitor (Figure 12g). This experiment was performed using OncoVex mGM-CSF They demonstrate that the combination of IL-16 with an anti-CTLA-4 inhibitor results in an increased presence of effector cells and a decreased presence of regulatory T cells, which in turn results in increased efficacy of the combination over either compound alone.

[0152] Example 7: OncoVex mGM-CSF Inhibits melanoma tumor growth in mouse models, alone or in combination with a CTLA-4 inhibitor B16F10 cells (5 x 10 4 -OncoVex due to lack of HSV-1 entry receptor mGM-CSF On day 0, mouse nectin-1-transfected B16F10 melanoma cells (OncoVex mGM-CSF The tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The subcutaneous tumors averaged approximately 100 mm 3 Once tumor volume reached 100 mg / kg, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume at the start of treatment and the variability in tumor volume were uniform across treatment groups. Animals were then administered OncoVex mGM-CSF (5×10 6 PFU / dose), four intraperitoneal injections of anti-CTLA-4 mAb, and OncoVex mGM-CSF (5×10 6 The mice were given three intratumoral injections of 1000 PFU / dose in combination with four intraperitoneal injections of anti-CTLA-4 mAb or vehicle alone. Clinical signs, body weight changes and survival time (when tumors reached 800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0153] OncoVex of B16F10 nectin-1 tumor-bearing micemGM-CSF or OncoVex mGM-CSF Treatment with a combination of OncoVex and anti-CTLA-4 mAb resulted in approximately 85% and 99% inhibition of tumor (subcutaneous) growth, respectively. No inhibition was observed with anti-CTLA-4 mAb alone (Figure 13a). Assessment of lung metastatic burden demonstrated the efficacy of OncoVex in inhibiting lung metastases. mGM-CSF showed that the combination of IL-1 and anti-CTLA-4 mAb was significantly more effective than either treatment alone (OncoVex mGM-CSF p=0.0008 for OncoVex and p=0.0007 for anti-CTLA-4 mAb (Figure 13b). Median survival was significantly increased in the combination group compared to vehicle (p<0.0001). Median survival was 30 days in the vehicle group and 10 days in the OncoVex group. mGM-CSF + 46 days in the anti-CTLA-4 mAb group (Figure 13c and Table 9).

[0154] [Table 11]

[0155] These results demonstrate that OncoVex is a novel therapeutic option for the treatment of established melanoma tumors in an in vivo mouse model. mGM-CSF or OncoVex mGM-CSF These results demonstrate that treatment with a combination of OncoVex and anti-CTLA-4 mAb results in potent inhibition of tumor growth, as evidenced by the ability of the combination to inhibit lung metastases. mGM-CSF We also show that the combination of IgG1 with anti-CTLA-4 mAb demonstrated significantly more robust systemic antitumor efficacy compared to either treatment alone. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune responses) and in non-injected contralateral tumors in the same host (presumably via adaptive immune responses).

[0156] Vehicle, OncoVex mGM-CSFIt was also observed that lung tumors in the anti-CTLA-4 mAb group rarely showed scattered T and B cells in the tumor periphery and mild intratumoral macrophages. mGM-CSF Tumors in the + anti-CTLA-4 mAb combination group showed a predominance of T cells in the tumor periphery as well as variable numbers of T cell infiltrates in the tumor. Macrophages predominated both in the tumor and as dense infiltrating cells at the tumor periphery. B cells remained exclusively in the tumor periphery (Figure 13d and data not shown).

[0157] The immune infiltration results of these lung tumors were analyzed using OncoVex mGM-CSF These results show that the combination of IL-16 with an anti-CTLA-4 inhibitor can convert poorly invasive B16F10 metastases (i.e., "cold" tumors) into well-invasive B16F10 metastases (i.e., "hot" tumors).

[0158] Example 8: OncoVex mGM-CSF inhibits triple-negative breast cancer tumor growth in mouse models 4T1 tumor cells were injected subcutaneously into the right flank of female BALB / c mice on day 0 (2 × 10 6 Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100 mg / kg, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume at the start of treatment and the variability in tumor volume were uniform across treatment groups. Animals were then administered OncoVex mGM-CSF (5×10 4 , 5×10 5 Or 5 x 10 6 The mice were given three intratumoral injections of either PFU / dose or vehicle. Clinical signs, body weight changes and survival time (tumors >800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

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

[0160] These results suggest that OncoVex mGM-CSF The results demonstrate that treatment with EGFR results in potent inhibition of tumor growth. Antitumor activity was measured only in directly injected tumors.

[0161] Example 9: OncoVex mGM-CSF Inhibits B-cell lymphoma tumor growth in mouse models, either alone or in combination with GITR agonists A20 tumor cells were injected subcutaneously into the right and left flanks of female BALB / c mice on day 0 (2 × 10 6 Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100 mg / kg, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume (in both flanks) at the start of treatment and the variability in tumor volume were uniform across treatment groups. Animals were then administered OncoVex mGM-CSF or OncoVex mGM-CSF The clinical signs, body weight changes, and survival time (tumors >800 mm) were evaluated. 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0162] Tumors treated intraperitoneally with anti-GITR mAb cured 30% of tumors (both in the injected tumor and in the contralateral tumor). OncoVex mGM-CSF cured 6 of 10 (60%) injected tumors, while the contralateral tumors showed a minor response with no cures. mGM-CSFThe combination of OncoVex with anti-GITR mAb caused regression of all tumors (both in the injected and contralateral tumors) and resulted in complete cure in 7 out of 10 mice. See Figure 15a. Median survival was significantly longer in the anti-GITR mAb group, OncoVex compared to vehicle. mGM-CSF Group and OncoVex mGM-CSF In addition, a significant extension in median survival was observed in the OncoVex combination arm compared with either agent alone. mGM-CSF and anti-GITR mAb combination group (OncoVex mGM-CSF p<0.0001 for OncoVex and p=0.039 for anti-GITR mAb. Median survival was 24 days in the vehicle group and 10 days in the OncoVex group. mGM-CSF The median survival time for the combination group was 26 days 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 of 10 mice showed no signs of tumor. See Figure 15b and Table 10.

[0163] [Table 12]

[0164] These results were published in OncoVex mGM-CSF and OncoVex mGM-CSF We demonstrate that treatment of B-cell lymphoma with a combination of IL-1 and anti-GITR mAb results in potent inhibition of tumor growth.

[0165] Example 10: OncoVex in a mouse colon (colorectal) adenocarcinoma (MC-38) tumor model muGM-CSF Trials evaluating the combination of anti-PD-1 blockade with The study was conducted in a murine MC-38 tumor model using OncoVex muGM-CSF , anti-PD-1 blockade or OncoVex muGM-CSF This study was designed to evaluate the tolerability and antitumor activity of combination of EGFR with anti-PD-1 blockade.

[0166] C57BL / 6 mice were inoculated with syngeneic MC-38 tumor cells in both the right and left flanks. Tumors averaged 5 mm in diameter (60 mm 3 On day 10 post-inoculation, animals were assigned to six groups (n=10 per group). OncoVEX muGM-CSF (5×10 6 Right tumors were injected intratumorally with 0.1% PFU / dose or formulation buffer control once daily, every 3 days for a total of 3 doses. Left tumors received no injections.

[0167] Anti-mouse PD-1 (clone RMP1-14, BioXCell) or isotype control antibody (rat IgG2a, clone 2A3, BioXCell) was administered intraperitoneally at either 1 mg / dose or 300 μg / dose twice weekly (starting on study day 10 and ending on study day 30 (7 doses)). Tumor volumes, body weights, and gross clinical findings of both injected (right) and non-injected (left) tumors were collected 2-3 times weekly. Animals were euthanized if total tumor volume (right + left) reached IACUC mandatory termination of >10% of body weight or if the animal showed signs of distress. Peripheral blood was collected for immunophenotyping analysis on study days 14 and 20 (4 and 10 days, respectively, after start of dosing). After red blood cell lysis, leukocytes 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 the duration of the experiment and showed no evidence of treatment-related adverse health effects as evidenced by body weight (Figure 16a) or survival time, and there were no notable adverse clinical signs identified by daily health monitoring examinations. The slightly lower body weights in the treated group compared to the control group are 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 either anti-mPD-1 antibody (at both tested doses of 300 μg and 1 mg per dose) or OncoVexmuGM-CSF (Figure 16b). Table 11 summarizes the number of animals that were tumor-free (regressed) at the end of the experiment on either the right (injected) or left (non-injected) flank. Single-agent activity with either agent was limited to 10-20% complete regression in injected tumors (and no complete regression of non-injected tumors), whereas the combination resulted in 80-90% regression in injected tumors (and 10-20% complete regression of non-injected tumors). These data were analyzed using OncoVex muGM-CSF We show that combination therapy with anti-PD-1 resulted in substantially improved tumor clearance in a murine MC-38 tumor model.

[0170] [Table 13]

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

[0172] Example 11: OncoVex in a mouse colon (colorectal) adenocarcinoma (MC-38) tumor model muGM-CSF Trials evaluating the combination of anti-PD-L1 blockade with The study was conducted in a murine MC-38 tumor model using OncoVex muGM-CSF , anti-PD-L1 blockade or OncoVex muGM-CSF This study was designed to evaluate the tolerability and antitumor activity of combination of EGFR-associated leukemia (EGFR-associated leukemia) with anti-PD-L1 blockade.

[0173] MC-38 tumor cells were injected subcutaneously into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3Once tumor volume reached 100%, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume at the start of treatment and the variability in tumor volume were uniform across treatment groups. mGM-CSF (5×10 6 Anti-PD-L1 mAb (clone MIH5, mouse IgG1) or control IgG1 were administered intratumorally every 3 days for a total of 3 injections (mAbs were administered a total of 4 times). The uninjected tumor (contralateral; on the left side of the animal) did not receive any injections. Clinical signs, body weight changes and survival times (tumors >800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0174] All animals survived for the duration of the study and showed no evidence of treatment-related adverse health effects as evidenced by body weight and no notable adverse clinical signs identified during daily health monitoring examinations.

[0175] Tumor growth inhibition was observed in both the injected and contralateral tumors with OncoVex mGM-CSF or anti-PD-L1 mAb (Figure 17), whereas mGM-CSF The combination of IL-17 with anti-PD-L1 mAb caused regression of all injected tumors and 7 of 10 injected tumors.

[0176] Example 12: OncoVex in a mouse melanoma (B16F10) tumor model muGM-CSF Trials evaluating the combination of anti-PD-1 blockade with This study evaluated the efficacy and safety of OncoVex in a mouse melanoma (B16F10) tumor model. muGM-CSF , anti-PD-1 blockade or OncoVex muGM-CSF This study was designed to evaluate the tolerability and antitumor activity of combination of EGFR with anti-PD-1 blockade.

[0177] B16F10 tumor cells engineered to express mNectin were injected subcutaneously into the right flank of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice weekly (Q2W) using electronic calipers. The tumors were approximately 100 mm 3 Once tumor volume reached 100%, animals were randomized into 4 groups (10 mice per group) such that the mean tumor volume at the start of treatment and the variability in tumor volume were uniform across treatment groups. mGM-CSF (5×10 6 PFU / dose) or formulation buffer control was administered intratumorally every 3 days for 3 doses, either alone or in combination with anti-PD-1 mAb (clone 29F1A12, mouse IgG1) or control IgG1 (mAbs were administered a total of 4 times). Clinical signs, body weight changes, and survival time (when tumors were 800 mm 3 Mice were excluded from the study if their blood glucose level reached 0.05g / kg / day. The blood glucose level was measured 2-3 times a week until the end of the study.

[0178] All animals survived for the duration of the study and showed no evidence of treatment-related adverse health effects as evidenced by body weight and no notable adverse clinical signs identified during daily health monitoring examinations.

[0179] Tumor growth inhibition was mGM-CSF While this was seen in response to monotherapy treatment with monotherapy (3 out of 10 mice showed tumor regression), anti-PD-1 mAb monotherapy did not have any inhibitory effect on tumor growth (Figure 18). OncoVex mGM-CSF The combination of OncoVex with anti-PD-1 mAb regressed 5 of 10 injected tumors (Figure 18). mGM-CSF demonstrated that the combination of EGFR-1 and anti-PD-1 mAb had superior antitumor activity compared with monotherapy.

[0180] Example 13: A Phase 1, Multicenter, Open-Label, Dose De-Escalation Study to Evaluate the Safety and Efficacy of Talimogene Laherparepvec in Pediatric Subjects with Advanced Non-Central Nervous System Tumors Amenable to Direct Infusion This example describes a Phase 1, multicenter, open-label, dose de-escalation study to evaluate the safety and efficacy of talimogene laherparepvec in pediatric subjects with advanced non-CNS tumors amenable to direct injection. See the National Institutes of Public Health website (clinicaltrials.gov), Study 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 assessed by the occurrence of dose-limiting toxicities (DLTs), in pediatric subjects with advanced non-CNS tumors amenable to direct infusion.

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

[0183] The oncology endpoint was to determine the safety and efficacy of talimogene laherparepvec, as assessed by the occurrence of dose-limiting toxicities (DLTs), in pediatric subjects with advanced non-central nervous system (CNS) tumors amenable to direct injection.

[0184] Secondary endpoints were: (1) to evaluate talimogene laherparepvec as assessed 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 modified Immune-related Response Criteria Simulating Response Evaluation Criteria in Solid Tumors (irRC-RECIST); and (2) to evaluate the association between granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor / subunits in archival tumor tissue and clinical outcomes (safety and efficacy endpoints, including ORR, DOR, TTR, TTP, PFS and OS).

[0185] Selection Criteria If the subject is too young legally to provide informed consent / assent, the subject's legally authorized representative has provided informed consent / assent and the subject has been provided with a written assent per regulations and / or guidelines before any study specific activities / procedures are initiated. -Male or female subjects must be between 0 and 18 years of age at the time of informed consent / assent. · Must be willing to provide local HSV-1 serostatus within 28 days prior to enrollment. Histologically or cytologically confirmed non-CNS solid tumors that have recurred following standard treatment or for which there is no available standard treatment. The presence of measurable (as defined by irRC-RECIST) or non-measurable disease as defined by modified irRC-RECIST. Subjects who are candidates for intralesional injections, defined as one or more of the following: - At least one injectable lesion ≥ 10 mm in longest dimension - Multiple injectable lesions, overall measuring ≥ 10 mm in longest dimension Note: Visceral lesions are not candidates for injection. Additionally, bone lesions are not candidates for injection unless there is a soft tissue component that is easily injectable. Performance Status: - For ages 12-18, Karnofsky performance status ≥ 70% - For children aged 0-12 years, the Lansky Play Scale must be ≥ 70% Life expectancy >4 months from the date of registration Adequate organ function as defined as: - Blood function (without the need for hematopoietic growth factors) Absolute neutrophil count (ANC) ≥ 1.0 × 10 9 / L Platelet count ≥ 75 × 10 9 / L Hemoglobin ≥ 8 g / dL (without transfusion support) - Kidney function For subjects with serum creatinine ≤ 1.5 × age upper limit of normal (ULN) or creatinine clearance ≥ 60 mL / min / 1.73 m for subjects with creatinine levels > 1.5 × age ULN, 2 (Note that creatinine clearance does not need to be determined if baseline serum creatinine is ≦1.5×ULN for age. Creatinine clearance should be determined according to institutional standards.) - Liver function Serum bilirubin ≤ 1.5 × ULN for age or direct bilirubin ≤ ULN for age for subjects with total bilirubin levels > 1.5 × ULN for age Aspartate aminotransferase (AST) ≦2.5×ULN for age or ≦5×ULN for subjects with liver metastases Alanine aminotransferase (ALT) ≦2.5×ULN for age or ≦5×ULN for subjects with liver metastases - Coagulation function International normalized ratio (INR) or prothrombin time (PT) ≤ 1.5 × ULN for age Partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT) ≤ 1.5 × ULN for age Female subjects of childbearing potential must have a negative urine or serum pregnancy test within 72 hours prior to dosing. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

[0186] Exclusion criteria Have been diagnosed with leukemia, non-Hodgkin's lymphoma, Hodgkin's disease or other hematological malignancies - Radiation therapy to bone marrow within 6 weeks prior to enrollment, or within 3 months if prior radiation to at least 60% of the craniospinal axis or pelvis was received, or within 2 weeks if local palliative radiation therapy was received -CNS tumor or clinically active brain metastases -Primary intraocular melanoma or mucosal melanoma History or evidence of giant congenital melanocytic nevus, dysplastic nevus syndrome, or xeroderma pigmentosum History of other malignancies within the past 5 years, with the following exceptions: - Malignancies that have been treated with curative intent, have no known active disease, have not received chemotherapy for >5 years prior to enrollment, and are felt by the treating physician to be at low risk of recurrence · History or evidence of active autoimmune disease requiring systemic treatment (i.e. with disease-modifying agents, corticosteroids, or immunosuppressants). 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, such as: - Primary immunodeficiency disorders such as severe combined immunodeficiency - Concurrent opportunistic infections - Receiving systemic immunosuppressive therapy (>2 weeks prior to enrollment) including oral steroids (maintenance physiological replacement therapy is excluded). Subjects requiring discontinuous use of inhaled steroids or local steroid injections are not excluded from the study. - Active herpetic skin lesions or previous complications of herpes infection (e.g. herpetic keratitis or herpetic encephalitis) Previous treatment with talimogene laherparepvec or any other oncolytic virus Previous treatment with a tumor vaccine The need for intermittent or chronic treatment with antiherpes drugs (e.g., acyclovir) other than intermittent topical use -Has received chemotherapy, radiation therapy, or biological cancer therapy within 28 days prior to enrollment, or has not recovered from an adverse event resulting from a cancer therapy administered more than 28 days prior to enrollment (Common Terminology Criteria for Adverse Events (CTCAE) Grade 1 or higher) Currently receiving treatment with another investigational medical device or drug, or have been treated with another investigational medical device or drug for less than 28 days. Receiving other investigational treatments while participating in this study is excluded. -Having undergone major surgery ≤28 days prior to enrollment - Patients are expected to require other cancer therapy during this study, except for local palliative radiation therapy. Have acute or chronic active hepatitis B or C virus infection, or have been treated with nucleotide analogues 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 of starting study treatment. Known or suspected human immunodeficiency virus (HIV) infection -Having received a live vaccine within 28 days prior to enrollment No approved antiplatelet or anticoagulant medications within 7 days prior to infusion of talimogene laherparepvec, except for low-dose heparin needed to maintain patency of venous catheters. Female subjects must be pregnant, nursing, or planning to become pregnant during study treatment and for 3 months after the last dose of talimogene laherparepvec. Female subjects of childbearing potential who are not willing to use an acceptable method of effective contraception throughout study treatment and for 3 months after the last dose of talimogene laherparepvec. NOTE: An acceptable method of effective contraception will be specified in the informed consent / assent form. If required by local laws and regulations, additional country-specific contraception requirements will be outlined in the country-specific protocol addendum at the end of the protocol appendix section. Sexually active subjects and their partners who do not want to use male or female latex condoms to avoid viral transmission during sexual contact during treatment and within 30 days after treatment with talimogene laherparepvec Subjects who are known to be sensitive to any of the administered formulations or components Subject who is unlikely to be able to complete all protocol-required study visits or procedures and / or who, to the best of the subject's and the investigator's knowledge, is likely to be unable to comply with all required study procedures. If consulted, has a history or evidence of any psychiatric disorder, substance abuse, or any other clinically significant disorder, condition, or disease (other than those outlined above) that, in the opinion of the investigator or an Amgen physician, would jeopardize the subject's safety or interfere with study evaluations, treatment, or completion. Subjects who do not wish to minimize exposure of their blood or body fluids to individuals at higher risk for HIV-1 induced complications (immunosuppressed individuals, HIV-positive individuals, pregnant women, or infants under 1 year of age) during treatment with Talimogene Laherparepvec and for 28 days after the last dose of Talimogene Laherparepvec.

[0187] As defined by the inclusion and exclusion criteria, talimogene laherparepvec will be administered by intralesional injection into injectable cutaneous, subcutaneous, lymph node and other non-visceral tumors only. Therefore, the anticipated eligible tumor types for this study are: Bone sarcomas: Ewing's sarcoma and osteosarcoma Soft tissue sarcomas: rhabdomyosarcoma and non-rhabdomyosarcoma soft tissue sarcomas Neuroblastoma Melanoma

[0188] The first dose of talimogene laherparepvec is administered on day 1. 6 The maximum PFU / mL is 4.0 mL. 8 A maximum of 4.0 mL of PFU / mL (or 10 for dose de-escalation cohorts) 6 The second injection of 1000 mg of 10000 PFU / mL (maximum of 4.0 mL) is administered on day 21 (+3) after the first injection (i.e., on day 22 or later, but should not be delayed more than 3 days from the day 21 time point). 8 A maximum of 4.0 mL of PFU / mL (or 10 for dose de-escalation cohorts) 6 All subsequent injections, with a maximum of 4.0 mL of PFU / mL, will be administered every 14 (± 3) days. Treatment cycle intervals may be extended due to toxicity. The maximum amount of talimogene laherparepvec administered at any dose is 4.0 mL for any individual lesion and for any treatment. The recommended amount of talimogene laherparepvec injected into the tumor will depend on the size of the tumor and will be determined according to the injection volume guidelines in Table 12. Using the priority model below and the injection volume guidelines based on tumor size, it is recommended that each lesion should receive the maximum volume possible to inject due to tumor characteristics at each visit before moving on to the next lesion.

[0189] [Table 14]

[0190] On each treatment day, the injection priority is recommended as follows: 1. Any new injectable tumors that have appeared since the previous injection 2. By tumor size, start with the largest tumor 3. Any tumors previously seen that were too small to inject at the time of tumor evaluation that are now large enough to inject

[0191] Subjects will receive talimogene laherparepvec until they achieve a complete response (CR), are free of injectable tumor, have confirmed progressive disease (PD) according to modified irRC-RECIST, are intolerant to study treatment, require alternative anticancer therapy or end of study 24 months after the date and time of the first dose of talimogene laherparepvec, whichever occurs first. Due to the mechanism of action, subjects may experience growth of existing tumors or the appearance of new tumors prior to the maximal clinical benefit of talimogene laherparepvec. Therefore, modified irRC-RECIST will be used for response assessment.

Claims

1. 1. A pharmaceutical composition for use in a method for treating B cell lymphoma, comprising: the pharmaceutical composition comprises an oncolytic virus; the oncolytic virus is a herpes simplex virus type 1 that (i) does not contain an intact ICP34.5 gene, (ii) does not contain an intact ICP47 gene, and (iii) contains a gene encoding human GM-CSF; The pharmaceutical composition, wherein the oncolytic virus is administered in combination with an anti-glucocorticoid-induced TNFR-related gene (GITR) antibody.

2. 2. The pharmaceutical composition according to claim 1, wherein the herpes simplex virus type 1 is talimogene laherparepvec.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the oncolytic virus is administered in combination with a checkpoint inhibitor and the anti-GITR antibody is AMG228 (9H6v3).

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