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

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts, under § 119(e), the benefits of the Provisional U.S. Patent Application No. 62 / 471,875, filed on 15 March 2017, which is incorporated herein by reference. [Background technology]

[0002] In light of advances in cancer treatment over the past few decades, cancer mortality rates for the most common types of cancer, including lung, colon, breast, and prostate cancer, continue to decline for both men and women. This improvement in survival rates is likely attributable to advances in the early diagnosis of certain cancers, improved treatments, and public health efforts that encourage preventive measures and screening.

[0003] Nevertheless, cancer remains a significant public health problem, with more than 16 million people diagnosed each year. In addition, a cancer diagnosis has profound consequences for patients, their families, and friends. Indeed, in the United States, cancer remains the second leading cause of death (only heart disease surpasses it), accounting for nearly one in four deaths. (See progressreport.cancer.gov / introduction, accessed March 8, 2017.)

[0004] The desired goal of cancer therapy is to preferentially kill cancer cells without causing harmful effects on normal cells. Several methods have been employed to achieve this goal, including surgery, radiation therapy, chemotherapy, and oncolytic virus therapy.

[0005] Local treatments such as radiotherapy and surgery provide ways to reduce tumor burden within accessible areas of the body through surgical techniques or high-dose radiotherapy. However, the primary approach to cancer treatment is chemotherapy. However, chemotherapy agents have limited effectiveness in treating many types of cancer, including many common solid tumors. This failure is partly due to drug resistance (whether acquired or endogenous) in many tumor cells. A major obstacle to the use of chemotherapy agents is their serious side effects. These include bone marrow suppression, nausea, vomiting, hair loss, and oral ulcers.

[0006] Proposed alternative therapies include the administration of oncolytic viruses and the use of viral vectors to deliver transgenes with anticancer activity. Genetic engineering of viruses for use as oncolytic agents initially focused on the use of replication-deficient viruses in an attempt to prevent viral-induced damage to non-tumor cells. The main limitation of this approach was that these replication-deficient viruses required helper viruses to enable integration and / or replication in host cells. These viruses have limited efficacy because each replication-deficient retroviral particle can only enter a single cell and thereafter cannot proliferate and infect other cells. Therefore, they cannot spread away from the producer cell and cannot completely invade many tumor cells in vivo. More recently, genetic engineering of oncolytic viruses has focused on producing "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 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 ICP47 deletion results in early expression of US11, a gene that promotes viral growth within tumor cells without reducing tumor selectivity. In addition, a coding sequence for human GM-CSF is inserted into the viral genome at the former ICP34.5 gene site. See Lie et al., Gene Ther., 10:292-303, 2003.

[0008] Therapeutic combinations of oncolytic viruses and checkpoint inhibitors are being explored. For example, the combination of tarimodine-laharpalepbek 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, nivolumab (anti-PD-1 antibody), and atezolizumab (anti-PD-L1 antibody) have demonstrated efficacy in various tumor types. See Grosso et al., Cancer Immun., 13:5 (2013); Pardoll, Nat Rev Cancer, 12:252-264 (2012); and Chen et al., Immunity, 39:1-10 (2013). [Prior art documents] [Non-patent 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) [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] However, there remains a need to further develop effective cancer therapies with reduced side effects (for example, compared to chemotherapy). There also remains a need to further develop cancer therapies that are effective against metastatic cancer. This invention addresses these and other needs. [Means for solving the problem]

[0011] In one embodiment, the present invention relates to 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 (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 dose of oncolytic virus. In some embodiments, the cancer is metastatic cancer. In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus may be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene. Herpes simplex virus type 1 can also be modified so that (i) it does not contain the intact ICP34.5 gene and (ii) it does not contain the intact ICP47 gene. In yet another embodiment, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene, (ii) it does not contain the intact ICP47 gene and (iii) it contains the gene encoding GM-CSF (e.g., human GM-CSF). In a particular embodiment, the oncolytic virus is talimogene laharpalebeck.

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

[0013] In one specific embodiment, the present invention also relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective dose of an oncolytic virus (e.g., tarimodine-laharpalepbek) and (ii) a therapeutically effective dose of a CTLA-4 blocker (e.g., an anti-CTLA-4 antibody, e.g., ipilimumab). In some embodiments, this cancer is metastatic. In another embodiment, the present invention relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective dose of an oncolytic virus (e.g., tarimodine-laharpalepbek) and (ii) a therapeutically effective dose of a PD-L1 blocker (e.g., an anti-PD-L1 antibody, e.g., atezolizumab). In other embodiments, the present invention relates to a method for treating B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer) by administering (i) a therapeutically effective dose of an oncolytic virus (e.g., tarimodyne-laharpalepbek) and (ii) a therapeutically effective dose 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 for treating B-cell lymphoma by administering (i) a therapeutically effective dose of an oncolytic virus and (ii) a therapeutically effective dose of a GITR agonist. In some embodiments, the cancer is metastatic B-cell lymphoma. In certain embodiments, the GITR agonist is AMG228 (also known as 9H6v3), TRX518, MEDI1873, or MK-4166. See PCT International Publication No. 2015031667 and U.S. Patent No. 9,464,139 (both of which are incorporated herein by reference in their entirety). In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus may be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene. Herpes simplex virus type 1 can also be modified so that (i) it does not contain the intact ICP34.5 gene and (ii) it does not contain the intact ICP47 gene. In yet another embodiment, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene, (ii) it does not contain the intact ICP47 gene and (iii) it contains the gene encoding GM-CSF (e.g., human GM-CSF). In a particular embodiment, the oncolytic virus is talimogene laharpalebeck.

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

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

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

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

[0019] [Figure 1] This study demonstrates the effect of intratumoral administration of talimogene laharpalebeck on tumor volume in A-673 Ewing sarcoma in Balb / c nude mice. [Figure 2] This study demonstrates the effect of intratumoral administration of talimogene laharpalebbek on tumor volume in SK-N-AS neuroblastomas in Balb / c nude mice. [Figure 3] This study demonstrates the effect of intratumoral administration of talimogene laharpalepbek on tumor volume in G-401 rhabdomyosarcoma-like tumors in Balb / c nude mice. [Figure 4] This study demonstrates the effect of intratumoral administration of talimogene laharpalepbek on tumor volume in SJSA-1 osteosarcoma in Balb / c nude mice. [Figure 5] This study demonstrates the effect of intratumoral administration of talimogene laharpalebbek on tumor volume in SJCRH30 rhabdomyosarcoma in Balb / c nude mice. [Figure 6] This shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimozine laharpalebeck in WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines. [Figure 7] This shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimodyne-laharpalebeck in HCT-116 (colorectal) and SK-MEL-5 (melanoma) cell lines. [Figure 8] This shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimodyne-laharpalebeck in HUT-78 (CTCL) and RPMI 8226 (multiple myeloma) cell lines. [Figure 9] This shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimodyne-laharpalebeck in CT-26 and MC-38 (colorectal) cell lines. [Figure 10a] This study demonstrates the effect of three doses of OncoVexmGM-CSF—3 × 10⁴ PFU, 3 × 10⁵ PFU, and 3 × 10⁶ PFU—on the volume of injected tumors in A20 tumor-bearing animals. [Figure 10b] This study demonstrates the effect of three doses of OncoVexmGM-CSF—3 × 10⁴ PFU, 3 × 10⁵ PFU, and 3 × 10⁶ PFU—on the volume of uninjected (contralateral) tumors in A20 tumor-bearing animals. [Figure 10c] This shows the effect of three doses of OncoVexmGM-CSF on the median survival time of A20 tumor-bearing animals treated with 3 × 10⁴ PFU, 3 × 10⁵ PFU, and 3 × 10⁶ PFU. [Figure 10d] This study demonstrates the effects of administering OncoVexmGM-CSF, anti-CTLA-4 mAb, and a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the body weight of mice. [Figure 10e] This study demonstrates the effects of administering OncoVexmGM-CSF, anti-CTLA-4 mAb, and a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the body weight of mice. [Figure 10f] This study demonstrates the effect of three doses of OncoVexmGM-CSF—5 × 10⁴ PFU, 5 × 10⁵ PFU, and 5 × 10⁶ PFU—on the volume of injected tumors in mice carrying neuro2a neuroblastoma tumors. [Figure 10g] This shows the effect on median survival time of mice carrying neuro2a neuroblastoma tumors treated with three different doses of OncoVexmGM-CSF: 5 × 10⁴ PFU, 5 × 10⁵ PFU, and 5 × 10⁶ PFU. [Figure 10h] This study demonstrates the effect of 5 × 10⁶ PFU of OncoVexmGM-CSF on the volume of injected (treated) and uninjected (contralateral / untreated) tumors in mice carrying neuro2a neuroblastoma tumors. [Figure 10i] This shows the effect of 5 × 10⁶ PFU of OncoVexmGM-CSF on the median survival time of mice carrying neuro2a neuroblastoma tumors. [Figure 11a] This study demonstrates the effects of treating A20 tumor-bearing animals with OncoVexmGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 11b] This study demonstrates the effect of treating A20 tumor-carrying animals with OncoVexmGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the median survival time of mice. [Figure 11c] This study demonstrates the effects of treating A20 tumor-bearing animals with OncoVexmGM-CSF, an anti-PD-L1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-L1 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 11d] This study demonstrates the effect of treating A20 tumor-carrying animals with OncoVexmGM-CSF, an anti-PD-L1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-L1 mAb on the median survival time of mice. [Figure 12a] This study demonstrates the effects of treating CT-26 tumor-bearing animals with OncoVexmGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 12b] This study demonstrates the effect of treating CT-26 tumor-carrying animals with OncoVexmGM-CSF, anti-CTLA-4 mAb, or a combination of OncoVexmGM-CSF and anti-CTLA-4 mAb on the median survival time of mice. [Figure 12c] This study demonstrates the effects of treating CT-26 tumor-bearing animals with OncoVexmGM-CSF, an anti-PD-L1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-L1 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 12d]This study demonstrates the effect of treating CT-26 tumor-carrying animals with OncoVexmGM-CSF, an anti-PD-L1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-L1 mAb on the median survival time of mice. [Figure 12e] This shows the quantification of systemic (splenic) anti-AH1 CD8+ T cells in CT-26 tumor-bearing mice treated with OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor, using ELISpot or dextramer staining with FACS. [Figure 12f] This shows the quantification of systemic (splenic) anti-AH1 CD8+ T cells in CT-26 tumor-bearing mice treated with OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor, using ELISpot or dextramer staining with FACS. [Figure 12g] This shows the quantification of localized (tumor) anti-AH1 CD8+ T cells in CT-26 tumor-bearing mice treated with OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor. [Figure 13a] This paper shows the effect on the volume of injected tumors of treatment in B16F10 nectin-1 tumor-carrying mice treated with control, OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor. [Figure 13b] This shows the evaluation of lung metastatic burden (demonstrated by the number of lung metastases) in tumor-bearing mice after treatment with a control, OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor. [Figure 13c] This study demonstrates the effect of treatment with a control group or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor on the median survival time of tumor-bearing mice. [Figure 13d]The results show that after treatment with the control, OncoVexmGM-CSF, a CTLA-4 inhibitor, or a combination of OncoVexmGM-CSF and a CTLA-4 inhibitor, B cells remained predominantly in the peritumoral region, while macrophages were prominent both within the tumor and within the dense cellular infiltration in the peritumoral region. [Figure 14] This study demonstrates the effect of treatment of 4T1 tumor-bearing mice with either a control or OncoVexmGM-CSF on the volume of injected tumors. [Figure 15a] This study demonstrates the effects of treating A20 tumor-bearing animals with OncoVexmGM-CSF, an anti-GITR mAb, or a combination of OncoVexmGM-CSF and an anti-GITR mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 15b] This study demonstrates the effect of treating A20 tumor-carrying animals with OncoVexmGM-CSF, an anti-GITR mAb, or a combination of OncoVexmGM-CSF and an anti-GITR mAb on median survival time in mice. [Figure 16a] This study demonstrates the effects of administering OncoVexmGM-CSF, an anti-PD-1 mAb, and a combination of OncoVexmGM-CSF and an anti-PD-1 mAb on the body weight of mice. [Figure 16b] This study demonstrates the effects of treating MC-38 tumor-carrying animals with OncoVexmGM-CSF, an anti-PD-1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-1 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 17] This study demonstrates the effects of treating MC-38 tumor-carrying animals with OncoVexmGM-CSF, an anti-PD-L1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-L1 mAb on the volume of directly injected and uninjected (contralateral) tumors. [Figure 18] This study demonstrates the effect of treating B16F10 tumor-carrying animals with OncoVexmGM-CSF, an anti-PD-1 mAb, or a combination of OncoVexmGM-CSF and an anti-PD-1 mAb on the volume of directly injected tumors. [Modes for carrying out the invention]

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

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

[0022] Oncolytic virus As discussed herein, the present invention demonstrates that oncolytic viruses can exert 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, for example, the antitumor effect is accompanied by less severe / negative side effects 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 for 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 another embodiment, the present invention relates to the use of a combination of oncolytic viruses and checkpoint inhibitors for the treatment of B-cell lymphoma, colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (for example, triple-negative breast cancer).

[0023] In some embodiments, the oncolytic virus is herpes simplex virus. This herpes simplex virus may be herpes simplex virus type 1. In some embodiments, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene. In other embodiments, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene and (ii) it does not contain the intact ICP47 gene. In yet another embodiment, herpes simplex virus type 1 is modified so that (i) it does not contain the intact ICP34.5 gene, (ii) it does not contain the intact ICP47 gene and (iii) it contains the gene encoding GM-CSF (e.g., human GM-CSF). In certain embodiments, the oncolytic virus is talimogene laharpalebeck.

[0024] Talimogene laharpalebeck, HSV-1 [JS1 strain] ICP34.5- / ICP47- / hGM-CSF (formerly OncoVex) GM-CSFTarimozine Laharpalebeck is an intratumor-delivered oncolytic immunotherapy containing an immunoenhancing HSV-1 that selectively replicates within solid tumors (Lui et al., Gene Therapy, 10:292-303, 2003; U.S. Patent Nos. 7,223,593 and 7,537,924). HSV-1 was derived from the JS1 strain deposited with the European Certified Cell Culture Agency (ECAAC) under registration number 01010209. In Tarimozine Laharpalebeck, the HSV-1 viral gene encoding ICP34.5 is functionally deleted. The loss of function of ICP34.5, which acts as a pathogenic factor during HSV infection, limits replication in non-dividing cells and makes the virus nonpathogenic. The safety of HSV with functionally deleted ICP34.5 has been demonstrated in multiple clinical trials (MacKie et al, Lancet 357:525-526, 2001; Markert et al, Gene Ther 7:867-874, 2000; Rampling et al, Gene Ther 7:859-866, 2000; Sundaresan et al, J. Virol 74:3822-3841, 2000; Hunter et al, J Virol Aug;73(8):6319-6326, 1999). In addition, ICP47 (which blocks viral antigen presentation to major histocompatibility complex class I and II molecules) is functionally deleted from talimozine laharpalebeck. Functional deletion of ICP47 also results in early expression of US11, a gene that promotes viral proliferation in tumor cells without reducing tumor selectivity. As used herein, “lacking a functional viral gene” means that a gene is partially or completely deleted, replaced, rearranged, or otherwise modified in the herpes simplex virus genome so 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, is inserted into the viral genome of talimogene laharpalebeck (at two previous sites in the ICP34.5 gene).The insertion of the gene encoding human GM-CSF is carried out in such a way that it replaces almost the entire ICP34.5 gene, ensuring that any potential recombination event between tarimozine-laharpalebeck and wild-type virus can only result in an incapacitated, non-pathogenic virus and cannot lead to the generation of wild-type viruses carrying the gene for human GM-CSF. The HSV thymidine kinase (TK) gene remains intact in tarimozine-laharpalebeck, making the virus susceptible to antiviral agents such as acyclovir. Therefore, acyclovir can be used to block tarimozine-laharpalebeck replication if necessary.

[0025] Examples of additional HSV genes that may be modified include ICP6, a large subunit of ribonucleotide reductase involved in nucleotide metabolism and viral DNA synthesis in non-dividing cells but not in dividing cells. The earliest genes encoding the thymidine kinase responsible for the phosphorylation of acyclovir to acyclovir monophosphate, the virion transactivator protein vmw65, glycoproteins H, vhs, ICP43 and ICP4, ICP27, ICP22, and / or ICP0 may also be modified.

[0026] Modifications can also be made to alter the timing of herpes simplex virus gene expression. 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. 5824318, Mohr & Gluzman (1996) EMBO 15:4759-4766).

[0027] As those skilled in the art will understand, 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] Tarimozine-Laharpalebeck produces a direct oncolytic effect through viral replication in tumors and induces an enhanced antitumor immune response through local expression of GM-CSF and release of tumor-derived antigens via lysis. Since many cancers exist as both primary and secondary (i.e., metastatic) tumors in patients, this dual activity is beneficial as a therapeutic treatment. Intended clinical effects include the destruction of injected tumors, the destruction of distant, uninjected tumors in the localized area, a reduction in the formation of new metastases, a reduction in the overall rate of disease progression and recurrence after treatment of the initial disease, and an extension of overall survival.

[0029] As used herein, the terms “patient” and “subject” are interchangeable and mean mammals, including but not limited to humans and non-human mammals such as cattle, horses, dogs, sheep, or cats. Preferably, the patient is human.

[0030] Tarimozine Laharpalebeck and OncoVex mGM-CSF Tarimozine-Laharpalebeck (an HSV-1 virus with the same genetic modification as Tarimozine-Laharpalebeck, except that human GM-CSF is replaced with mouse GM-CSF) has been tested for efficacy in various in vitro (cell line) and in vivo mouse tumor models, demonstrating that it eradicates tumors or substantially inhibits their growth at doses comparable to those used in clinical trials. Nonclinical evaluations have also confirmed that GM-CSF enhances the resulting immune response, enhancing both the response of injected and uninjected tumors, and that increased surface levels of MHC class I molecules result from the deletion of ICP47. Tarimozine-Laharpalebeck has been injected into normal and tumor-bearing mice, and its safety has been evaluated. In general, the virus has shown good tolerance, up to 1 × 10⁶ 8 The PFU / dosage ratio did not indicate any safety concerns. (See, for example, Liu et al., Gene Ther 10:292-303, 2003).

[0031] Clinical trials are underway or currently underway in several advanced tumor forms, with over 400 patients treated with tarimodine-laharpalepbek (see, for example, 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 suggest that tarimodine-laharpalepbek may offer overall clinical benefit in patients with advanced melanoma. In particular, a high rate of complete response was achieved in stage IIIc to IV melanoma (Scenzer et al., J.Clin.Oncol.271(12):907-913, 2009). In addition, responses were observed in both injected and uninjected areas, including visceral tissue.

[0032] The virus of the present invention may also be derived from herpes simplex virus type 2 (HSV-2) strains or derivatives thereof. Derivative strains include intertyped recombinants containing DNA from HSV-1 and HSV-2 strains. Such intertyped 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] Herpes simplex virus strains may originate from clinical isolates. Such strains are isolated from infected individuals, such as those with recurrent herpes simplex. As described in U.S. Patent Nos. 7,063,835 and 7,223,593 (each of which is incorporated in whole by reference), clinical isolates may be screened against standard laboratory strains for desired capabilities or characteristics, such as enhanced replication in tumors and / or other cells in vitro and / or in vivo. In one embodiment, the herpes simplex virus is a clinical isolate from recurrent herpes simplex.

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

[0035] Further examples of modified herpes simplex viruses include, but are not limited to, the Seprehvir(trademark)(HSV1716)17+ strain of herpes simplex virus type 1, which has a 759 bp deletion located within each copy of the BamHI fragment (0-0-02 and 0-81-0.83 map units) of the long repeat region of the HSV genome, removing one complete copy from the 18 bp DR to the "a" sequence element, and terminating 1105 bp upstream of the 5' end of the earliest (IE) gene 1 (see MacLean et al., (1991) Journal of General Virology 79:631-639).

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

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

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

[0039] M032 is a herpes simplex virus that has deletions in 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 loss of function in the genes encoding vhs, ICP47, ICP34.5, UL43, and US5.

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

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

[0043] Herpesvirus strains and methods for producing such strains are described in U.S. Patent Nos. 5,824,318, 6,764,675, 6,770,274, 7,063,835, 7,223,593, 7,749,745, 7,744,899, 8273,568, 8420,071, and 8470,577, WIP. This information is also found in International Publication Nos. 199600007, 199639841, 199907394, 200054795, 2006002394, and 201306795, Chinese Patent 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 regulate certain types of immune system cells, such as T cells (which play a central role in cell-mediated immunity). While immune checkpoints help suppress the immune response, they can also prevent 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 kill cancer cells more effectively.

[0045] As used herein, the terms “immune checkpoint inhibitor” or “checkpoint inhibitor” refer to molecules that completely or partially reduce, inhibit, interfere with, or modulate one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer 12:252-264, 2012). These proteins are responsible for co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins control and maintain autoimmune tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors may contain or be derived from antibodies.

[0046] Checkpoint inhibitors may include small molecule inhibitors, or antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that 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 (belonging to the CD2 family of molecules and expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. Examples of 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 antibodies or antibody-binding fragments thereof, other binding proteins, biopharmaceuticals, or small molecules that bind to one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049 and block or inhibit the activity of one or more of these proteins.

[0047] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint molecule that downregulates the T cell activation pathway. CTLA-4 is a negative regulator of T cell activation. Blocking 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 enhance T cell activation through two distinct mechanisms to enhance the anti-tumor immune response against tumor antigens released after cytolytic replication of the virus within the tumor. Therefore, the combination of herpes simplex virus and anti-CTLA-4 antibody can increase the destruction of injected and uninjected / distal tumors, improve the overall tumor response, and, in particular, extend overall survival when compared to that obtained 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, playing a role in their fate / differentiation. PD-1's two ligands, PD-L1 and PD-L2, are members of the B7 family. PD-1 restricts T cell activity in peripheral tissues during inflammatory responses to infection, and limiting autoimmune PD-1 blockade in vitro enhances T cell proliferation and cytokine production in response to challenge by specific antigen targets or allogeneic cells during mixed lymphocyte reactions. A strong correlation between PD-1 expression and response has been demonstrated by PD-1 blockade (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). PD-1 blockade can be achieved through various mechanisms, including antibodies that bind to PD-1 or PD-L1.

[0049] Programmed cell death ligand 1 (PD-L1), also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene. See Entrez Gene:CD274 and the CD274 molecule. PD-L1 is a 40 kDa type 1 transmembrane protein that plays a role in the suppression of 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 activator gene-3 (LAG-3) inhibitors, such as IMP321 and soluble Ig fusion proteins (Brignone et al., 2007, J.Immunol.179:4202-4211). Also included are B7 inhibitors, such as B7-H3 and B7-H4 inhibitors (e.g., anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin.Cancer Res.July 15(18)3834)). Another checkpoint inhibitor is TIM3 (T cell immunoglobulin domain and mucin domain 3) (Fourcade et al., 2010, J. Exp.Med.207:2175-86 and Sakuishi et al., 2010, J.Exp.Med.207:2187-94).

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

[0052] Accordingly, in one aspect of the present invention, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4, PD-1, PD-L1, or PD-L2. In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4 such as tremelimumab, ipilimumab (also known as 10D1, MDX-D010), BMS-986249, AGEN-1884, and an anti-CTLA-4 antibody (described in U.S. Patents No. 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, respectively, which are incorporated herein by reference).In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of PD-L1 or PD-1 (e.g., a molecule that inhibits the interaction between PD-1 and PD-L1 and / or PD-L2 inhibitors), 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 antibody). -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 PD-L2 / PD-1 interaction), MEDI4736 (durvalumab; anti-PD-L1 antibody), MSB0010718C (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 the brochures of International Publication Nos. 03042402, 2008156712, 2010089411, 2010036959, 2011066342, 2011159877, 2011082400, and 2011161699 (each of which is incorporated herein by reference).Additional anti-PD-1 antibodies include PDR-001, SHR-1210, BGB-A317, BCD-100, JNJ-63723283, PF-06801591, BI-754091, JS-001, AGEN-2034, MGD-013, LZM-009, GLS-010, MGA-012, AK-103, genolimzumab, dostarlimab, semiprimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

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

[0054] BMS 936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1. Phase 1 trial results showed that bi-weekly administration of this drug resulted in sustained responses, particularly in patients with melanoma. Objective response rates ranged from 6% to 17% depending on the type of cancer in patients with advanced NSCLC, melanoma, RCC, or ovarian cancer, with some patients experiencing responses lasting for more than one year.

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

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

[0057] GITR Agonist The glucocorticoid-inducible TNFR-related gene (GITR:TNFRSF18), sometimes called an activation-inducible TNFR family member (AITR), is a receptor belonging to the TNF receptor superfamily (TNFRSF). It is activated by its congener ligand, the GITR ligand (GITRL, TNFSF18). GITR is a type I transmembrane protein containing a cysteine-rich extracellular domain, a 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 investigated as a means of promoting the proliferation of CD8+ T effector memory cell populations, while simultaneously promoting the elimination or inhibition of Treg cells.

[0058] The suppression of responder T cell co-stimulation and regulatory T cell suppression means that GITR activation leads to an enhancement of the immune response. Such activation has the potential to restore the immune response to infection and tumors. Therefore, molecules that can activate GITR may have value as immunostimulants in situations where inducing an enhancement of the immune response is desirable.

[0059] As further described herein, in one aspect, the present 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 present 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-inactive 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 for NCT02583165 (progressive 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 patients 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 patients with advanced solid tumors).

[0066] INCAGN01876 is an anti-GITR agonist monoclonal antibody. INCAGN01876 is currently being investigated in clinical trials, including NCT02697591 (in patients 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 patients with advanced cancer or lymphoma).

[0068] Treatment methods for diseases or disorders The present invention also relates to methods for treating diseases or disorders such as cancer. In some embodiments, 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, 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, cancer is metastatic cancer.

[0069] The term "metastatic cancer" refers to cancer that has spread from the part of the body where it originated (i.e., the primary site) to other parts of the body. When cancer spreads to new areas (i.e., metastasizes), it is still named according to the part of the body where it originated. For example, colon cancer that has metastasized to the pancreas is not called pancreatic cancer, but rather "metastatic colon cancer to the pancreas." Treatment is also based on where the cancer originated. Even if colon cancer metastasizes to the bone, it is still colon cancer, and the relevant physician will recommend treatment that has been 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 for treating a disease or disorder, such as cancer, by administering (i) a therapeutically effective amount of oncolytic virus and (ii) a therapeutically effective amount of 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 may be any of those described herein. In some embodiments, the oncolytic virus is herpes simplex virus (e.g., herpes simplex virus type 1). In other embodiments, herpes simplex virus type 1 is modified so that it does not contain the intact ICP34.5 gene. In yet another embodiment, herpes simplex virus type 1 is modified so that it does not contain the intact ICP34.5 gene and does not contain the intact ICP47 gene. In yet another embodiment, herpes simplex virus type 1 is modified so that it does not contain the intact ICP34.5 gene and does not contain the intact ICP47 gene and does contain a gene encoding GM-CSF (e.g., human GM-CSF). In a specific embodiment, the oncolytic virus is talimogene laharpalebeck.

[0073] Checkpoint inhibitors can be any molecule that blocks or inhibits an inhibitory pathway in 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 (all NK, γδ and memory CD8 molecules belonging to the CD2 family). + (Expressed on αβ)T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. Examples of 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 their antibody-binding fragments), biopharmaceuticals, or small molecules that bind to one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049 and block or inhibit the activity of one or more of these proteins.

[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. Patents 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, respectively, which are incorporated herein by reference).Examples of molecules that inhibit the interaction between PD-1 and 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), and 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 PD-L2 / PD-1 interaction), MEDI4736 (durvalumab; anti-PD-L1 antibody), MSB0010718C (avelumab; anti-PD-L1 antibody), and U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8, Examples include those described in Specifications No. 168,757, No. 8,217,149, and the PCT Published Patent Application International Publication Brochures No. 03042402, No. 2008156712, No. 2010089411, No. 2010036959, No. 2011066342, and No. 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, semiprimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

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

[0076] In many cases, cancer exists in patients both as a primary tumor (i.e., a tumor that grows in an anatomical site where tumor progression begins and continues to result in a cancerous mass) and as a secondary tumor or metastasis (i.e., the spread of the tumor from its primary site to other parts of the body). The oncolytic viruses of the present invention may be effective in treating tumors through lytic and systemic immune effects. For example, with respect to talimogene-Laharpalebeck, the virus physically lyses the tumor, causing primary tumor cell death. In addition, [1] the lysis of tumor cells subsequently releases tumor-derived antigens that are recognized by the immune system, and [2] the production of GM-CSF assists in inducing an anti-tumor immune response. It is thought that both of these mechanisms result in a systemic immune response, thereby enabling the immune system to recognize and attack both primary and secondary / metastatic tumors. In embodiments in which the oncolytic virus is combined with a checkpoint inhibitor, the checkpoint inhibitor is thought to further enhance the systemic immune response by enhancing priming and reducing the inhibitory effect of immune checkpoint proteins on immune system cells such as T cells. Furthermore, in embodiments in which an oncolytic virus is combined with a GITR agonist, the GITR agonist is thought to further enhance the systemic immune response by promoting the proliferation of CD8+ T effector memory cell populations, thereby promoting the elimination or inhibition of Treg cells. Therefore, the present invention aims to treat primary tumors, metastatic tumors (i.e., secondary tumors), or both, using an oncolytic virus (e.g., tarimodyne laharpalepbek) alone or in combination with a checkpoint inhibitor.

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

[0078] The method of the present invention can be used to treat cancer at several different stages. Most disease classification systems include information on whether the cancer has spread near 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 malignancy of the tumor (i.e., the level of cellular abnormality, the likelihood of the tumor growing and spreading). For example, stage 0 refers to the presence of abnormal cells that have not spread near tissues—i.e., cells that could become cancerous. Stages I, II, and III cancers refer to the presence of cancer. As the stage increases, the cancerous tumor grows larger and spreads more near 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 composition 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 compositions may contain, for example, formulation materials for modifying, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or osmosis of the composition. Pharmacoactive agents can be administered to patients by various routes, such as oral or parenteral, including intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intracisional, intratumoral, intravascular, and intradermal, or by passive or enhanced absorption through the skin, for example, using a skin patch or transcutaneous iontophoresis. In one embodiment, an oncolytic virus (e.g., tarimodyne laharpalepbek) is injected into the tumor (i.e., via intratumoral injection). In another embodiment, a 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 will be able to determine the dosage and duration of treatment according to any aspect of this disclosure. For example, a person skilled in the art can monitor the patient to determine whether treatment should be initiated, continued, interrupted, or resumed. The effective dose 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 dosage of administration. Clinicians use parameters known in the art to determine the appropriate dose. The effective dose of a therapeutically used pharmaceutical composition will depend, for example, on the background and purpose of the treatment. A person skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the molecules thus delivered, the indications for which the conjugated drug molecules are used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age and overall health). Therefore, clinicians may potentiate the dose and modify the route of administration to obtain the optimal therapeutic effect.

[0081] Clinical studies have demonstrated that talimogene laherparepvec can be injected directly into visible and palpable skin lesions, subcutaneous lesions, or lymph node lesions, or can be injected using ultrasound guidance. Thus, in one aspect, a pharmaceutical composition comprising talimogene laherparepvec is administered via intralesional injection. Talimogene laherparepvec is currently provided in 1 mL disposable vials at a fixed dosing concentration: 10 6 pfu / mL for the initial dose and 10 8 pfu / mL for subsequent doses (Reske, et al. J Immunol, 2008, 180(11): p.7525 - 36). The volume injected can vary depending on the tumor type. For example, talimogene laherparepvec is administered at a maximum dose of 4.0 mL at 10 6 plaque - forming units / mL (PFU / mL) on day 1 of week 1, followed by a maximum dose of 4.0 mL at 10 8 PFU / mL on day 1 of week 4, and then administered by intratumoral injection every 2 weeks (±3 days) in injectable skin tumors, subcutaneous tumors, and lymph node tumors. In another embodiment, talimogene laherparepvec is administered at a maximum dose of 4.0 mL at 10 6 plaque - forming units / mL (PFU / mL) on day 1 of week 1, followed by a maximum dose of 4.0 mL at 10 7 PFU / mL on day 1 of week 4, and then administered by intratumoral injection every 2 weeks (±3 days) in injectable skin tumors, subcutaneous tumors, and lymph node tumors. The recommended volume of talimogene laherparepvec injected into the tumor can be determined according to the size of the tumor, in accordance with the injection volume guidelines shown in Table 1 (and as shown in Patent Application PCT / US Patent Application Publication No. 2013 / 057542, which is incorporated herein by reference).

[0082]

Table 1

[0083] In general, all appropriately injectable lesions should be injected at the maximum effective dose for each individual administration case. On each treatment day, the following is recommended for prioritizing injections: any new injectable tumors that have appeared since the last injection; starting with the largest tumor by size; and any currently injectable, previously uninjected tumors.

[0084] The compositions of the present invention may include one or more additional components, including physiologically acceptable carriers, excipients, or diluents. For example, the composition may include one or more of the following: a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., having fewer than 10 amino acids), a protein, amino acids, carbohydrates such as glucose, sucrose, or dextrin, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient. Acceptable carriers include, for example, neutral buffered saline or saline mixed with certain serum albumin. Preservatives such as benzyl alcohol may also be added. The composition may be formulated as a lyophilized product using a suitable excipient solution (e.g., sucrose) as a diluent.

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

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

[0087] In one embodiment, an anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of approximately 1 mg / kg to 3 mg / kg every two weeks, for example, approximately 1 mg / kg, 2 mg / kg, or 3 mg / kg. In another embodiment, an anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of approximately 2 mg / kg at three-week intervals. In yet another embodiment, nivolumab may be administered at a dose of approximately 1 mg / kg to 5 mg / kg, for example, 3 mg / kg, over a period of 60 minutes, approximately once a week to once every two, three, or four weeks.

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

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

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

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

[0092] In other embodiments, the present invention relates to a therapeutically effective dose of 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 yet another embodiment, the present invention relates to a therapeutically effective dose of oncolytic virus and checkpoint inhibitor for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), colorectal cancer, melanoma, head and neck squamous cell carcinoma, or breast cancer (e.g., triple-negative breast cancer). In an additional embodiment, the present invention relates to a therapeutically effective dose of oncolytic virus and GITR agonist for use in the treatment of B-cell lymphoma.

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

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

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

[0096] In some embodiments, the pharmaceutical compositions described herein are not used in combination with or with radiation therapy. In other embodiments, the pharmaceutical compositions described herein do not contain chemotherapeutic agents (e.g., cisplatin). In yet another embodiment, the pharmaceutical compositions described herein are not used in combination therapy with radiation and chemotherapeutic agents (e.g., cisplatin).

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

[0098] In another embodiment, the present invention relates to a kit comprising [1] an oncolytic virus in combination with an optional GITR agonist, and [2] instructions for administration to a patient. In other embodiments, the kit of the present invention may comprise an oncolytic virus (e.g., tarimodyne laharpalepbek), a GITR agonist (e.g., AMG228 (also known as 9H6v3), TRX518, MEDI1873, or MK-4166), and instructions for treating a patient with cancer (e.g., in a package insert or label).

[0099] In some embodiments, the kit containing talimogene laharpalebeck is administered on day 1 of week 1. 6 A maximum dose of 4.0 ml of PFU / mL, followed by 10 on the first day of the fourth week. 8 The instructions (e.g., in the package insert or label) include a maximum dose of 4.0 ml of PFU / mL, followed by intratumoral injection every two weeks (e.g., until complete response). In some embodiments, the kit containing talimogene-Laharpalepbek is administered on day 1 of week 1 with 10 6 A maximum dose of 4.0 ml of PFU / mL, followed by 10 on the first day of the fourth week. 7 The instructions (e.g., in the package insert or label) include a maximum dose of 4.0 ml of PFU / mL, followed by administration by intratumoral injection every two weeks (e.g., until complete response).

[0100] In embodiments in which the kit contains an anti-PD-1 antibody, the kit includes instructions (e.g., in the accompanying leaflet or label) for intravenous administration at the doses specified herein. Examples of anti-PD-1 antibodies include pembrolizumab and nivolumab.

[0101] In embodiments in which the kit contains an anti-PD-L1 antibody, the kit includes instructions (e.g., in the accompanying leaflet or label) for intravenous administration at the doses specified herein. An example of an anti-PD-L1 antibody is atezolizumab.

[0102] In embodiments in which the kit contains an anti-CTLA-4 antibody, the kit includes instructions (e.g., in the accompanying leaflet or label) for intravenous administration at the doses specified herein. An example of an anti-CTLA-4 antibody is ipilimumab.

[0103] In embodiments in which the kit includes a GITR agonist, the kit includes instructions (e.g., in the accompanying leaflet or label) for intravenous administration at the doses specified herein. Examples of anti-GITR antibodies include AMG 228, TRX518, MEDI1873, or MK-4166.

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

[0105] In some embodiments, the kits described herein are not used in combination with or with radiation therapy. In other embodiments, the kits described herein do not contain chemotherapeutic agents (e.g., cisplatin). In yet another embodiment, the kits described herein are not used in combination therapy with radiation and chemotherapeutic agents (e.g., cisplatin). [Examples]

[0106] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

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

[0108] The antitumor efficacy of talimogene-laharpalebeck was evaluated in several mouse xenograft studies in Balb / c nude mice. Tumor cells (A-673 human pediatric Ewing's sarcoma, SJCRH30 human pediatric rhabdomyosarcoma, G-401 human pediatric rhabdomyosarcoma-like tumor, SK-N-AS human pediatric neuroblastoma, or SJSA-1 human pediatric osteosarcoma) were transplanted into the right flank of each mouse by subcutaneous injection. In all cases, 5 × 10¹⁴ cells were administered in 100–200 μL of 50% Matrigel / 50% DMEM. 6 ~1 × 10 7 Individual cells were transplanted into mice.

[0109] Tumor measurements were taken twice a week. Treatment with tarimodine-laharpalepbek was initiated when the tumor reached an average diameter of 4–6 mm. Three different doses of tarimodine-laharpalepbek were administered (5 × 10). 4 , 5×10 5 or 5 x 10 6 PFU / dose (50 μL dose) was administered intratumorally over three days. Body weight, overall clinical findings, and tumor measurements were obtained twice a week. Animals were euthanized when the tumor weight exceeded 10% of body weight.

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

[0111] Ewing's sarcoma A-673 Ewing sarcoma-carrying mice were treated with talimogene-laharpalebeck in 5 × 10⁶ doses. 4 , 5×10 5 or 5 x 10 6 Therapeutic treatment was performed in terms of PFU / dose. Tarimodine / Laharpalepbek was administered once daily by intratumor injection on days 8, 11, and 14 of the study (red arrows in Figure 1). Tumors were measured 2-3 times per week. Results were expressed as a function of time in mm, with day 0 being the day of tumor intake. 3The mean tumor volume of each group is expressed as the mean tumor volume with respect to the standard error of the mean (n=10 per group unless otherwise noted for the vehicle control group on days 16, 20, and 23). An asterisk indicates that all talimogene-laharpalebeck groups had p<0.0001 compared to the vehicle control on day 23 of the trial.

[0112] The results are shown in Figure 1.

[0113] Neuroblastoma SK-N-AS neuroblastoma-carrying mice were treated with talimogen-laharpalebbek 5 × 10⁻¹⁵ 4 , 5×10 5 or 5 x 10 6 The tumors were therapeutically treated with PFU / dose. Tarimodine / Laharpalepbek was administered once daily by intratumor injection on days 6, 9, and 12 of the study (red arrows in Figure 2). Tumors were measured 2-3 times per week. Results were expressed as a function of time in mm, with day 0 being the day of tumor intake. 3 Expressed as the mean tumor volume of the group with ± the standard error of the mean (5 × 10⁻¹⁰ 4 Unless otherwise noted for the PFU / dose group, n=10 per group. Asterisks indicate 5 × 10 compared to the formulation buffer control on day 23 of the study. 6 and 5×10 5 For the PFU / dose group, p<0.0001, and 5×10 4 This shows that p=0.0001 for the PFU / dose group.

[0114] The results are shown in Figure 2.

[0115] Rhabdomyosarcoma-like tumor G-401 rhabdomyosarcoma-like tumor-carrying mice were treated with talimogene-laharpalebeck 5 × 10⁶ times. 4 , 5×10 5 or 5 x 10 6Therapeutic treatment was performed in terms of PFU / dose. Tarimodine / Laharpalepbek was administered once daily by intratumor injection on days 14, 17, and 20 of the study (red arrows in Figure 3). Tumors were measured 2-3 times per week. Results were expressed as a function of time in mm, with day 0 being the day of tumor intake. 3 The mean tumor volume of each group is expressed as the standard error of the mean (n=10 per group unless otherwise noted). An asterisk indicates that all tarimozine / laharpalepbek-treated groups had a p<0.0001 ratio compared to the vehicle control at day 40 of the trial.

[0116] The results are shown in Figure 3.

[0117] Osteosarcoma SJSA-1 osteosarcoma-carrying mice were treated with talimogen-laharpalebeck 5 × 10⁶ times. 4 , 5×10 5 or 5 x 10 6 Therapeutic treatment was performed in terms of PFU / dose. Tarimodine / Laharpalepbek was administered once daily by intratumor injection on days 9, 12, and 15 of the study (red arrows in Figure 4). Tumors were measured 2-3 times per week. Results were expressed as a function of time in mm, with day 0 being the day of tumor intake. 3 This is expressed as the mean tumor volume of the group with ± the standard error of the mean (n=10 per group).

[0118] The results are shown in Figure 4.

[0119] Rhabdomyosarcoma SJCRH30 rhabdomyosarcoma-carrying mice were treated with talimogene-laharpalebbek 5 × 10⁶ times. 4 , 5×10 5 or 5 x 10 6 Therapeutic treatment was performed in terms of PFU / dose. Tarimodine / Laharpalepbek was administered once daily by intratumor injection on days 8, 11, and 14 of the study (red arrows in Figure 5). Tumors were measured 2-3 times per week. Results were expressed as a function of time in mm, with day 0 being the day of tumor intake. 3Expressed as the mean tumor volume of the group with ± the standard error of the mean (5 × 10⁻¹⁰ 4 The PFU / dose group and the 5×10 group on days 30 and 33. 6 Unless otherwise noted, n=10 per group for each PFU / dose group. An asterisk indicates that p<0.0001 was observed for all tarimozine-laharpalebeck-administered groups compared to the vehicle control on day 26 of the study (the last day of the study in which all control animals were still in the study).

[0120] The results are shown in Figure 5.

[0121] Example 2: Talimogene Laharpalebeck 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-Laharpalepbek was started at 100 MOI and serially diluted in nine wells (1:4 serial dilution). After 72 hours of incubation, the number of cells remaining in each well was quantified using the CellTiter-Glo Luminescent cell viability assay (Promega, Madison, WI).

[0122] Tarimozine Laharpalebbek was effective in 14 of 21 DLBCL cell lines at MOIs below 100. Five cell lines (SU-DHL-2, OCI-LY-3, TMD8, RI-1 (ABC subtype), and WSU-NHL (GCB subtype)) showed an MOI of 1 or less IC. 50The most sensitive cells were found at 1 MOI (Table 2). In contrast, OCI-LY-1, KARPAS422, WSU-DLCL2, SU-DHL-4, SU-DHL-10, and OCI-LY-7 (all GCB subtypes) showed resistance to talimozine-laharpalepbek at a maximum MOI of 100 MOI (Table 2). Inhibition of cell proliferation was observed at an MOI of 1 IC. 50 The sensitivity was lower than that observed in most cell lines, with the highest level observed in most cell lines. Figure 6 shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimozine-laharpalebeck in WSU-NHL (GCB subtype) and TMD8 (ABC subtype) DLBCL cell lines. These results demonstrate that treating DLBCL cell lines with tarimozine-laharpalebeck results in potent inhibition of DLBCL tumor cell proliferation.

[0123] [Table 2]

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

[0125] Tarimozine-Laharpalebeck was effective against all 13 melanoma and cancer cell lines tested. All tested cell lines had an MOI IC of less than 1. 50This was shown (Table 3). Figure 7 shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimozine-laharpalebeck 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 tarimozine-laharpalebeck results in potent inhibition of tumor cell proliferation.

[0126] [Table 3]

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

[0128] Tarimodine-Laharpalebeck was effective against all five cell lines tested (Table 4). Multiple myeloma cell lines showed higher sensitivity than cutaneous T-cell lymphoma cell lines. Figure 8 shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimodine-Laharpalebeck 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 tarimodine-Laharpalebeck results in potent inhibition of tumor cell proliferation.

[0129] [Table 4]

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

[0131] Tarimodine-Laharpalebeck was effective against four of the five cell lines tested (Table 5). The B16F10 melanoma cell line demonstrated resistance to Tarimodine-Laharpalebeck. This resistance is mediated by the absence 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 cancer) cell lines showed an MOI of approximately 0.2 IC. 50 Similar sensitivity was observed. Figure 9 shows the degree of cell proliferation inhibition achieved by increasing the concentration of tarimodine-laharpalebeck in CT-26 and MC-38 (colorectal cancer) cell lines. These results demonstrate that treatment of mouse tumor cell lines (melanoma, colorectal cancer, and B-cell lymphoma) with tarimodine-laharpalebeck results in potent inhibition of tumor cell proliferation.

[0132] [Table 5]

[0133] Example 4: OncoVex mGM-CSF It inhibits the proliferation of B-cell lymphoma and neuroblastoma in mouse models. A20 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2 × 10⁻¹⁰ 6 Individual cells). Tumor volume (mm²). 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3 Once the mice reached this stage, they were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) and the variability of tumor volume at the start of treatment were uniform across the treatment group. Then, the mice were given OncoVex. mGM-CSF (3×10 4 ~3×10 6 Three intratumoral injections of PFU / dosage or vehicle were administered on days 10, 13, and 16. Clinical signs, weight changes, and survival time (tumor size 800 mm) were monitored. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0134] A20 OncoVex in tumor-carrying animals mGM-CSF The treatment involves all three dosages: 3 x 10 4 PFU, 3x10 5 PFU, 3x10 6 PFU resulted in 100% complete regression of all directly injected tumors (Figure 10a). Colorectal tumors were 3 × 10 4 PFU dose did not show a response, 3 × 10 5 PFU doses showed 50% tumor growth, and 3 × 10 6 PFU doses showed 100% inhibition of tumor growth (Figure 10b). Median survival was 3 × 10⁻⁶ compared to the vehicle. 5 PFU (p=0.0054) and 3×10 6 The PFU dose (p=0.0004) group showed a significantly longer duration (38 days vs. 21 days, respectively - Figure 10c). No weight loss was observed, indicating that the tested treatment was safe and tolerable (Figures 10d and 10e). Table 6 shows the percentage of tumor-free subjects in each group.

[0135] In addition, mice carrying neuro2a neuroblastoma tumors were used in OncoVex mGM-CSF 5 x 104 , 5×10 5 or 5 x 10 6 Treatment was administered using PFU / dose (n=10 per group). OncoVex mGM-CSF The drug was administered once daily by intratumor injection on days 10, 13, and 16 of the study (Figure 10f). The injected tumor was measured twice a week. The results were expressed as a function of time in mm, where day 0 is the day of tumor intake. 3 It is expressed as the individual tumor volume. The median survival time is 5 × 10 compared to the vehicle. 4 PFU / dosage group (p=0.0056), 5×10 5 PFU / dosage group (p<0.0001) and 5×10 6 The PFU / dosage group showed a significantly longer duration (p<0.0001) (Figure 10g).

[0136] OncoVex effect on untreated tumors in mice carrying neuro2a neuroblastoma tumors mGM-CSF The effectiveness of the treatment was also evaluated. Neuro2a tumor cells were subcutaneously implanted into the left and right flanks of female A / J mice on day 0. Tumor volume (mm3) was measured twice a week (Q2W) using electronic calipers. The tumors averaged approximately 100 mm. 3 Once the mice reached this stage, they were randomized into groups (10 mice per group) such that the mean tumor volume (in both flanks) and the variability of tumor volume at the start of treatment were uniform across the treatment group. Then, the mice were given OncoVex. mGM-CSF (5×10 6 Three intratumor injections of PFU / dosage or vehicle were administered to the right side ("treated" side) on days 10, 13, and 16. Tumor volume and survival time (tumor volume 800 mm on either side) 3 If the threshold was reached, the mouse was removed from the test. The threshold was measured twice a week until the end of the test (Figure 10h). OncoVex of neuro2a tumor-carrying animals. mGM-CSF Treatment with OncoVex resulted in complete regression in 8 out of 10 directly injected tumors (Figure 10h). Contralateral uninjected ("untreated") tumors showed a significant delay in tumor growth (Figure 10h). Median survival was better with OncoVex compared to the vehicle.mGM-CSF It significantly extended in the treatment group (32 days vs. 18 days, p < 0.0001, Figure 10i).

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

[0138] [Table 6]

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

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

[0141]

Table 7

[0142] Treatment of A20 tumor-bearing animals with OncoVex, anti-PD-L1 mAb or the combination of OncoVex mGM-CSF and anti-PD-L1 mAb resulted in complete regression of all directly injected tumors (Figure 11c). Tumors treated with intraperitoneal anti-CTL4-4 alone (bilateral flanks) showed no effect on tumor growth. The contralateral tumors showed some consistent effect with OncoVex alone, while the combination of OncoVex mGM-CSF and anti-PD-L1 mAb showed some consistent effect with OncoVex alone, while the combination of OncoVex mGM-CSF showed some consistent effect with OncoVex alone, while the combination of OncoVex mGM-CSFThe combination with an anti-PD-L1 mAb caused all tumors to regress and resulted in complete cure in 10 out of 10 mice. Median survival time was better with OncoVex compared to the vehicle. mGM-CSF Survival time was significantly extended in both the single-dose and combination groups. In addition, a significant extension in median survival time was measured in the combination group compared to anti-PD-L1 mAb monotherapy. The combination was performed using OncoVex. mGM-CSF When compared to other studies, an increase in the trend in overall survival was observed, but no statistical significance was observed (OncoVex). mGM-CSF (p=0.067 for [compared to] and p=0.0013 for anti-PD-L1 mAb). The median survival time was 23 days in the vehicle and 23 days in the OncoVex group. mGM-CSF The median survival time for the combined group was 48 days, compared to 26 days for the anti-PD-L1 mAb group. The median survival time for the combined groups remained indeterminate beyond day 40, at which point all 10 mice showed no signs of tumor (Figure 11d and Table 7b).

[0143] [Table 8]

[0144] These results suggest that OncoVex, when combined with either a PD-L1 or CTLA-4 inhibitor, is effective in treating established B-cell lymphoma tumors in an in vivo mouse model. mGM-CSF Treatment with [agent] has demonstrated better inhibition of tumor growth compared to any single agent alone. Antitumor activity was observed in directly injected tumors (presumably via oncolysis and immune response) and in uninjected contralateral tumors in the same host (presumably via adaptive immune response).

[0145] Example 6: OncoVex mGM-CSF It inhibits the growth of colorectal tumors in mouse models, either alone or in combination with CTLA-4 or PD-L1 inhibitors. CT-26 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2 × 10⁻¹⁰ 6Individual cells). Tumor volume (mm²). 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3 Once the animals reached this point, they were randomized into four groups (10 mice per group) so that the mean tumor volume (in both flanks) and tumor volume variability at the start of treatment were uniform across the treatment group. The animals were then divided into a) PBS + IgG control and b) OncoVex mGM-CSF +IgG control, c) PBS + anti-CTTLA-4 mAb or PBS + anti-PD-L1 mAb, or d) OncoVex mGM-CSF + Anti-CTLA-4 mAb or OncoVex mGM-CSF +Anti-PD-L1mAb was administered. Clinical signs, weight change, and survival time (tumor size 800mm) 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0146] OncoVex in CT-26 tumor-carrying animals mGM-CSF Treatment with anti-CTLA-4 mAbs or a combination thereof resulted in tumor growth inhibition in approximately 75% of all directly injected tumors by day 20 (data not shown). Contralateral tumors were treated with OncoVex. mGM-CSF While the anti-CTLA-4 mAb alone did not show a response (approximately 25% TGI), tumor growth inhibition was observed in approximately 75% of the anti-CTLA-4 mAb and combination groups. Median survival was significantly longer in the anti-CTLA-4 mAb and combination groups compared to the vehicle-only group (Figure 12b). The median survival was 20 days in the vehicle-only group and 20 days in the OncoVex group. mGM-CSF The median survival time was 22 days in the group and 41 days in the anti-CTLA-4 group. The median survival time for the combination group was longer than 50 days and remained undetermined when the experiment was discontinued. The median survival time for the combination group was as follows: OncoVex mGM-CSF The response time was significantly longer than in either the monotherapy group (p=0.0001) or the anti-CTLA-4 mAb monotherapy group (p=0.0031) (Figure 12b and Table 8a).

[0147] [Table 9]

[0148] OncoVex in CT-26 tumor-carrying animals mGM-CSF or OncoVex mGM-CSF Treatment with a combination of [unclear] and an anti-PD-L1 mAb resulted in tumor growth inhibition in approximately 75% of all directly injected tumors by day 18 (data not shown). Intraperitoneal injection of anti-PD-L1 mAb had little effect on tumor growth (either flank). Contralateral tumors were treated with OncoVex. mGM-CSF While the combination group did not show a response to either anti-PD-L1 mAb monotherapy, approximately 75% of tumor growth inhibition was observed (Figure 12c). Median survival was significantly longer in the combination group compared to either drug monotherapy (OncoVex). mGM-CSF (p=0.012 for vehicle and p=0.007 for anti-PD-L1 mAb). The median survival time was 21 days for vehicle and anti-PD-L1 mAb, compared to OncoVex. mGM-CSF The duration was 23 days for the group and 34 days for the combined group (Figure 12d and Table 8b).

[0149] [Table 10]

[0150] These results indicate that, 1) OncoVex, a method for treating established colorectal tumors in an in vivo mouse model. mGM-CSF 2) OncoVex mGM-CSF 3) A combination of this and an anti-CTLA-4 inhibitor, or 3) OncoVex mGM-CSF Treatment with a combination of this and an anti-PD-L1 inhibitor has been shown to provide potent inhibition of tumor growth. Antitumor activity is demonstrated by 1) OncoVex mGM-CSF 2) OncoVex mGM-CSF and combination with anti-CTLA-4 inhibitors, and 3) OncoVex mGM-CSF Antitumor activity was observed in tumors directly injected with a combination of 1) OncoVex and an anti-PD-L1 inhibitor (presumably via tumor lysis and immune response). mGM-CSF2) Combination with an anti-CTLA-4 inhibitor, and 2) OncoVex mGM-CSF This was observed in uninjected contralateral tumors (presumably via an adaptive immune response) in the same host treated with a combination of anti-PD-L1 inhibitors.

[0151] OncoVex mGM-CSF To further understand the antitumor activity of the combination of 1) OncoVex mGM-CSF 2) Anti-CTLA-4 inhibitors, and 3) OncoVex mGM-CSF The ability of the combination of [anti-CTLA-4 inhibitor] to release tumor antigens and stimulate an antitumor-specific T cell response 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. OncoVex mGM-CSF Splenocytes (8×10) at day 10 of CT-26 tumor-carrying mice treated with anti-CTLA-4 mAb or a combination thereof. 5 The ) was incubated with a control peptide (GFP) or AH1 peptide (SPSYVYHQF) at a final concentration of 1 μM at 37°C for 20 hours. The AH1 peptide is an immunodominant Ag derived from the envelope protein (gp70) of endogenous murine leukemia virus presented by the MHC class ILd molecule (25). Spots were counted using a CTLS6 Fluorospot analyzer (CTL, Shaker Height, OH). Systemic (splenic) anti-AH1 CD8 + Quantification of T cells by dextramer staining using ELISpot or FACS is performed by 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 a combination of [specific agent] and an anti-CTLA-4 inhibitor (Figure 12e, 12f). [Specific agent] Focal (tumor) anti-AH1 CD8 +Quantification of T cells showed a significant increase only in the combination group. A significant decrease in Tregs was also observed in the CT-26 model. This effect was greater in combination with the CTLA-4 inhibitor (Figure 12g). This experiment was conducted by OncoVex. mGM-CSF The combination of this compound with an anti-CTLA-4 inhibitor resulted in an increase in the presence of effector cells and a decrease in the presence of regulatory T cells, which in turn demonstrated an increase in the efficacy of the combination compared to either compound alone.

[0152] Example 7: OncoVex mGM-CSF It inhibits the growth of melanoma tumors in mouse models, either alone or in combination with CTLA-4 inhibitors. B16F10 cells (5 x 10 4 - Due to the lack of HSV-1 entry receptors, OncoVex mGM-CSF A lysozyme (resistant to lysis) was administered intravenously on day 0. On day 2, B16F 10 melanoma cells transfected with mouse nectin 1 (OncoVex) were administered. mGM-CSF A drug (sensitive to lysis) was subcutaneously injected into the right flank of female BL6 mice. Tumor volume (mm 3 The subcutaneous tumor was measured twice a week (Q2W) using an electronic caliper. The average subcutaneous tumor size was approximately 100 mm. 3 Once the animals reached this point, they were randomized into four groups (10 mice per group) so that the mean tumor volume and tumor volume variability at the start of treatment were uniform across all treatment groups. The animals were then given OncoVex. mGM-CSF (5×10 6 Three intratumoral injections of PFU (per dose), four intraperitoneal injections of anti-CTLA-4 mAb, OncoVex mGM-CSF (5×10 6 A combination of three intratumor injections of PFU / dose and four intraperitoneal injections of anti-CTLA-4 mAb, or vehicle alone, was administered. Clinical signs, weight change, and survival time (tumor size 800 mm) were monitored. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0153] OncoVex mice carrying B16F10 nectin 1 tumorsmGM-CSF or OncoVex mGM-CSF Treatment with a combination of 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). Lung metastasis burden was assessed, and OncoVex was used to inhibit lung metastases. mGM-CSF The combination of [unclear] and an anti-CTLA-4 mAb was shown to be 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 longer in the combination group compared to the vehicle (p<0.0001). The median survival was 30 days in the vehicle group and 30 days in the OncoVex group. mGM-CSF In the anti-CTLA-4 mAb group, the duration was 46 days (Figure 13c and Table 9).

[0154] [Table 11]

[0155] These results indicate that OncoVex is effective in treating engrafted melanoma tumors in an in vivo mouse model. mGM-CSF or OncoVex mGM-CSF Treatment with a combination of this and an anti-CTLA-4 mAb has been shown to provide potent inhibition of tumor growth. These results are evident from the ability of the combination to inhibit lung metastases, as demonstrated by OncoVex. mGM-CSF The combination of the drug and an anti-CTLA-4 mAb also demonstrates a significantly more robust systemic antitumor effect compared to either treatment alone. Antitumor activity was observed in directly injected tumors (presumably via tumor lysis and immune response) and in uninjected contralateral tumors in the same host (presumably via adaptive immune response).

[0156] Vehicle, OncoVex mGM-CSFFurthermore, in the anti-CTLA-4 mAb group, lung tumors were rarely observed to show scattered T and B cells in the peritumoral region and mild intratumoral macrophages. Interestingly, OncoVex mGM-CSF In the group treated with the anti-CTLA-4 mAb combination, tumors showed a variable number of T cells predominantly in the peritumoral region and T cell infiltration into the tumor. Macrophages were dominant in both the tumor and densely infiltrating cells in the peritumoral region. B cells remained exclusively in the peritumoral region (Figure 13d and data not shown).

[0157] The results of these lung tumors' immune infiltration were found in OncoVex. mGM-CSF This demonstrates that the combination of this drug and an anti-CTLA-4 inhibitor can convert converted, insufficiently invasive B16F10 metastases (i.e., "cold" tumors) into fully invasive B16F10 metastases (i.e., "hot" tumors).

[0158] Example 8: OncoVex mGM-CSF This inhibits the growth of triple-negative breast cancer tumors in a mouse model. 4T1 tumor cells were subcutaneously injected into the right flank of female BALB / c mice on day 0 (2 × 10⁻¹⁰ 6 Individual cells). Tumor volume (mm²). 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3 Once the animals reached this point, they were randomized into four groups (10 mice per group) so that the mean tumor volume and tumor volume variability at the start of treatment were uniform across all treatment groups. The animals were then given OncoVex. mGM-CSF (5×10 4 , 5×10 5 or 5 x 10 6 Three intratumor injections of PFU / dosage or vehicle were administered. Clinical signs, weight changes, and survival time (tumor size 800 mm) were monitored. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0159] OncoVex in 4T1 tumor-carrying animals mGM-CSF The treatment by 5 × 106 OncoVex demonstrated approximately 75% inhibition of tumor growth at the PFU / dose level (p<0.0001). mGM-CSF 5 x 10 4 and 5×10 5 The dose did not result in any measurable inhibition of tumor growth (Figure 14).

[0160] These results are from OncoVex for established triple-negative breast cancer tumors. mGM-CSF This study demonstrates that treatment with [the substance] results in potent inhibition of tumor growth. Antitumor activity was measured only in tumors directly injected with the substance.

[0161] Example 9: OncoVex mGM-CSF It inhibits the growth of B-cell lymphoma tumors in mouse models, either alone or in combination with a GITR agonist. A20 tumor cells were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0 (2 × 10⁻¹⁰ 6 Individual cells). Tumor volume (mm²). 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3 Once the animals reached this point, they were randomized into four groups (10 mice per group) so that the mean tumor volume (in both flanks) and the variability of tumor volume at the start of treatment were uniform across the treatment group. The animals were then given OncoVex. mGM-CSF or OncoVex mGM-CSF The drug was administered in combination with an anti-GITR mAb. Clinical signs, weight changes, and survival time (tumor size 800 mm) were monitored. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

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

[0163]

Table 12

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

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

[0166] Syngeneic MC-38 tumor cells were inoculated into both the right and left flanks of C57BL / 6 mice. The tumors grew to an average diameter of 5 mm (60 mm). 3 (Tumor volume) Animals were assigned to 6 groups 10 days after inoculation (n=10 per group). OncoVEX muGM-CSF (5×10 6 PFU / dose) or a formulation buffer control was administered intratumorally to the right-sided tumor once daily, every three days for a total of three doses. The left-sided tumor did not receive injections.

[0167] Anti-mouse PD-1 (clone RMP1-14, BioXCell) or isotype control antibody (rat IgG2a, clone 2A3, BioXCell) was administered intraperitoneally twice a week at either 1 mg / dose or 300 μg / dose (started on day 10 of the study and ended on day 30 (7 doses)). Tumor volume, body weight, and overall clinical findings were collected 2-3 times a week for both the injected (right side) and uninjected (left side) tumors. Animals were euthanized when the total tumor volume (right side + left side) reached >10% of body weight, or when the animal showed signs of distress. Peripheral blood was collected for immunophenotyping analysis on days 14 and 20 of the study (4 and 10 days after the start of administration, respectively). After erythrocyte 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 experimental period and showed no evidence of adverse health effects associated with the treatment, as is evident from 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 weight in the treatment group compared to the control group is attributable to more invasive tumor growth in the control animals compared to the treated animals, as will be discussed below.

[0169] Tumor growth inhibition is achieved with anti-mPD-1 antibody (in both tested doses of 300 μg and 1 mg per dose) or OncoVex.muGM-CSF This was observed in response to monotherapy treatment with either of the drugs (Figure 16b). Table 11 summarizes the number of animals with no tumor (regressed) at the end of the experiment, either on the right (injected) or left (uninjected) flank. Monotherapy activity with either drug was limited to 10–20% complete regression in injected tumors (and no complete regression in uninjected tumors), while the combination resulted in 80–90% regression in injected tumors (and 10–20% complete regression in uninjected tumors). These data were compiled using OncoVex. muGM-CSF This study demonstrates that combination therapy with anti-PD-1 resulted in substantially improved tumor clearance in a mouse MC-38 tumor model.

[0170] [Table 13]

[0171] These results are from OncoVex muGM-CSF It has been shown to inhibit the growth of colorectal MC-38 tumors, either alone or in combination with a PD-1 inhibitor.

[0172] Example 11: OncoVex in a mouse colon (colorectal) adenocarcinoma (MC-38) tumor model muGM-CSF A trial to evaluate combinations of groups and anti-PD-L1 inhibitors. This study used OncoVex in a mouse MC-38 tumor model. muGM-CSF , anti-PD-L1 inhibitor or OncoVex muGM-CSF The study was designed to evaluate the tolerability and antitumor activity of the combination of this drug and an anti-PD-L1 inhibitor.

[0173] MC-38 tumor cells were subcutaneously injected into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm²) 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3Once the target was reached, the animals were randomized into four groups (10 mice per group) so that the mean tumor volume and tumor volume variability at the start of treatment were uniform across all treatment groups. (OncoVex) mGM-CSF (5×10 6 PFU / dosage) or formulation buffer control was administered intratumorally in three injections every three days, either alone or in combination with an anti-PD-L1 mAb (clone MIH5, mouse IgG1) or control IgG1 (the mAb was administered a total of four times). Tumors that were not injected (contralateral; on the left side of the animal) did not receive injections. Clinical signs, weight change, and survival time (tumor was 800 mm) were monitored. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0174] All animals survived the experimental period and showed no evidence of adverse health effects related to the treatment, as evident from their body weight, nor were there any notable adverse clinical signs identified by daily health monitoring examinations.

[0175] Tumor growth inhibition is achieved with OncoVex in both the injected tumor and the contralateral tumor. mGM-CSF This was observed in response to monotherapy with either an anti-PD-L1 mAb or (Figure 17), whereas OncoVex mGM-CSF The combination with an anti-PD-L1 mAb caused regression in all injected tumors, as well as 7 out of 10 injected tumors.

[0176] Example 12: OncoVex in a mouse melanoma (B16F10) tumor model muGM-CSF A trial evaluating combinations of the group and anti-PD-1 inhibitor. This study used OncoVex in a mouse melanoma (B16F10) tumor model. muGM-CSF , anti-PD-1 inhibitor or OncoVex muGM-CSF The design was intended to evaluate the tolerability and antitumor activity of the combination of this drug and an anti-PD-1 inhibitor.

[0177] B16F10 tumor cells, engineered to express mNectin, were subcutaneously injected into the right flank of female C57BL / 6 mice on day 0. Tumor volume (mm²) 3 The tumor size was measured twice a week (Q2W) using an electronic caliper. The tumor size averaged approximately 100 mm. 3 Once the target was reached, the animals were randomized into four groups (10 mice per group) so that the mean tumor volume and tumor volume variability at the start of treatment were uniform across all treatment groups. (OncoVex) mGM-CSF (5×10 6 PFU / dosage) or formulation buffer control was administered intratumorally every 3 days for 3 doses, either alone or in combination with an anti-PD-1 mAb (clone 29F1A12, mouse IgG1) or control IgG1 (mAbs were administered a total of 4 times). Clinical signs, weight change, and survival time (tumor size 800 mm) were assessed. 3 (If the threshold was reached, the mouse was removed from the test.) Measurements were taken 2-3 times per week until the end of the test.

[0178] All animals survived the experimental period and showed no evidence of adverse health effects related to the treatment, as evident from their body weight, nor were there any notable adverse clinical signs identified by daily health monitoring examinations.

[0179] Tumor growth inhibition is achieved through OncoVex. mGM-CSF Tumor regression was observed in response to monotherapy (3 out of 10 mice showed tumor regression), while anti-PD-1 mAb monotherapy had no inhibitory effect on tumor growth (Figure 18). OncoVex mGM-CSF The combination with an anti-PD-1 mAb caused regression in 5 out of 10 injected tumors (Figure 18), and OncoVex mGM-CSF We demonstrated that the combination of this drug and an anti-PD-1 mAb exhibits superior antitumor activity compared to monotherapy.

[0180] Example 13: Phase 1, multicenter, open-label, dose-deescalation study to evaluate the safety and efficacy of tarimodine laharpalepbek in pediatric patients with progressive non-central nervous system tumors that are easily administered by direct injection. This example describes a Phase 1, multi-center, open-label, dose-escalation trial to evaluate the safety and efficacy of talimogene laherparepvec in pediatric subjects with progressive non-central nervous system tumors that are easily injectable. See the website of the US National Institutes of Health (clinicaltrials.gov), trial identifier: NCT 02756845 (which is incorporated herein by reference).

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

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

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

[0184] Secondary endpoints include (1) evaluating tarimodine-laharpalepbek, assessed by overall response rate (ORR), duration of response (DOR), time to first response (TTR), time to progression-free survival (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) evaluating the association between granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors / subunits in archived tumor tissue and clinical outcomes (safety endpoints such as ORR, DOR, TTR, TTP, PFS, and OS, as well as efficacy endpoints).

[0185] Selection Criteria If the subject is too young to submit informed consent / ascent, a legally authorized representative of the subject has submitted informed consent / ascent, and the subject has been provided with an ascent written in accordance with the regulations and / or guidelines before any trial-specific activity / procedure is initiated. • The subject, whether male or female, must be between 0 and 18 years of age at the time of informed consent / ascent. • Applicants should be willing to submit their local HSV-1 serological status within 28 days prior to registration. • The tumor is a histologically or cytologically confirmed non-CNS solid tumor that has recurred after standard treatment or for which no standard treatment is available. • The presence of measurable (as defined by irRC-RECIST) or unmeasurable lesions as defined by modified irRC-RECIST. • Candidates for intralesional injection defined as one or more of the following: - At least one injectable lesion with a maximum diameter of ≥10 mm - Multiple injectable lesions with a total longest diameter of ≥10 mm. Note: Internal organ lesions are not candidates for injection. Furthermore, bone lesions are not candidates for injection unless there are soft tissue components that are easily injected. • Performance status: - For ages 12 to under 18, the Karnovsky Performance Status must be ≥70%. - For children aged 0 to under 12 years, the Lansky Play Scale score must be ≥70%. • The average life expectancy must be >4 months from the registration date. • Having sufficient organ function as defined below: - Blood function (without requiring hematopoietic growth factors) Absolute neutrophil count (ANC) ≥ 1.0 × 10 9 / L Platelet count ≥ 75 × 10 9 / L Hemoglobin level must be ≥8 g / dL (without transfusion support). - Kidney function For subjects with serum creatinine levels ≤ 1.5 × age upper limit of normal (ULN) or > 1.5 × age ULN, creatinine clearance ≥ 60 mL / min / 1.73 m 2 (Note that creatine clearance does not need to be determined if baseline serum creatinine is ≤1.5 × age ULN. Creatinine clearance should be determined according to institutional standards.) - Liver function Serum bilirubin is ≤1.5 × age ULN, or direct bilirubin is ≤age ULN for subjects with total bilirubin levels >1.5 × age ULN. Aspartate aminotransferase (AST) levels are ≤2.5 × age ULN, or ≤5 × age ULN for subjects with liver metastases. Alanine aminotransferase (ALT) levels are ≤2.5 × age ULN, or ≤5 × age ULN for subjects with liver metastases. - Coagulation function The International Normalized Ratio (INR) or Prothrombin Time (PT) must be ≤1.5 × age (ULN). The partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT) is ≤ 1.5 × age (ULN). This treatment is for women of childbearing age who must have a negative urine or serum pregnancy test within 72 hours prior to administration. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required.

[0186] Exclusion criteria • Diagnosed with leukemia, non-Hodgkin lymphoma, Hodgkin's disease, or other hematological malignancies. • Bone marrow radiation therapy must have been received within the last six weeks prior to registration, or within the last three months prior to registration if prior radiation was received to at least 60% of the craniospinal axis or pelvis, or within two weeks prior to registration if local palliative radiation therapy was received. • The presence of a CNS tumor or clinically active brain metastases. • The presence of primary intraocular melanoma or mucosal melanoma. • A history or evidence of giant congenital pleochromocytic nevus, dysplastic nevus syndrome, or xeroderma pigmentosum. • Except for the following exceptions, a history of other malignant tumors within the past five years: - Malignant tumors treated for therapeutic purposes, without known active disease, without chemotherapy in the prior 5 years prior to registration, and treated by the treating physician who perceives a low risk of recurrence. • A history or evidence of active autoimmune disease requiring systemic treatment (i.e., with disease modifiers, corticosteroids, or immunosuppressants). Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary dysfunction) is not considered a form of systemic treatment. • The following clinically significant immunosuppressions are present: - Primary immunodeficiency disorders such as severe combined immunodeficiency - Simultaneous opportunistic infections - Receiving systemic immunosuppressive therapy, including oral steroids, within the two weeks prior to enrollment (excluding maintenance physiological replacement therapy). Subjects requiring discontinuous use of inhaled steroids or topical steroid injections are not excluded from this study. • Having active herpetic skin lesions, or a history of complications from herpes infection (e.g., herpetic keratitis or herpetic encephalitis) • Previous treatment with talimogene, laharpalebeck, or any other oncolytic virus. • Previously received treatment with a tumor vaccine. • The need for discontinuous or chronic treatment with anti-herpes drugs (e.g., acyclovir) other than discontinuous topical use. • The individual must not have previously received chemotherapy, radiotherapy, or biological cancer therapy within 28 days prior to registration, or have recovered from an adverse event resulting from cancer therapy administered more than 28 days prior to registration to a Common Terminology Criteria for Adverse Events (CTCAE) grade of 1 or higher. • You are currently receiving treatment with another investigational medical device or drug, or have completed treatment with another investigational medical device or drug less than 28 days ago. Receiving other investigational treatment while participating in this study is excluded. • The applicant must have undergone major surgery ≤ 28 days prior to registration. • During this trial, it is anticipated that patients will require other cancer therapies besides local palliative radiotherapy. • Having acute or chronic active hepatitis B virus or hepatitis C virus infection, or having received treatment with a nucleotide analog such as those used for treating hepatitis B virus (e.g., lamivudine, adefovir, tenofovir, terbivudine, and entecavir), ribavirin, or interferon alfa within 12 weeks of the start of the test treatment. • Known or suspected human immunodeficiency virus (HIV) infection • Must have received a live vaccine within 28 days prior to registration. • No approved antiplatelet or anticoagulant medications have been taken within 7 days prior to the infusion of Tarimodine Laharpa Repvek, except for low-dose heparin necessary to maintain patency of the venous catheter. • During the study treatment and throughout the 3 months following the last dose of tarimodine / laharpalepbek, the female subject must be pregnant, breastfeeding, or planning to become pregnant. • The study must be for women of childbearing age who do not wish to use an acceptable method of effective contraception during the study procedure and throughout the three months following the last dose of tarimodine-laharpalepbek. Note: Acceptable methods of effective contraception are defined in the informed consent / ascent form. Any additional country-mandated contraceptive requirements, where required by ordinance and regulations, are outlined in the country-mandated protocol supplement at the end of the protocol appendix section. Sexually active individuals and their partners who do not wish to use male or female latex condoms to avoid virus transmission during sexual contact during treatment or within 30 days after treatment with Tarimozine Laharpalebeck. • Subjects known to be sensitive to either the administered formulation or its components during medication administration. • Subjects who are unable to attend the clinic or complete the required procedures for the trial as required by all protocols, and / or who are likely to be unable to comply with all required trial procedures to the best of the knowledge of the subject and the investigator. • When seeking consultation, the investigator or an Amgen physician must have a history or evidence of any mental disorder, substance abuse, or any other clinically significant disorder, condition, or illness (other than those outlined above) that they believe may pose a risk to the safety of the subject or interfere with the evaluation, treatment, or completion of the study. • During treatment with Tarimodine-Laharpalepbek and throughout the 28 days following the last dose of Tarimodine-Laharpalepbek, for subjects who do not wish to minimize exposure of their blood or bodily fluids to individuals at higher risk of HIV-1-induced complications (immunosuppressed individuals, HIV-positive individuals, pregnant women, or infants under 1 year of age).

[0187] Based on the definitions of the inclusion and exclusion criteria, tarimozine laharpalepbek will be administered by intralesional injection only to injectable cutaneous, subcutaneous, lymph node, and other non-visceral tumors. Therefore, the expected eligible tumor types for this study are as follows: • Bone sarcomas: Ewing's sarcoma and osteosarcoma • Soft tissue sarcoma: Rhabdomyosarcoma and non-rhabdomyosarcoma soft tissue sarcoma • Neuroblastoma • Melanoma

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

[0189] [Table 14]

[0190] On each treatment day, the recommended priority for injections is as follows: 1. Any new injectable tumors that have appeared since the last injection. 2. Start with the largest tumor, depending on its size. 3. Any tumors previously observed that were too small to be injected at the time of tumor evaluation, but are now large enough to be injected now.

[0191] Tarimodine-laharpalepbek will be administered to subjects until they achieve a complete response (CR), no injectable tumors remain, disease progression (PD) is confirmed according to the modified irRC-RECIST, tolerability of the study treatment deteriorates, or 24 months have passed since the first dose of tarimodine-laharpalepbek, whichever comes first, necessitating alternative anticancer therapy or termination of the study. Due to the mechanism of action, subjects may experience proliferation of existing tumors or the appearance of new tumors before the maximum clinical benefit of tarimodine-laharpalepbek is realized. Therefore, the modified irRC-RECIST will be used to evaluate response.

Claims

1. A pharmaceutical composition for use in the treatment of B-cell lymphoma, The aforementioned pharmaceutical composition contains an oncolytic virus, The oncolytic virus is herpes simplex virus type 1, which (i) does not contain the intact ICP34.5 gene, (ii) does not contain the intact ICP47 gene, and (iii) contains the gene encoding human GM-CSF. A pharmaceutical composition in which the oncolytic virus is administered together with a checkpoint inhibitor, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

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

3. The pharmaceutical composition according to claim 1 or 2, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

4. The pharmaceutical composition according to claim 3, wherein the anti-CTLA-4 antibody is ipilimumab.

5. The pharmaceutical composition according to claim 1 or 2, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

6. The pharmaceutical composition according to claim 5, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

7. The pharmaceutical composition according to claim 1 or 2, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

8. The pharmaceutical composition according to claim 7, wherein the anti-PD-L1 antibody is atezolizumab.

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